ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group

Transcription

ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Anatomy/Pathology
149 Ocular Tumors: Experimental therapeutics
Sunday, May 04, 2014 3:15 PM–5:00 PM
S 330CD Paper Session
Program #/Board # Range: 846–851
Organizing Section: Anatomy/Pathology
Program Number: 846
Presentation Time: 3:15 PM–3:30 PM
NFkB Inhibition in Uveal Melanoma – construction of a new
delivery system
Shahar Frenkel1, Dudi Shneor1, 2, Alik Honigman2, Jacob Pe’er1.
1
Ophthalmology, Hadassah-Hebrew University Medical Center,
Jerusalem, Israel; 2Biochemistry and Molecular Biology, IMRIC, The
Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Purpose: We have previously shown that NFkB inhibition leads to
diminished uveal melanoma cell viability in vitro, and to necrosis of
tumors in vivo in the mouse intrahepatic direct injection model. Here
we describe the design of a targeted delivery system which affects
only dividing cells - a recombinant Murine leukemia virus (MuLV)based replication-competent retroviruses (RCR) delivery system.
Methods: We exchanged the GFP sequence in the IRES-GFP cassette
of the MuLV-based RCR pACE with the NFkB nuclear localization
sequence VQRKRQKLMP (pACE-NFkB-NLS). Five uveal
melanoma cell lines were infected with the pACE-NFkB-NLS vector.
Infected cells were grown on “chamber-slides” without or with the
TNFα activator of NFkB, and the subcellular localization of NFkB
was analyzed with a laser scanning confocal microscope (Nikon).
Activity of NFkB was analyzed by transfecting infected cell with a
luciferase based reporter gene. Cell viability and caspase 3 activity
were determined using the Fluorescent Cell Viability and CaspaseGlo 3/7 Assays (Promega).
Results: Expression of the NLS peptide inside the cells created
a competition with the activated NFkB, and reduced its nuclear
localization and its ability to trans-activate target genes.
Conclusions: Inhibition of NFkB reduces cell viability and increases
apoptosis. This new modality for NFkB inhibition which can spread
through solid tumors is expected to better affect tumors in vivo than
IV/IP administration of chemotherapeutics.
Commercial Relationships: Shahar Frenkel, None; Dudi Shneor,
None; Alik Honigman, None; Jacob Pe’er, None
Support: ISF clinician-researcher, MOH Chief Scientist, HadassahHebrew University joint fund
Program Number: 847
Sensitivity and Resistance of Uveal Melanoma (UM) cells to PKC
inhibition: Role of the Steroid Receptor Coactivator (SRC)-3
Vassiliki Poulaki1, Sue Anne Chew2, 3, Bert W. O’Malley3, Nicholas
Mitsiades2, 3. 1VA Boston Hlthcare Sys, Ophtal, Boston University,
Boston, MA; 2Medicine, Baylor College of Medicine, Houston,
TX; 3Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX.
Purpose: We have previously reported that mutant G proteins (Gαq
and Gα11) in UM promote, via the protein kinase C (PKC) pathway,
the post-translational stabilization of SRC-3, a multifunctional
transcriptional coactivator that is frequently overexpressed and
confers poor prognosis in many types of cancers, including
melanoma. G protein-mutant UM cells are exquisitely dependent
on PKC activity and SRC-3 expression for their survival in vitro.
We now examined the role of SRC-3 as a therapeutic target and as a
downstream mediator of the PKC pathway in animal models of UM.
Methods: We transfected GNAQ-mutant Mel202 cells with a
plasmid expressing SRC-3 shRNA (2 different sequences) or control
vector, under a tetracycline-inducible promoter. Cells were injected
subcutaneously (sc) into immunocompromized mice and treated
with doxycycline (provided in the drinking water). We also treated a
xenograft model of 92.1 GNAQ-mutant UM cells with the PKC small
molecule inhibitor (SMI) PKC412 (midostaurin).
Results: Doxycycline-induced SRC-3 shRNA suppressed SRC-3
expression and exerted potent anticancer activity against UM cells in
vitro and in vivo. The PKC SMI PKC412 suppressed SRC-3 protein
expression in our UM xenograft model (92.1 cells) and exerted potent
anticancer activity in vivo, prolonging median survival (not reached
yet after 8 months of PKC412 treatment) compared to the vehicletreated cohort (2 weeks, see Fig. A). Acute restoration of SRC-3
expression by adenovirus rescued UM cells from the anticancer effect
of PKC412. UM (92.1) cells with acquired resistance to PKC412
that emerged after prolonged treatment in vivo were found to express
restored SRC-3 protein levels.
Conclusions: SRC-3 is an important therapeutic target in UM in vitro
and in vivo. We propose that SRC-3 is a downstream mediator of
PKC signaling and that SRC-3 inhibition could restore sensitivity to
PKC SMIs.
Treatment of 92.1 UM cells xenografted sc into
immunocompromized mice with PKC412 (100mg/kg/day, 5 days/
week) prolonged median survival (not reached yet after 8 months of
treatment) compared to vehicle-treated controls (2 weeks, A). IHC
revealed loss of SRC-3 expression in UM xenografts from PKC412treated mice (C) compared to the vehicle-treated mice (B). In the
PKC412-treated mice that have died so far, IHC revealed that the
PKC412-resistant UM xenografts express significantly higher levels
of SRC-3 (D).
Commercial Relationships: Vassiliki Poulaki, None; Sue Anne
Chew, None; Bert W. O’Malley, None; Nicholas Mitsiades, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
Program Number: 848
Inhibition of STAT3 suppresses growth of retinoblastoma
Dong Hyun Jo1, 2, Jin Hyoung Kim1, 3, Seung Ho Yoo1, 4, Young
Suk Yu1, 5, Jeong Hun Kim1, 2. 1Fight against Angiogenesis-Related
Blindness (FARB) Laboratory, Clinical Research Institute, Seoul
National University Hospital, Seoul, Republic of Korea; 2Department
of Biomedical Sciences, College of Medicine, Seoul National
University, Seoul, Republic of Korea; 3Tumor Microenvironment
Research Center, Global Core Research Center, Seoul National
University, Seoul, Republic of Korea; 4Department of Advanced
Education for Clinician-Scientists, College of Medicine, Seoul
National University, Seoul, Republic of Korea; 5Department of
Ophthalmology, College of Medicine, Seoul National University,
Seoul, Republic of Korea.
Purpose: To investigate the therapeutic potential of STAT3 inhibition
in the suppression of growth of retinoblastoma
Methods: We evaluated the expression of activated STAT3 in human
retinoblastoma tissues and mouse orthotopic transplantation models
of retinoblastoma. Relative expressions of activated and total STAT3
were compared among normal and retinoblastoma cells (SNUOT-Rb1
and Y79) by Western blot and the levels of expression of target genes
of STAT3 among cells were estimated by real time polymerase chain
reaction. Retinoblastoma cells were transfected with small interfering
RNA targeting STAT3 to identify the effect of STAT3 inhibition.
Furthermore, we evaluated in vivo therapeutic effect of STAT3
inhibition in mouse retinoblastoma models.
Results: We showed that phosphorylated form of STAT3 was
expressed in human retinoblastoma tissues and mouse retinoblastoma
models. Retinoblastoma cells also demonstrated relatively high
expression of activated STAT3 and target genes of STAT3 compared
to those of other normal cells. STAT3 inhibition via small interfering
RNA led to decreased expression of target genes of STAT3
and suppressed the growth of retinoblastoma in in vivo mouse
retinoblastoma models.
Conclusions: Our results demonstrated that STAT3 inhibition might
be a valuable therapeutic option against retinoblastoma.
The proportion of disfigured eyes according to the status of STAT3
inhibition. STAT3 KD, STAT3 inhibition via small interfering RNA
targeting STAT3.
Commercial Relationships: Dong Hyun Jo, None; Jin Hyoung
Kim, None; Seung Ho Yoo, None; Young Suk Yu, None; Jeong
Hun Kim, None
Program Number: 849
Verteporfin (VP) inhibits human retinoblastoma cell growth in
vitro without light activation.
Katarzyna Brodowska, Ahmad Al Moujahed, Anna Marmalidou,
Melissa Meyer zu Horste, Joanna Cichy, Joan W. Miller, Evangelos
S. Gragoudas, Demetrios G. Vavvas. Angiogenesis Lab, Harvard
Medical School, Boston, MA.
Purpose: Verteporfin (VP), a benzoporphyrin derivative, is clinically
used as a photosensitizer in photodynamic therapy for neovascular
macular degeneration and ocular tumors. Recent studies indicate
that VP may inhibit growth of tumor cells without photoactivation
through inhibition of YAP-TEAD complex. In this study, we
examined the effects of VP without light activation on human
retinoblastoma Y79 and WERI cell growth in vitro.
Methods: Human retinoblastoma cell line Y79 and WERI was
treated with VP. Cell growth was assessed by trypan blue exclusion
test and MTT assay. Western blot analyses were performed to
determine the expression of cell cycle regulators as well as potential
YAP-TEAD downstream molecules and pathways such as mTOR/
autophagy and Akt.
Results: VP treatment without light activation inhibited Y79 and
WERI cell growth, proliferation and viability in a dose-dependent
manner. VP increased doubling time and induced a mild G0/G1phase cell cycle arrest and downregulation of cyclins. Treatment of
retinoblastoma cells with VP was associated with downregulation of
YAP-TEAD associated downstream signaling molecules. In addition
there was an inhibition of mammalian target of rapamycin (mTOR)
pathway, decreased phosphorylation of Akt.
Conclusions: VP without light activation is a potent inhibitor of cell
growth in retinoblastoma Y79 and WERI cells and may provide a
novel, non-chemotherapeutic approach for retinoblastoma treatment.
Commercial Relationships: Katarzyna Brodowska, None;
Ahmad Al Moujahed, None; Anna Marmalidou, None; Melissa
Meyer zu Horste, None; Joanna Cichy, None; Joan W. Miller,
Alcon (C), Imagen Biotech, Inc. (C), ISIS Pharmaceuticals, Inc. (C),
KalVista Pharmaceuticals (C), Maculogix, Inc. (C), Massachusetts
Eye and Ear Infirmary (P), ONL Therapeutics, LLC (C), Regeneron
Pharmaceuticals, Inc. (C); Evangelos S. Gragoudas, QLT
Phototherapeutics, Inc. (R); Demetrios G. Vavvas, None
Support: National Eye Institute grant EY014104 - MEEI Core Grant
Program Number: 850
Pharmacologic Targeting of Skp2 in Retinoblastoma
Xiaoliang L. Xu1, 3, Timothy Cardozo4, Dan-Ning Hu6, David
Cobrinik5, David H. Abramson2, Suresh Jhanwar1. 1Department of
Pathology, Memorial Sloan Kettering Cancer Center, New York,
NY; 2Ophthalmic Oncology Service, Memorial Sloan Kettering
Cancer Center, New York, NY; 3Sloan-Kettering Institute, Memorial
Sloan-Kettering Cancer Center, New York, NY; 4Department of
Biochemistry and Molecular Pharmacology, New York University,
New York, NY; 5The Saban Research Institute, Children’s Hospital
Los Angeles, University of South California, Los Angeles, NY; 6New
York Eye and Ear Infirmary, New York Medical College, New York,
NY.
Purpose: Retinoblastoma is the most common primary intraocular
malignancy in children. Significant numbers of patients still lose their
eyes and even their lives in developing countries, in spite of current
advances in the treatment of retinoblastoma. Previously our studies
showed that retinoblastomas exhibit retinal cone precursor properties
and depend on cone-specific thyroid hormone receptor beta2 (TRB2)
and SKP2 signaling (Xu et al, 2009; Wang et al, 2010). In this study,
we sought to suppress retinoblastoma cell growth by chemical SKP2
inhibitors as a prelude to targeted therapy in vitro and in vivo.
Methods: We knocked down TRB2 and SKP2 or over-expressed
p27 in retinoblastoma cells to investigate SKP2 and p27 signaling
alterations. The retinoblastoma cell lines Y79, WERI, and RB177
were treated with SKP2 inhibitors C1, C2 (Wu et al, 2012), #25, and
#25-9(Chan et al, 2013) at different concentrations, following which
Western blotting was performed to study SKP2 and p27 expression as
well as ubiquitination.
Results: TRB2 knockdown in Y79 and RB177 caused SKP2
downregulation and degradation, p27 up-regulation, and S phase
arrest, whereas, SKP2 knockdown or p27 over-expression caused
p27 accumulation and G1-S arrest. In the cell lines Y79, WERI,
and RB177, treatment with C1 caused SKP2 ubiquitination and
degradation, p27 de-ubiquitination and accumulation, and cell growth
arrest. Compounds C2, #25, and #25-9, on the other hand exhibited
only moderate effects on suppressing retinoblastoma growth without
significant p27 level changes.
Conclusions: Retinoblastoma tumorigenesis depends on conespecific TRB2 and SKP2 signaling. SKP2 is the synthetic lethal gene
in retinoblastoma with RB1 loss. SKP2 inihibitor C1 significantly
suppresses retinoblastoma cell growth by SKP2 degradation and p27
accumulation. In vivo animal model investigation of the effects of C1
on retinoblastoma cell growth is therefore warranted, and, in the long
term, SKP2 targeted therapy, may be a promising therapeutic strategy
for the treatment of retinoblastoma.
Commercial Relationships: Xiaoliang L. Xu, None; Timothy
Cardozo, None; Dan-Ning Hu, None; David Cobrinik, None;
David H. Abramson, None; Suresh Jhanwar, None
Support: Clinical Development Grant of Pathology, MSKCC;
Gerber Foundation; Cycle for Survival
Program Number: 851
Identification of vascular endothelial growth factor protein in
eyelid basal cell carcinomas: A potential novel, non-surgical
therapy
Christine Law, Ashley Minuk, Isabella Irrcher, James Farmer,
Vladimir Kratky. Ophthalmology, Queen, Kingston, ON, Canada.
Purpose: The gold-standard treatment for eyelid basal cell
carcinomas (BCCs) is complete excision. Due to limited eyelid
surface area, excisions may lead to extensive surgical reconstruction.
Anti-vascular endothelial growth factor (VEGF) agents have been
commonly used as targeted therapy of other systemic cancers. No
research has quantified skin BCC VEGF levels relative to their
pathologic subtype. We investigated the potential role of VEGF as a
non-surgical treatment target by quantifying VEGF levels in eyelid
BCCs, and testing the safety of bevacizumab eyelid injections in an
animal model.
Methods: All patients with biopsy-proven BCCs undergoing lesion
excision from April to October 2013 were included. A 2.5mm punch
biopsy was obtained from both the BCC and uninvolved skin away
from the lesion. All BCC lesions were sent to pathology for final
histopathologic diagnosis. The punch biopsy samples were processed
for tissue VEGF levels through a commercially available VEGF
enzyme-linked immunosorbent assay. To assess safety, bevacizumab
was injected subcutaneously into the left eyelid of New Zealand
white rabbits. Each right rabbit eyelid received volume-matched
saline control. Eyelid punch biopsies for histological examination of
inflammation were obtained pre-injection, at week 1 and 5 postinjection. Adverse events were recorded, and photographs taken
weekly to assess eyelids for gross clinical changes.
Results: A total of 14 patients with pathology-proven BCCs were
included. Histopathologic types included infiltrative (8/14), nodular
(5/14), and micronodular (1/14). BCC versus uninvolved skin
VEGF levels were not different as a whole (1219 pg/mL vs. 974
pg/mL; P=0.400). However, according to their histopathologic
type, infiltrative BCCs displayed significantly higher VEGF levels
compared to non-cancerous skin (1334 pg/mL vs. 343 pg/mL;
P=0.001). Nodular BCC VEGF levels were not elevated (949 pg/mL
vs. 1709 pg/mL; P=0.204).
No adverse events, significant inflammation, or gross clinical change
of rabbit eyelids were seen at any time point after bevacizumab
injection.
Conclusions: We show (1) eyelid infiltrative BCCs, the most
aggressive of our subtypes, are VEGF-positive tumors, and (2)
bevacizumab eyelid injections are safe through our animal model.
Thus anti-VEGF therapy may offer a novel, targeted, non-surgical
treatment option for infiltrative BCCs.
Commercial Relationships: Christine Law, None; Ashley Minuk,
None; Isabella Irrcher, None; James Farmer, None; Vladimir
Kratky, None
211 Ocular Tumors: Molecular genetics
Monday, May 05, 2014 8:30 AM–10:15 AM
Program Number: 1282
Presentation Time: 8:30 AM–8:45 AM
Analysis of the Hedgehog signalling pathway in periocular
sebaceous gland carcinoma
John Bladen1, 2, Caroline Thaung3, Michele Beaconsfield2, Edel
O’Toole1, Michael Philpott1. 1Centre for Cutaneous Research, Barts
& The London School of Medicine, London, United Kingdom;
2
Adnexal Oncology, Moorfields Eye Hospital, London, United
Kingdom; 3Pathology, UCL Institute of Ophthalmology, London,
United Kingdom.
Purpose: Sebaceous gland carcinoma (SGC) is a masquerader of
benign conditions leading to significant eye morbidity, including
destructive surgical treatment such as exenteration, and mortality
in up to 22% of patients. Little is known about the genetic or
molecular basis of SGC. Aberrant Hedgehog (Hh) signalling has been
implicated, however, the pathway has not been assessed in detail.
In contrast, basal cell carcinoma (BCC) is known to involve the Hh
pathway, often through mutational inactivation of the Patched-1
(PTCH1) gene. This makes BCC a good tumour for comparison of
abnormal Hh activation. The aim is to identify any canonical Hh
pathway aberrance in SGC along with its magnitude by comparing it
directly to nodular BCC.
Methods: Periocular SGC and nodular BCC tissue were obtained
from the Moorfields Biobank; SGC (n=15) samples were compared
to nodular BCC (n=10) and control tissue (testes, n=4). Expression
of PTCH1, Smoothened (SMO), and Glioblastoma transcription
factors (GLI1 and GLI2) were assessed in histological sections using
immunohistochemistry and immunofluorescence techniques. Semiquantification was carried out comparing tumours and control tissue
using Image J.
Results: Upregulation of the canonical Hh signalling pathway in
SGC was demonstrated by overexpression of PTCH1, SMO, GLI1
and GLI2 compared to nodular BCC and control tissue.
Conclusions: The Hh signalling pathway is significantly more
upregulated in periocular SGC compared to nodular BCC, a known
aberrant Hh pathway tumour. This raises the possibility of potential
treatment targets that are already in clinical practice (such as SMO
antagonists) to avoid or reduce destructive surgical treatment,
preserve vision and prevent mortality.
Commercial Relationships: John Bladen, None; Caroline Thaung,
None; Michele Beaconsfield, None; Edel O’Toole, None; Michael
Philpott, None
Support: Fight for Sight
Copy number variations in conjunctival melanoma detected by
Single Nucleotide Polymorphism array
Nihal Kenawy1, 3, Bertil E. Damato2, 3, Sarah E. Coupland1, Sarah
L. Lake1. 1Molecular and clinical cancer medicine, University of
Liverpool, Liverpool, United Kingdom; 2Ocular Oncology, University
of California, San Fransisco, CA; 3Liverpool Ocular Oncology
Centre, Royal Liverpool University Hospital, Liverpool, United
Kingdom.
Purpose: Despite the evolving research into the molecular changes
in conjunctival melanoma (CoM), the molecular etiology of this
disease is still uncertain. In this study, we aimed to determine gene
copy number variations (CNVs) occurring in CoM with a view to
ultimately, identifying disease-specific biomarkers that could be
used to develop prognostic testing, as has been achieved in uveal
melanoma, and improve patient care.
Methods: In this study, 56 patients with primary CoM were recruited
from five centres in Europe and the USA between 2005 and 2012.
DNA was extracted from formalin-fixed, paraffin-embedded excised
tumors. Sufficient DNA for analysis using the Affymetrix 6.0 Single
Nucleotide Polymorphism (SNP) microarray was obtained from 33
samples. Data analysis was performed using the Partek Genomic
suite of software.
Results: Amplification of PIK3CA (3q26.32) and LRRC16A
(6p22.2) was observed in 52% of patient samples. Less commonly,
amplification of CDK6 (7q21.2), WISP1 (8q24.22) and KRAS
(12p12.1) were detected in 48%, 45% and 36% of tumors,
respectively.
Recurrent deletions were detected in GPRIN2 (10q11.22) and JAK2
(9p24.1) in 39% of CoM. In addition, RET (10q11.21), WWOX
(16q23.1), CELF4 (18q12.2) and BTNL8 (5q35.3), were deleted in
36% of the patient cohort.
There was no statistical correlation between the frequency of genetic
alterations in the CoM and whether they metastasized, were of
epithelioid cell type, or arose in the caruncle. Amplification of TAF6L
(11q12.3) was statistically significantly associated with non-bulbar
tumor location (p=0.048, Fisher’s exact test).
Conclusions: This study improves our understanding of CoM
pathogenesis by identifying CNVs, in individual genes, not
previously detected in CoM. The observed alterations differ
from those seen in both uveal and cutaneous melanomas, further
suggesting different etiological mechanisms for each of these tumor
types.
Validation of the identified CNVs by an alternative methodology is
needed, in addition to investigation of their biological significance
to further improve our understanding of the molecular regulation of
CoM development. This will facilitate the identification of patients
at high-risk of recurrence or metastasis and, potentially, allow for the
development of targeted therapies in CoM.
Commercial Relationships: Nihal Kenawy, None; Bertil E.
Damato, None; Sarah E. Coupland, None; Sarah L. Lake, None
Support: Eye Tumour Research Fund JXR10418
Activation of the MAP kinase pathway is involved in tumor
progression of conjunctival melanocytic proliferations
Alexandre P. Moulin1, Maya Bucher2, Francois Majo3, Michael J.
Nicolas3. 1Department of Ophthalmology, University of Lausanne,
Jules Gonin Eye Hospital, Lausanne, Switzerland; 2Dermatology,
University of Lausanne, CHUV, Lausanne, Switzerland; 3Department
of Ophthalmology, University of Lausanne, Jules Gonin Eye
Hospital, Lausanne, Switzerland.
Purpose: The molecular events leading to the development of
conjunctival melanoma have not been extensively studied. As
activating mutations of BRAF and NRAS have recently been identified
in a mutually exclusive way in 47% of conjunctival melanoma, we
decided to evaluate the activation status of the MAP kinase pathway
and PI3K/mTOR pathway in benign and malignant conjunctival
melanocytic proliferations.
Methods: The phosphorylation status of MEK, ERK and S6
ribosomal protein was evaluated by immunohistochemistry in 35
conjunctival naevi and 31 conjunctival melanoma. Statistical analysis
was performed with JUMP 8,0 software. Immunohistochemistry was
assessed independently by three observers.
Results: There was a concordance in 85%, 87,9% and 96,5% of
the cases in the evaluation of the activation of MEK, ERK and S6
respectively. Discrepant cases were simultaneously reviewed to
achieve complete agreement.
There were 13 subepithelial nevi and 22 compound nevi. There were
14 females and 21 males with a mean age was 36.9 ± 3.6 yo (SEM).
P-MEK was found in 20 nevi (59%). P-ERK was found 17 nevi (49
%). P-S6 was found in 11 nevi (49%).
The melanoma group was composed of 17 females and 14 males
with a mean age of 67.1 ± 3.38 yo (SEM). Using TNM classification,
10 tumors were belonging to the T1, 10 to the T2 and 11 to the T3
categories. P-MEK and p-ERK were both identified in 27 melanoma
(87%). P-S6 was found in 29 melanoma (94%). The phosphorylation
of MEK and ERK and S6 was significantly elevated in the melanoma
compared to the nevi (p=0,0109; 0,0009 and p> 0,0001 respectively).
There was also a significant correlation between the activation of
MEK and ERK (p=0,0045).
Conclusions: Our results demonstrate in vivo a significant increase in
the activation of the MAP kinase pathway in malignant conjunctival
melanocytic proliferations. We also identified a significant increase
in the phosphorylation of S6 ribosomal protein suggesting a
contribution of the P13K/mTOR pathway to conjunctival melanoma
development. The inhibition of these pathways might represent
potential therapeutical options for the treatment of conjunctival
melanoma.
Commercial Relationships: Alexandre P. Moulin, None; Maya
Bucher, None; Francois Majo, None; Michael J. Nicolas, None
Melanoma Inhibitor of Apoptosis (ML-IAP) – Driven Expression
of the Pro-Apoptotic Protein BAD in Uveal Melanoma Cells
Jon R. Backstrom1, Stephanie M. Evans1, Ashwath Jayagopal1,
2 1
. Ophthalmology and Visual Sciences, Vanderbilt Eye Institute,
Nashville, TN; 2Molecular Physiology and Biophysics, Vanderbilt
University Medical Center, Nashville, TN.
Purpose: The paucity of effective treatments and low survival rate
of patients with metastatic uveal melanoma warrant new targeting
strategies. Our goal is to develop adeno-associated viruses that
selectively express cargo genes (such as the pro-apoptotic gene,
BAD) in uveal melanoma cells that have metastasized to the liver.
Expression of ML-IAP (birc7/livin) is restricted to melanomas and
RPE. Therefore, we evaluated the promoter activity of ML-IAP in
uveal melanoma and RPE cell lines.
Methods: The ML-IAP promoter and BAD cDNA were cloned
from the human melanoma cell line SK-MEL-28. Site-directed
mutagenesis was used to mutate the ML-IAP promoter at consensus
transcription factor binding sites and BAD cDNA at previously
characterized phosphorylation sites. Plasmids with ML-IAP/BAD
upstream of either CMV/Renilla luciferase or CMV/mCherry were
introduced into primary and metastatic uveal melanoma cell lines
derived from the same patient. A CMV/Renilla plasmid without MLIAP/BAD was used in separate wells as a negative control for BAD
activity since ML-IAP-driven expression of BAD results in cell death
and concomitant lower luciferase activity. Immunocytochemistry
with antibodies against cleaved caspase-3 was used to monitor
apoptosis of cells transiently expressing BAD-FLAG and mCherry.
Results: Higher levels of endogenous ML-IAP protein were detected
on immunoblots prepared from primary melanoma cells than from
metastatic cells. The effectiveness of introduced ML-IAP/BAD was
abrogated (highest luciferase activity) with a single serine-to-aspartic
acid mutation at the Bcl2/Bcl-XL interaction site of BAD and
increased (lowest luciferase activity) with serine-to-alanine mutations
at three phosphorylation sites (BAD-3SA). Therefore, BAD-3SA was
utilized for subsequent analyses of the ML-IAP promoter. The MLIAP/BAD-3SA construct was most effective in primary melanoma
cells and least effective in the RPE cell line ARPE-19. Mutations at
all of the E-box sites in the ML-IAP promoter (MITF, USF, and AP4)
prevented BAD-3SA activity in primary melanoma cells, but not
metastatic cells.
Conclusions: The ML-IAP promoter is capable of driving expression
of BAD at levels that promote apoptosis of uveal melanoma cells.
Furthermore, the data raise the intriguing possibility that transcription
factor interactions at the ML-IAP promoter change as cells transition
from primary to metastatic.
Commercial Relationships: Jon R. Backstrom, None; Stephanie
M. Evans, None; Ashwath Jayagopal, None
Support: R01EY023397, P30EY008126, Research to Prevent
Blindness
Relationships between clinical features, molecular classification,
4-gene mutation profile and outcomes in uveal melanoma
Daniel L. Chao, J William Harbour. Ocular Oncology Service,
Bascom Palmer Eye Inst, Univ of Miami, Miami, FL.
Purpose: To analyze the relationships between clinical features, gene
expression profile (GEP) molecular classification, and mutation status
of four driver mutations in uveal melanoma.
Methods: 70 primary untreated uveal melanomas underwent GEP
classification and mutational analysis for GNAQ, GNA11, BAP1,
SF3B1 mutations. GEP and mutation status were analyzed for
association with patient age and sex, tumor diameter and thickness,
ciliary body involvement, extraocular tumor extension, histological
cell type, and metastasis.
Results: GNAQ and GNA11 mutations were found in a mutually
exclusive fashion in 85% of samples. BAP1 mutations were found
almost exclusively in class 2 tumors and were associated with class
2 GEP, older patient age, ciliary body involvement, increased tumor
diameter and thickness and epithelioid cell type. SF3B1 mutations
were inversely associated with class 2 GEP, BAP1 mutations, and
epithelioid cell type.
Conclusions: The relationships between GEP classification and
the four driver mutations point out the two major pathways of
uveal melanoma progression (class 1/SF3B1 versus class 2/BAP1
pathways), and provide a basis for rational drug selection for treating
metastatic uveal melanoma.
Commercial Relationships: Daniel L. Chao, None; J William
Harbour, Castle Biosciences (C), Castle Biosciences (P)
Support: This work was supported by grants to J.W.H. from the
National Cancer Institute (R01 CA125970), Melanoma Research
Alliance, Melanoma Research Foundation and Tumori Foundation,
as well as NIH Core Grant P30EY014801, Research to Prevent
Blindness Unrestricted Grant, and Department of Defense Grant
#W81XWH-09-1-0675 to the Bascom Palmer Eye Institute.
Quantitative Label-free LC-MS Proteomic Analysis of Uveal
Melanoma Identifies Proteins Associated with Metastasis
Pathma Ramasamy3, 2, Michael Henry3, Martin Clynes3, Conor
Murphy2, 1, Anne-Marie Larkin3, Stephen Beatty4, Paul Moriarty1,
Susan Kennedy1, 5, Paula Meleady3, 5. 1Ophthalmology, Royal
Victoria Eye and Ear Hospital Dublin Ireland, Dublin 2, Ireland;
2
Ophthalmology, Royal College of Surgeons Ireland, Dublin, Ireland;
3
National Institute of Cellular Biotechnology, Dublin City University,
Dublin, Ireland; 4Macular Pigment Research Group, Waterford
Institute of Technology, Waterford, Ireland; 5Joint senior authors,
Dublin City University, Dublin, Ireland.
Purpose: To further the molecular biological understanding of
the events governing the development of metastatic disease and
to identify therapeutic targets for patients at risk of developing
metastasis/patients with metastatic disease by comparing the protein
expression profile between primary uveal melanoma (UM) tissues of
patients who developed metastatic disease compared to those that did
not.
Methods: 8 fresh frozen primary UM tissues of patients who
developed metastasis vs. 8 who did not were subjected to
quantitative, label-free LC-MS proteomic analysis. These patients
had a minimum of 7 years follow-up. UM tissue samples were
homogenised, lysed and proteins were digested into peptides using
trypsin prior to mass spectrometry (MS) analysis. Progenesis LC-MS
software was used to analyse the protein expression profile. Criteria
applied to the data prior to exporting the MS output file for peptide
identification were peptide features with p < 0.01, charge states +1
to +3 and > 3 isotopes per peptide. Peptides were identified with
MASCOT searched against the UniProtKB–SwissProt database.
Proteins with < 3 peptides and proteins with peptide conflicts were
excluded. Only differentially expressed proteins with p < 0.05
between the two patient groups were considered.
Results: A total of 401 proteins were identified (global proteome). 50
proteins were found to be differentially expressed between metastatic
and non-metastatic primary UM tissues. 28 proteins were upregulated
and 22 were downregulated in UM tissues that developed metastasis.
Increased expression of FABP3 and TPI1, and decreased expression
of HSP-27, among other differentially expressed proteins were found.
These proteins potentially are associated with the development of
metastatic disease. A number of proteins of interest are currently
being evaluated by immunohistochemistry using tissue microarrays
and FFPE sections on patient groups.
Conclusions: Proteomic analysis of 8 metastatic vs. 8 non-metastatic
primary uveal melanoma tissue has identified proteins that are
associated with the development of metastatic disease.
Commercial Relationships: Pathma Ramasamy, None; Michael
Henry, None; Martin Clynes, None; Conor Murphy, None; AnneMarie Larkin, None; Stephen Beatty, None; Paul Moriarty, None;
Susan Kennedy, None; Paula Meleady, None
Support: Royal Victoria Eye and Ear Research Foundation
MACC1 gene expression in Retinoblastoma
Geeta K. Vemuganti1, Rohini M. Nair1, Adit Gupta2, Swathi Kaliki2,
Mohammad J Ali2, Santosh Honavar3, Dilip Mishra4. 1School of
Medical Sciences, University of Hyderbad, Hyderabad, India;
2
Ophthalmic Plastic Surgery, Orbit & Ocular Oncology, L.V.Prasad
Eye Institute, Hyderabad, India; 3Ocular Oncology Service,
Centre for Sight Superspeciality Eye Hospital, Hyderabad, India;
4
Ophthalmic Pathology Laboratory, L.V.Prasad Eye Institute,
Hyderabad, India.
Purpose: Histopathologic high risk factors(HRF) are well established
prognostic markers in retinoblastoma(Rb).Metastasis Associated in
Colon Cancer1(MACC1) gene is reported as an important prognostic
indicator for systemic metastasis and metastasis-free survival in many
cancers like Colon,Breast,and Gastric.This study aims to evaluate
MACC1 expression in Rb cases,with and without HRF,and its
possible association with clinical outcome.
Methods: After IRB approval,medical records of histopathologically
proven Rb cases with(n=12) and without HRF(n=11) were retrieved
from the Ocular Oncology Service and Ophthalmic Pathology
Laboratory,LVPEI.Of these,19(83%) cases had undergone primary
enucleation and 4(17%) secondary enucleation following poor
response to systemic chemotherapy.Patients with HRF underwent
bone marrow and cerebro-spinal fluid evaluation for metastasis and
treated using the standard clinical protocol with 6 cycles of adjuvant
systemic chemotherapy(Vincristine,Etoposide,Carboplatin).RNA
was isolated from FFPE tissues(central calotte only) and evaluated
for MACC1 expression by semi-quantitative PCR.The relative gene
expression was quantified using Image Lab software(Bio-Rad).
Expression levels were correlated with HRF and clinical outcome of
patients.
Results: Mean age of patients at presentation was 30 months.
Bilateral Rb was noted in 47%(11/23) cases with 65% being male.
Leucokoria was the chief presenting clinical feature(78%).In HRF
cases,the average MACC1 expression was increased(3.46 vs 2.68,
p=0.09) with 50% showing more than 3 fold increase compared to
negative controls.In no HRF cases,≥ 3 fold expression of MACC1
was seen in 18% of cases.MACC1 expression was marginally
increased in poorly differentiated cases(48%) compared to well and
moderately differentiated cases(3.15 vs 3.02fold,p=0.4).It was also
higher in cases that underwent primary enucleation(83%), compared
to those who underwent secondary enucleation post-systemic
chemotherapy (4/23)(3.15 vs 2.8fold,p=0.67).At a mean follow-up of
27 months(3-48 months),except 1 patient who died at 3 months post
enucleation,none of them showed evidence of local recurrence or
metastasis.
Conclusions: This study shows that high MACC1 expression in Rb
correlates with histologic risk factor,thus indicating its possible role
in tumor invasion and metastasis.However,further studies on larger
cohort and multivariate analysis is warranted to establish its role as a
potential prognostic marker in retinoblastoma.
Commercial Relationships: Geeta K. Vemuganti, None; Rohini M.
Nair, None; Adit Gupta, None; Swathi Kaliki, None; Mohammad
J Ali, None; Santosh Honavar, None; Dilip Mishra, None
Support: Indian Council of Medical Research, Hyderabad Eye
Research Foundation
240 Immunohistichemistry
Monday, May 05, 2014 11:00 AM–12:45 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 1843–1854/A0399–A0410
Program Number: 1843 Poster Board Number: A0399
Presentation Time: 11:00 AM–12:45 PM
Establishment and Regulation of the B lymphocytes and Orbital
Fibroblasts Co-culture in Thyroid Associated Ophthalmopathy
Renyan Wang. 1Ophthalmology, Beijing Tsinghua Chang Gung
hospital, Beijing, China; 2Ophthalmology, Peking Union Medical
College Hospital, Beijing, China.
Purpose: 1.To establish and to identify an in vitro experimental
model of autoimmune TAO by co-culture the orbital fibroblasts and
peripheral B lymphocytes.
2.To investigate the Insulin-like growth factor-1(IGF-1R) receptor
expressing on OFs, observe the inflammatory responses in the model,
and explore the important role of B cells in TAO.
3.Exploring pathogenesis and therapeutic approaches for TAO.
Methods: 1.Orbital fibroblasts obtained from 15 patients with TAO
and from 15 control subjects were used to set up primary cultures,
and were identified by immunohistochemistry. B lymphocytes
were isolated from peripheral blood obtained from 10 patients
with TAO and 10 controls. B cells were enriched, purified using
immunomagnetic beads separation techniques and analyzed by folw
cytometry.
2.The IGF-1R of OFs were evaluated by flow cytometry and
observed under confocal microscopy. The expression of interleukin-6
and chemoattractants RANTES of were quantified by ELISA at
24hours,48 hours and 72 hours after co-culture.
3.The depleting effects of Rituximab on B cells at several
concentrations in different time were measured by MTS. The
inhibition on the expression of IL-6 and RANTES after using RTX
and IGF-1 binding protein on the co-culture model were analyzed by
ELISA.
Results: 1.The co-culture of orbital fibroblasts and B lymphocytes
was established.
2.The expression of IGF-1R on OFs in patients with TAO was
significantly higher than normal’s.
3.The expression of IL-6 and RANTES in each co-culture group was
increased at 24 hours, especially in the T+T group (B lymphocytes
and orbital fibroblasts all obtained from the TAO patients) is the
highest.
4.RTX and IGF-1 binding protein significantly inhibited the
expression of IL-6 and RANTES in the T+T co-culture group at 48
hours.
Conclusions: 1.An in vitro model that partially represents TAO, as
an autoimmune inflammatory disease, was established by co-culture
of orbital fibroblasts and peripheral B lymphoctes. The interactions
between the two cells may play a role in TAO pathogenesis.
2.IGF-1R may be the pathway in the interactions between OFs and
B cells. It may mediate the process of TAO. Inflammatory responses
may be depressed after blocking the recptor.
3.B cell depleting agents RTX have a good anti-inflammatory effects
in the vitro model. Monoclonal anti-CD20 antibody therapy may be a
novel treatment option in TAO in the future.
Commercial Relationships: Renyan Wang, None
Catestatin-like immunoreactivity in the rat eye
Maren Kriechbaum1, Oliver W. Gramlich1, Katrin Lorenz1, Franz
H. Grus1, Daniela Ehrlich2, Christian Humpel2, Reiner Fischer
Colbrie3, Nikolaos . E. Bechrakis4, Josef Troger4. 1Experimental
Ophthalmology, University Medical Center Mainz, Mainz, Germany;
2
Department of Psychiatry and Psychotherapy, Laboratory of
Psychiatry and Experimental Alzheimers Research, 6020 Innsbruck,
Austria; 3Department of Pharmacology, Medical University of
Innsbruck, 6020 Innsbruck, Austria; 4Department of Ophthalmology,
Medical University of Innsbruck, 6020 Innsbruck, Austria.
Purpose: The aim of the study was to investigate the presence and
distribution of the chromogranin A-derived peptide catestatin in the
rat eye and trigeminal ganglion.
Methods: Western blots were performed in an attempt to characterize
the immunoreactivities detected by the catestatin antiserum and the
distribution pattern was explored by immunofluorescence.
Results: Sparse immunoreactive nerve fibers were visualized in the
corneal stroma, in the chamber angle, in the sphincter muscle but
also in association with the dilator muscle, in the stroma of the ciliary
body and processes, but dense in the irideal stroma, around blood
vessels at the limbus and in the choroid and in cells of the innermost
retina representing amacrine cells as identified by colocalization
with substance P. Furthermore, catestatin-immunoreactivity was
detected in the trigeminal ganglion in small to medium-sized cells
and there were abundant catestatin-positive nerve fibers stained
throughout the stroma of the ganglion. Double immunofluorescence
of catestatin with substance P revealed colocalization both in cells
of the trigeminal ganglion as well as in nerve fibers in the choroid.
The immunoreactivities are present obviously as free catestatin and/
or small-sized catestatin-containing fragments in the retina and
ocular nerves but as large processed fragments as well, weak in the
retina and more prominent in remaining ocular tissues, possibly in
endothelial cells.
Conclusions: The results indicate that this peptide is a constituent
of sensory neurons innervating the rat eye and the presence in
amacrine cells in the retina is typical for neuropeptides. Catestatin
is biologically highly active and might be of significance in the
pathophysiology of the eye.
Commercial Relationships: Maren Kriechbaum, None; Oliver
W. Gramlich, None; Katrin Lorenz, None; Franz H. Grus, None;
Daniela Ehrlich, None; Christian Humpel, None; Reiner Fischer
Colbrie, None; Nikolaos . E. Bechrakis, None; Josef Troger, None
Spheroidal degeneration in H626R TGFBI corneal
stromal dystrophy: clinical, genetic, histopathologic,
immunohistochemical, and ultrastructural analysis
Kevin Lai1, Jason Reidy1, Benjamin Bert2, Tatyana Milman1.
1
Ophthalmology, New York Eye and Ear Infirmary, New York,
NY; 2Ophthalmology, University of California, San Francisco, San
Francisco, CA.
Purpose: To describe the clinical, imaging, histopathologic,
immunohistochemical, and ultrastructural characteristics of coexistent
amyloid and spheroidal degeneration-type deposits in a family with
Histidine-626-Argenine Transforming Growth Factor Beta Inducible
Gene (H626R TGFBI) corneal stromal dystrophy.
Methods: Retrospective clinical-pathologic and genetic analysis of
one family with H626R lattice dystrophy.
Results: The disease showed autosomal dominant inheritance pattern
by pedigree analysis. The affected individuals presented in 4th or 5th
decades with progressive visual impairment and recurrent erosions.
Ophthalmic examination of the 3 affected family members revealed
asymmetric, thick, branching lattice-like deposits, associated with
corneal haze. Sequencing of the TFGBI gene revealed a highpenetrance disease causing sequence variation (H626R CAT>CGT
heterozygous). Optical coherence tomography demonstrated fusiform,
poorly demarcated, hyper-echoic stromal deposits, consistent with
amyloid, with focal hypo-echoic central region. Histologic evaluation
of the corneal buttons from the 2 affected family members showed
stromal fusiform Periodic acid-Schiff (PAS)-positive, Congo redpositive, birefringent, and keratoepithelin antibody-immunoreactive
deposits, consistent with TGFBI amyloid. Few amyloid deposits
contained a central nidus of spheroidal degeneration-type material.
This material demonstrated autofluorescence, stained with elastic and
Masson-trichrome stains, did not stain with PAS or Congo red stains,
was non-birefringent, and did not immunoreact with keratoepithelin
antibodies. Transmission electron microscopy confirmed the presence
of peripheral amyloid fibrils with central electrodense, homogeneous,
discrete spheroidal degeneration-type deposits.
Conclusions: Presence of spheroidal degeneration-type deposits in
a subset of affected patients, the variability in presentation within an
individual and between the family members, the predominant anterior
corneal stromal location and the non-immunoreactivity of deposits
for keratoepithelin suggest that these deposits are degenerative
in nature. The deposits may arise from ultraviolet light-altered
proteins diffused from the limbus, which form a nidus for mutant
keratoepithelin deposition in patients with the late onset H626R
lattice dystrophy variant.
Commercial Relationships: Kevin Lai, None; Jason Reidy, None;
Benjamin Bert, None; Tatyana Milman, None
Superficial Conjunctival Epithelium as the Main Producer of
Protective Tear Component Cystatin SN
An-Katrien De Roo1, 3, Beatrijs Foets4, 2, Joost J. van den Oord1, 3.
1
Department of Imaging & Pathology, Unit of Translational Cell &
Tissue Research, KU Leuven, Leuven, Belgium; 2Department of
Neurosciences, Unit of Research Group Ophthalmology, KU Leuven,
Leuven, Belgium; 3Department of Pathology, UZ Leuven, Leuven,
Belgium; 4Department of Ophthalmology, UZ Leuven, Leuven,
Belgium.
Purpose: CST1 is a gene known to be expressed in lacrimal glands.
Its corresponding protein cystatin SN, is a component of human tear
fluid, that is thought to play a protective role by inhibiting cysteine
proteases. We investigated whether the conjunctival and corneal
epithelia contribute to the production of cystatin SN and whether this
production increases in response to injury.
Methods: From our Pathology files we selected formalin-fixed,
paraffin-embedded tissues from 13 corneas from enucleated uveal
melanomas, 5 donor corneal rims, 9 corneas with acute keratitis,
2 pterygia, 1 conjunctival naevocellular naevus, 1 conjunctival
intraepithelial neoplasia, 1 conjunctival squamous cell carcinoma,
and 3 lacrimal glands. Permissions from the local Ethics Committee
and Biobank were obtained. Tissue sections were manually stained
for cystatin SN.
Results: All normal corneas showed strong and diffuse
immunoreactivity for cystatin SN in the superficial layers of
the conjunctival epithelium and corneal limbus, with an abrupt
loss of staining in the more central corneal epithelium. Scarce
immunoreactivity was observed in a proportion of epithelial cells
lining the main excretory ducts and in a minority of secretory cells
of the lacrimal glands. Gain of immunoreactivity occurred in the
central corneal epithelium of injured corneas. The basal layer of
conjunctival and corneal epithelium did not stain. Intraepithelial
conjunctival neoplasia showed hyperplasia of the basal cell layers,
lacking immunoreactivity. A minority of tumor cells within the nests
of squamous cell carcinoma were stained. No reactivity was seen in
naevus cells.
Conclusions: In contrast to current belief, these findings show that
the conjunctiva, and not the lacrimal gland, is the main producer
of cystatin SN in the anterior eye. Furthermore, the gain of corneal
epithelial immunoreactivity in injured corneas supports the presumed
role of CST1 in protecting the anterior ocular surface. The absence of
staining in basal epithelial cells suggests that production of cystatin
SN requires a certain level of cell maturation. And finally, cystatin SN
can be proposed as a new and robust marker to distinguish between
peripheral and central corneal epithelium in the normal cornea.
Polymerase chain reaction will be conducted to confirm that this
immunoreactivity is due to CST1 expression and not to uptake of the
protein from the surrounding tear fluid.
Commercial Relationships: An-Katrien De Roo, None; Beatrijs
Foets, None; Joost J. van den Oord, None
Support: PhD fellow for Research Foundation – Flanders
(11C7513N)
Clinicopathologic Features of Ophthalmic Neoplasms Arising in
the Setting of Xeroderma Pigmentosum
Maria J. Suarez B, Fausto J. Rodriguez. Ophthalmic Pathology,
Johns Hopkins University, Baltimore, MD.
Purpose: To determine the clinical, pathologic and
immunohistochemical (IHC) features of neoplasms involving the
ocular surface and/or adnexa in three patients satisfying clinical
criteria for Xeroderma Pigmentosum (XP).
Methods: We retrieved formalin-fixed paraffin-embedded material
from tumors involving the ocular surface and ocular adnexa from
3 patients with XP who underwent clinical evaluation at our
institution. Clinical information was obtained by retrospective chart
review. Histopathological evaluation was performed, as well as
immunohistochemistry in all available cases using antibodies directed
against the most common mutated proteins in XP patients (XPA,
XPC, and XPD). Scoring of nuclear immunoreactivity was performed
in 3-tiered scale: 2=moderate to strong staining; 1=weak/focal
staining; 0=negative staining.
Results: Three (9, 13, 28 years old) patients of African descent
with XP (2 males, 1 female) and ocular and adnexal tumors were
studied. Patient 1 had two squamous cell carcinomas (SCC) in the
conjunctiva (one in situ and one invasive). Patient 2 had invasive
basal cell carcinoma (BCC), SCC in situ of the eyelid, and orbital
malignant melanoma with recurrence. Patient 3 had invasive SCC
of the eyelid. Nuclear expression of XPA, XPC, and XPD proteins
in normal eyelid skin, particularly in basal epithelium and adnexal
glands, was noted in controls. Nuclear staining for XPD was also
present in normal conjunctiva. In patient 1, immunoexpression of
XPA and XPC was present in both tumors, while XPD was lost in the
invasive (but not in situ) SCC. Conversely, patient 2 had XPA loss
in invasive tumors (BCC, melanoma) and retained XPC and XPD.
Positive immunoreactivity for XPA, XPC and XPD in SCC in situ
was present. Finally, patient 3 showed retained XPA, XPC, and XPD
expression in SCC.
Conclusions: Our study outlines our early experience with pathology
of ocular neoplasms in XP patients. XPC immunoreactivity in all
tumors suggests that XPC genetic alterations may not be a common
feature in our population. Immunohistochemistry for XPA and XPD
may be more useful in the study of invasive tumors compared to
in situ carcinoma. These findings deserve further exploration with
genetic studies and additional patients.
Commercial Relationships: Maria J. Suarez B, None; Fausto J.
Rodriguez, None
Novel ultrastructural patterns of corneal immunoglobulin
deposition in monoclonal gammopathy of undetermined
significance
Andrew A. Kao1, Jason Reidy3, David S. Chu4, Ira J. Udell5, Anne
Steiner5, Carrie Zaslow5, Tatyana Milman1, 2. 1Ophthalmology,
The New York Eye and Ear Infirmary, New York, NY; 2Pathology,
The New York Eye and Ear Infirmary, New York, NY; 3Beth Israel
Medical Center, New York, NY; 4The Institute of Ophthalmology &
Visual Science, UMDNJ-New Jersey Medical School, Newark, NJ;
5
Ophthalmology, North Shore-Long Island Jewish Health System,
Great Neck, NY.
Purpose: To describe two novel ultrastructural patterns of corneal
immunoglobulin deposition in monoclonal gammopathy of
undetermined significance (MGUS).
Methods: The corneal buttons of two patients with monoclonal
gammopathy were studied. Histologic sections of corneal tissue were
stained with hematoxylin and eosin, periodic acid-Schiff (PAS),
Masson trichrome, and Congo red stains. Immunohistochemistry and
in situ hybridization (ISH) studies for immunoglobulin heavy and
light chains were performed on the histologic sections. Transmission
electron microscopy (TEM) was also performed. Pertinent literature
was reviewed.
Results: Patient 1: A 76-year-old woman presented with bilateral
corneal haze and vascularization believed to be secondary to
old interstitial keratitis. Penetrating keratoplasty was performed.
Histologic analysis revealed eosinophilic deposits within the corneal
stroma which stained strongly with Masson trichrome. ISH failed
to demonstrate the presence of light chains. TEM demonstrated
extracellular electron-dense stromal scroll-like deposits ranging
in size from 100-300 nm in diameter. Further clinical workup
demonstrated MGUS.The patient is being monitored without
treatment, with no recurrence noted at 16-month follow-up.
Patient 2: A 53-year-old man presented with bilateral crystalline
corneal opacities in the epithelium and superficial stroma in 2008.
Serum protein electrophoresis (SPEP) was negative. The haze
worsened in 2013 and repeat SPEP showed an M spike, presumed
MGUS. Corneal biopsy demonstrated non-birefringent eosinophilic,
PAS-positive, and Masson trichrome-positive deposits within the
epithelium, Bowman layer, and superficial stroma. The deposits
immunoreacted with antibodies to lambda light chains. TEM
demonstrated round, electron-dense deposits consistent with immune
complexes. Systemic workup including bone marrow biopsy is
pending.
Conclusions: To our knowledge, the ultrastructural patterns of
large scroll-like deposits and immune complexes have not been
previously described in the cornea but have been reported in the
kidneys of patients with paraproteinemia. Our findings underscore
the importance of careful clinical, histopathologic and ultrastructural
evaluation of patients with corneal opacities suggestive of
paraproteinemia.
Commercial Relationships: Andrew A. Kao, None; Jason Reidy,
None; David S. Chu, None; Ira J. Udell, None; Anne Steiner,
None; Carrie Zaslow, None; Tatyana Milman, None
Fluorescence Microscopy Identification of Retinal Pigment
Epithelium-65 and Zonular Occludens-1 Expression in
Spontaneously Differentiated Human Embryonic Stem Cell
derived Retinal Pigment Epithelium Cells
Lee R. Ferguson, K V Chalam. Ophthalmology, University of Florida,
Jacksonville, FL.
Purpose: To evaluate cellular expression profiles of retinal pigment
epithelium-65 (RPE65) and zonular occludens-1 (ZO-1) markers
during spontaneous differentiation human embryonic stem cell into
retinal pigment epithelial cells (hESC-RPE)
Methods: Human embryonic stem cells (hESC), from the WA09DL-11 feeder dependent line, were removed from liquid nitrogen
and grown on a confluent layer of inactivated mouse embryonic
fibroblast. Differentiated pigmented embryoid body (EB) clusters
were dissected from undifferentiated hESC colonies. After dissection,
EBs were isolated onto 6-well gelatin coated plates for further
monolayer expansion into hESC-RPE cells. Confluent monolayers
were passaged 2 – 3 weeks following EB isolation and plated onto
gelatin-coated slides. Each slide was placed into a 10 mm petri dish
filled with RPE maintenance media and allowed to grow to either 7,
21, or 35 days post passage. Slides were extracted on designated time
points and prepared for immunohistocytochemistry with antibodies
targeted to RPE65 and ZO-1. Immunofluorescence was performed
under 10x magnification on an inverted Olympus IX51-IX2-SL
microscope with Olympus U-RFL-T fluorescence lamp.
Results: Day seven following hESC-RPE precursor cell plating
showed no evidence of RPE65 or ZO-1 expression. Two weeks later
on day 21, maturation of hESC-RPE precursor cells demonstrated
an enhanced fluorescence of RPE65 and a slight fluorescence
increase in ZO-1 expression. Four weeks after precursor cell plating,
fluorescence intensity for both RPE65 and ZO-1 was more intense
than the prior two time points.
Conclusions: This study shows enhanced in vitro cellular expression
patterns of RPE65 and ZO-1 during differentiation of hESC-RPE
precursor cells into a more mature state. Based on the above findings,
precursor hESC-RPE cells demonstrate RPE marker patterns between
one to three weeks after plating. This information can assist with
therapeutic implementation especially when attempting to identify
points of hESC-RPE precursor maturation.
Commercial Relationships: Lee R. Ferguson, None; K V Chalam,
None
Alzheimer-induced changes in biomarkers in the Human Lateral
Geniculate Nucleus
Elizabeth Couser1, Steven L. Bernstein2. 1Gerontology Doctoral
Program, University of Maryland, Baltimore, Baltimore, MD;
2
Ophthalmology & Visual Sciences, University of Maryland School
of Medicine, Baltimore, MD.
Purpose: While Alzheimer’s disease (AD) patients are known to
experience histological retinal changes, the thalamic intermediaries
connecting eye and cortex have been understudied. We wanted to
examine whether Alzheimer’s disease (AD) biomarkers are expressed
in the normal human lateral geniculate nucleus (LGN) and whether
AD alters the presence of these markers. We also wanted to determine
whether these markers are expressed in pre-clinical AD.
Methods: Following IRB approval, we obtained human tissue
samples from the Maryland Brain and Tissue Bank. These included
normal, pre-clinical, and severe AD. We immunohistochemically
evaluated human LGN for the expression of both AD markers
(phosphorylated tau, amyloid precursor protein (APP) and amyloid-β
(Aβ)) and inflammation using standard immunohistochemistry (IHC)
procedures. Cellular inflammation was compared using IBA1. Slides
were analyzed using a confocal fluorescent microscope and Fluoview
Software.
Results: While normal tissue showed minimal expression of AD
biomarkers, there was a progressive increase in Tau, Aβ, APP, and
inflammation in the lateral geniculate nucleus through the different
stages of AD severity. Interestingly, the inflammatory response and
deposits of AD biomarkers were shown in significant amounts in the
pre-clinical AD samples of the LGN.
Conclusions: The LGN-thalamic structure shows AD-induced
alterations as the disease progresses and very early in the disease
process. This may result in a decreased ability to process visual
information, and visual problems. The presence of inflammation and
AD-related biomarkers in pre-clinical AD patients suggests that for
at least some older adults, changes in visual processing ability and
speed may be due to AD onset, and not normal age-related visual
decline.
Commercial Relationships: Elizabeth Couser, None; Steven L.
Bernstein, None
Müller Cell Glial Fibrillary Acidic Protein (GFAP) Upregulation
in Experimental Glaucoma is not dependent on the Presence of
Retinal Ganglion Cells (RGCs)
Jeffrey C. Ockuly1, Charlene B Y. Kim1, 2, Brian J. Christian3, Mélissa
C. De Lombaert1, T Michael Nork1, 2. 1University of Wisconsin School
of Medicine and Public Health, Madison, WI; 2Ocular Services On
Demand, LLC (OSOD), Madison, WI; 3Covance, Inc., Madison, WI.
Purpose: To determine the relationship of GFAP upregulation in
Müller cells to elevated intraocular pressure (IOP) and the presence
of RGCs in experimental glaucoma and axotomy in monkeys.
Methods: Immunohistochemistry for GFAP was performed on the
retinas of both eyes of 13 monkeys. In all of the animals, the right eye
was the experimental eye and the left eye was the untreated control.
6 cynomolgus macaques had glaucoma induced by laser trabecular
destruction (LTD) from 3 to 9 years prior to sacrifice. The mean
IOPs during the last 14 months of life varied from 23 to 54 mmHg in
the treated eyes (43 mean for all animals). 4 cynomolgus macaques
had inferior hemiretinal endodiathermy axotomy (HEA) either 3 or
4 months prior to sacrifice with no elevation in IOP (Dashek et al,
IOVS, 2013;54:3479). 3 rhesus macaques had HEA axotomy and 9.5
months later underwent LTD (mean IOP varied from 34 to 56 mmHg)
and were sacrificed 4.5 months after LTD.
Results: Although the retinal astrocytes and optic nerve
oligodendrocytes were strongly positive for GFAP, there was little
or no upregulation of GFAP in the Müller cells in any of the 6 eyes
with chronic glaucoma. Likewise, there was no upregulation of GFAP
in the Müller cells of any of the 4 eyes that underwent HEA alone.
However, there was marked Müller cell GFAP upregulation in both
the superior (no axotomy) and inferior (axotomy) areas of all 3 eyes
that had HEA plus 4.5 months of experimental glaucoma.
Conclusions: Müller cell GFAP upregulation is not dependent on the
presence of RGCs since upregulation occurred even though RGCs
were essentially absent from the axotomized areas of retina in the 3
HEA animals with elevated IOP. However, elevated IOP alone is not
a sufficient condition for sustained Müller cell GFAP upregulation
because it was not readily apparent in any of the 6 eyes with chronic
glaucoma. One possibility is that Müller cell GFAP upregulation
is the result of retinal ischemia, such as from decreased choroidal
blood flow. Compensatory mechanisms, e.g. increased blood flow or
decreased retinal oxygen need due to retinal cell death and/or lowered
metabolism, may have allowed the Müller cell GFAP to return to
nearly normal levels in the eyes with chronic glaucoma.
Commercial Relationships: Jeffrey C. Ockuly, None; Charlene B
Y. Kim, None; Brian J. Christian, None; Mélissa C. De Lombaert,
None; T Michael Nork, None
Support: NIH Grant P30 EY016665, Research to Prevent Blindness,
The Wisconsin National Primate Research Center P51RR000167/
P51OD011106
Elevated photoreceptor COX-2 expression correlates with aging
retinas
Carlos Quezada1, 2, Patrick Logan1, Ana Beatriz T. Dias1, Francisco
Ceballos1, Lisa Jagan1, Tiago Briccoli1, Miguel N. Burnier1.
1
Ophthalmology & Pathology, The Henry C. Witelson Ocular
Pathology Laboratory, McGill University, Montreal, QC, Canada;
2
California Retina Consultants, Santa Barbara, CA.
Purpose: While COX-2 is often associated with inflammation and
neoplasia, published reports have shown COX-2 to be expressed in
normal tissue. Our aim was to evaluate expression of COX-2 in all
layers of the normal human retina and determine whether correlations
with age or gender exist
Methods: 130 formalin-fixed, paraffin-embedded normal human
eyes were selected to investigate the expression of COX-2.
Immunohistochemistry was performed using anti-human COX2 antibody. Immunostaining was classified based on intensity
(negative=0, weak=1, strong=2) and extent (negative=0, staining
≤50% of cells=1, staining >50% of cells=2). An immunoreactive
score (IRS) was calculated to describe the expression of COX-2
using the following equation: 2 x expression x intensity. Results
were expressed in the following manner: 0-2=negative, 3-4=weak,
≥5=strong. Logistic regression analysis was conducted to estimate
the association between age and staining score of any layer. Linear
regression analysis was used to determine possible correlations
between IRS and time elapsed between death and enucleation or
enucleation and tissue processing p-value <0.05 was considered
statistically significant
Results: Of the 130 eyes evaluated, 72 were from female donors, 53
from male donors, and 5 from donors with no gender information.
Mean age was 62.1 years. A total of 78 cases had RPE and 66
had sufficient neurosensory retina for evaluation. The number of
cases with positive COX-2 expression by retinal layer was: retinal
pigmented epithelium (RPE): 61/78; nerve fiber layer: 33/66;
ganglion cell layer: 58/66; inner plexiform layer: 55/66; inner nuclear
layer: 36/66; outer plexiform layer: 56/66; outer nuclear layer: 31/66;
photoreceptors: 55/66. COX-2 expression in the photoreceptors was
significantly correlated with older donor age (p=0.045). COX-2 IRS
was strong in the ganglion cell, photoreceptor and RPE layers. No
significant correlations were found between IRS and time elapsed
between death and enucleation or IRS and time elapsed between
enucleation and tissue processing
Conclusions: COX-2 is expressed throughout the normal human
retina with strong expression on ganglion cell, photoreceptor
and RPE layers. Its expression in photoreceptors is significantly
correlated with older age. These results demonstrate that
consideration of a role for COX-2 in normal retinal physiology is
warranted, particularly when anti-COX-2 medications are being
prescribed
Commercial Relationships: Carlos Quezada, None; Patrick
Logan, None; Ana Beatriz T. Dias, None; Francisco Ceballos,
None; Lisa Jagan, None; Tiago Briccoli, None; Miguel N. Burnier,
None
SIRTUINS ARE DIFFERENTIALLY EXPRESSED IN
DISTINCT RETINAL LAYERS
Natàlia Vilà, Pablo Zoroquiain, Shawn C Maloney, Ana Beatriz T.
Dias, Emilia Antecka, Miguel N. Burnier. Ophthalmology - Ocular
Pathology, McGill University, Montreal, QC, Canada.
Purpose: While recent studies have indicated a role for Sirtuins
(SIRT) in biological processes, including metabolic diseases, cancer,
diabetes and aging, the exact nature of SIRT functions has not been
elucidated. Moreover, their function and expression in normal ocular
tissues remain unknown. The aim of this study is to evaluate the
expression of all sirtuin proteins (SIRT1-7) in normal human retinas.
Methods: Twenty-three formalin-fixed, paraffin-embedded normal
donor eyes (mean age = 72+/- 21.7 years), were evaluated in this
study. Immunohistochemistry was performed on serial sections
of all eyes with antibodies against each of the seven individual
sirtuins (SIRT1-7). Staining was graded semi-quantitatively in the
macula area and peripheral retina based on the intensity (negative=0,
weak=1, strong=2) and extent (negative=0, staining ≤50% of cells=1,
staining >50% of cells=2). A combined score was calculated to
describe the expression for each sirtuin using the following equation:
2 x expression x intensity. Results were expressed in the following
manner: 0 to 2=negative, 3 to 4= weak expression, ≥5 = strong
expression.
Results: All sirtuins were expressed in all of the studied retinas;
however, the staining score differed for each sirtuin across the
various retinal layers assessed. The retinal pigment epithelium (RPE)
expressed SIRT-4, 6 and 7. SIRT-6 was strongly positive only in the
macula, and both SIRT-4 and -7 were also strongly positive in the
macula and peripheral retina. The inner nuclear layer (INL) weakly
expressed SIRT-3 throughout the retina. The outer nuclear layer
(ONL) was the sole structure that was negative for all sirtuins. No
significant differences were found between macular and peripheral
retina in the same layer, except for SIRT-6 in RPE, which was
strongly expressed in the macula while negative in the peripheral
retina (p<0.05).
Conclusions: Our data provides an overview of the expression of
sirtuins in normal human retinas. SIRT-3 was the only sirtuin protein
found in the inner nuclear layer, while no sirtuins were expressed
in the outer nuclear layer of the retina. These results serve as a
foundation for further research into the roles of sirtuins in normal and
diseased retina.
Commercial Relationships: Natàlia Vilà, None; Pablo Zoroquiain,
None; Shawn C Maloney, None; Ana Beatriz T. Dias, None;
Emilia Antecka, None; Miguel N. Burnier, None
Expression of amyloid and tau proteins in the Octodon degus
retina
Monica L. Acosta1, Lily Chang1, Alvaro Ardiles2, Adrian Palacios2.
1
Optometry & Vision Science, The University of Auckland,
Auckland, New Zealand; 2Centro Interdisciplinario de Neurociencias,
Universidad de Valparaiso, Valparaiso, Chile.
Purpose: To determine whether the Octodon degus, an animal model
of Alzheimer’s disease (AD) expresses amyloid and tau proteins
indicative of AD in the eye as a function of development.
Methods: The retina from developing Octodon degus aged less
than 6 months (2-6 months-old; n=6) and adults older than 5 years
old (n=7) were employed. Eyes were collected immediately postmortem and immersion fixed in 4% paraformaldehyde in PBS
(pH 7.4) for over an hour. Immunocytochemical methods were
applied to cross-sections of the retina and retinal whole mounts.
Horseradish peroxidase (HRP-DAB) staining was carried out on
similar sections. The retina was immunolabeled against the major AD
proteins using antibodies against amyloid-beta (APPA4), Aβ peptide
(Aβ4G8, Aβ6E10), Aβ oligomers (A11), tau (Tau5A6) and hyperphosphorylated tau (PHF-tau). The area occupied by the antibody
mark in young and old retina was quantified. Congo red staining was
used to determine the presence of Aβ plaques.
Results: AD proteins were predominantly expressed in the ganglion
cell layer in the adult but not in the young retina. Tau and hyperphosphorylated tau were expressed in the central and peripheral
retina. Normal amyloid proteins were expressed in both young and
old retina while Aβ oligomers were only seen in old animals central
retina. Congo red staining revealed no apple-green birefringence.
Conclusions: There was an age-related expression of AD proteins
in O. degus eyes and the existence of common factors in the ocular
and brain tissues involved in AD etiology/pathogenesis. This further
supports the idea that non-invasive eye tests could be developed
for early AD diagnosis and that O. degus is a suitable model for
developing and validating diagnostic tests.
Commercial Relationships: Monica L. Acosta, None; Lily Chang,
None; Alvaro Ardiles, None; Adrian Palacios, None
Support: Neurological Foundation
309 New Insights into the Anatomy of Blood and Lymphatic
Vasculature of the Eye - Minisymposium
Tuesday, May 06, 2014 8:30 AM–10:15 AM
S 330CD Minisymposium
Immunhistochemical Detection of Blood and Lymphatic Vessels:
Potency and Limitations of Novel Markers
Falk Schroedl. 1Ophthalmology, Paracelsus Medical University,
Salzburg, Austria; 2Anatomy, Paracelsus Medical University,
Salzburg, Austria.
Presentation Description: The lymphatic system has major
impact on the drainage of extracellular fluid, immune response and
tumor dissemination. The detection of lymphatic vessels in routine
histological sections was long time not unequivocally possible
since appropriate markers were lacking. This changed with the
introduction of LYVE-1 or podoplanin as markers for lymphatic
endothelium. However, the specificity of a single marker is not 100%,
and depends also on the tissue or tumor investigated. While in the
immunprivileged and normally alymphatic eye, lymphatic vessels
were detected under pathological conditions, their existence is still
controversially discussed in normal conditions. Therefore we here
summarize various markers of lymphatic vessels and discuss their
relation to vascular endothelial markers with special emphasis on the
ocular situation.
Commercial Relationships: Falk Schroedl, None
Support: University grant PMU-FFF R13-/01/042-KAS
In Vivo Imaging of Blood Vessels: Potential and Limitations of
OCT-based methods at the Ocular Surface and Fundus
Robert J. Zawadzki. Ophthalmology & Vision Science, UC Davis,
Sacramento, CA.
Presentation Description: Improving our ability of non-invasive
imaging of blood vasculature in the eyes remains a subject of
intensive research. Over last several years substantial progress has
been made in developing Optical Coherence Tomography - based
methods allowing in-vivo evaluation of the vasculature of the anterior
and posterior segments of the eye.
In this talk I will briefly present review of the current imaging
methods followed by example images of blood vasculature acquired
in-vivo in the eyes of human subjects and animal models.
Commercial Relationships: Robert J. Zawadzki, None
Support: National Eye Institute (EY 014743 and EY 021054), UC
Davis RISE Grant, NSF I/UCRC Grant and Research to Prevent
Blindness.
Vasculature of the Cornea and Ocular Surface: New Implications
for Corneal Transplantation and Dry Eye Disease
Claus Cursiefen. Dept of Ophthalmology, Koln, Germany.
Presentation Description: Pathologic hem- and especially
lymphangiogenesis have recently been shown to be involved in
mediating immune responses at the ocular surface such as dry eye
disease and corneal allograft rejection. Here the role of normal
and pathologic vasculature of cornea and conjunctiva in mediating
immune responses and new therapeutic strategies aimed at these
pathologic vascular events are outlined. Finally, the interaction
between blood and lymphatic vessels and immune cell poplations at
the ocular surface are discussed.
Commercial Relationships: Claus Cursiefen, Allergan (C), Gene
Signal (C), Novaliq (C)
Support: DFG Cu 47/4-1
331 Myopia
Tuesday, May 06, 2014 11:00 AM–12:45 PM
Vasculature of the Conjunctiva: New Implications for Ocular
Tumors
Ludwig M. Heindl. Department of Ophthalmology, University of
Cologne, Cologne, Germany.
Presentation Description: The human conjunctiva is physiologically
well-endowed with blood and lymphatic vessels. The induction of
angiogenesis with sprouting of new capillary blood vessels from
preexisting ones is generally considered as one of the six hallmarks
of cancer, since tumors require sustenance in the form of nutrients
and oxygen as well as an ability to evacuate metabolic wastes and
carbon dioxide. Furthermore, the outgrowth of new from preexisting
lymphatic vessels, the so-called tumor-associated lymphangiogenesis,
is regarded as the initial step in lymphogenic metastasis of
several non-ocular malignant tumors. Recently, tumor-associated
lymphangiogenesis could be detected in malignant tumors affecting
the ocular surface, and associated with prognostic significance for
the risk of local recurrence, lymphatic spread, distant metastasis
and tumor-related death. In the future, novel antihemangiogenic and
antilymphangiogenic therapies might help to enhance survival of
these ocular tumor patients.
Commercial Relationships: Ludwig M. Heindl, None
Support: None.
Zebrafish as a Model to Study Emmetropization, Refractive
Error, and Retinal Substructure using Spectral Domain-Optical
Coherence Tomography
Ross F. Collery1, Francie Moehring1, Robert F. Cooper3, Adam
M. Dubis1, Joseph Carroll1, 2, Brian A. Link1. 1Cell Biology,
Neurobiology and Anatomy, Medical College of Wisconsin,
Milwaukee, WI; 2Ophthalmology, Medical College of Wisconsin,
Milwaukee, WI; 3Biomedical Engineering, Marquette University,
Milwaukee, WI.
Purpose: Spectral-domain optical coherence tomography (SDOCT) accurately measures the anatomy and dimensions of the eye in
vivo. Here, we characterize emmetropization of wild-type zebrafish,
myopia onset in bugeye/lrp2 mutants, and visualize the highly
ordered cone photoreceptor mosaic by SD-OCT. We combine high
resolution visualization with an animal model amenable to genetic
manipulation that can be used to study candidate genes for refractive
error and other ocular diseases.
Methods: Eye axial length, focal length and lens diameter were
measured in wild-type and bugeye/lrp2 mutant zebrafish throughout
their lifespan using a Bioptigen SD-OCT system. Cone photoreceptor
mosaics were visualized using en face summed volume projection
(SVP) images derived from the SD-OCT volume scans. Melanin
synthesis was ablated in a subset of RPE cells using TALENmediated inactivation of tyrosinase.
Results: We found that wild-type zebrafish became emmetropic by
1 month, while bugeye/lrp2 mutants were myopic, and worsened as
they aged. Wild-type fish maintained emmetropia, and our data show
that their lenses grow to balance the focusing power required as eye
size increases. By generating SVP images at different retinal depths,
we visualized the UV and S cone submosaics. Density measurements
of these submosaics agreed with published values from histology.
SVP images focused on the RPE layer showed regional melanin
inhibition provided by the TALEN technique, with improved
discrimination of the cone-RPE interface and underlying choroid and
sclera in B-scans of ‘windows’ of non-pigmented RPE.
Conclusions: As the zebrafish eye uses only lens refraction and
axial length to control emmetropia, we can assay the effects of genes
associated with myopia specifically on axial length modulation,
the largest single contributor to refractive error. Changes in retinal
morphology can be assessed during induction of blinding disorders
by SD-OCT, and changes in cone density or patterning can be used to
assess photoreceptor damage in visual disorders.
Vasculature of the Retina and Choroid: New Implications for
retinal diseases
Richard F. Spaide. Vitreous Retina Macula Consultants, New York,
NY.
Presentation Description: .
Commercial Relationships: Richard F. Spaide, Topcon (C),
Topcon (P)
In Vivo Imaging of Lymphatics: Challenges and Opportunities
Yeni H. Yucel. Ophthal & Vision Sciences, Univ of Toronto/St
Michael Hosp, Toronto, ON, Canada.
Presentation Description: New insights into the recently discovered
ocular lymphatics suggest a role in aqueous humor drainage in
addition to eye immunity. Non-invasive in vivo imaging techniques
have been used to track systemic and ocular lymphatic drainage
in animal models. Available novel detection systems, advantages,
limitations, and application to the study of lymphatics in health and
diseases of the eye, will be reviewed.
Commercial Relationships: Yeni H. Yucel, None
Support: Canadian Institutes of Health Research, Leaders
Opportunity Fund - Canada Foundation for Innovation, Glaucoma
Research Society of Canada
A. B-scan showing anatomy of adult zebrafish eye; B. B-scan of adult
zebrafish retina; C. en face (SVP) showing photoreceptor mosaic; D.
merged SVPs from different depths showing UV cone and S cone
submosaics (magenta, green); E. SVP of mosaic RPE showing nonpigmented cell areas (dark) surrounded by normal pigmented areas
(bright)
Commercial Relationships: Ross F. Collery, None; Francie
Moehring, None; Robert F. Cooper, None; Adam M. Dubis, None;
Joseph Carroll, None; Brian A. Link, None
Support: NIH/NEI R01EY016060; P30EY001931; Research to
Prevent Blindness
The role of cell-cell coupling in myopia development and light
adaptation
Michelle Teves1, Qing Shi2, William K. Stell3, Derek Eng1. 1B.Sc.
Neuroscience Program, University of Calgary, Calgary, AB, Canada;
2
Graduate Neuroscience Program, University of Calgary, Calgary,
AB, Canada; 3Cell Biology and Anatomy, and Hotchkiss Brain
Institute, University of Calgary, Calgary, AB, Canada.
Purpose: Myopia is a refractive disorder in which excessive
elongation of the eye causes blurring of distance-vision. Dopamine
(DA) and nitric oxide (NO), which are released in the retina under
light-adaptation, are critical for myopia-prevention and light
adaptation. How they work is still poorly understood; but both have
been shown to uncouple gap junctions in retinal networks. Therefore,
cell-cell coupling in the retina may play a key role in the regulation
of ocular growth, e.g., by mediating the effects of light, DA, and
NO on spatial contrast sensitivity (CS) in the retina. We tested this
hypothesis using a gap junction blocker, meclofenamic acid (MFA),
to determine its effect on myopia development and spatial CS.
Methods: Form-deprivation myopia (FDM) was induced in 6 day
old (P6) White Leghorn cockerels by diffusers over the right eyes
(treated, “T”) leaving the left eyes uncovered as controls (C). We
injected intravitreally 20 mL of 20 mM MFA in dH2O, or dH2O
alone, on P7, P9, and P11. On P12 we measured refractive error, axial
length, equatorial length, and eye weight. Treatment effects were
expressed as interocular differences (T-C; one-way ANOVA, Tukey
post-hoc).
The role of cell-cell coupling in light-adaptation in retinal circuitry
was assessed by the optokinetic response, using OptoMotry®, before
and after injection of 20 mM MFA (as above). Mean intensities were
in the low to intermediate photopic range (-0.7 to 2.0 log cd/m2).
Results: In dH2O controls, goggles induced a myopic shift in
refractive error (-9 ± 3D, mean ± SD) and excessive axial length
(0.9 ± 0.3mm) [T-C]. MFA significantly reduced the induced
increases in refractive error (0 ± 2D; p<0.001) and axial length (0.0
± 0.3mm, p<0.001) [T-C]. Equatorial length and eye weight were not
significantly affected by treatment.
At the highest luminance, MFA increased CS at medium-high spatial
frequencies (SFs) (SF=0.32 cyc/deg, p<0.001; SF=0.5 cyc/deg,
p<0.05; SF=0.8 cyc/deg, p<0.01; SF=1.0 cyc/deg, p<0.05, n=8-9,
paired t-test), whereas it did not alter CS under dim light (-0.7 log cd/
m2).
Conclusions: Intravitreal MFA mimics the actions of increased light
intensity, DA, and NO, on form-deprivation myopia and optokinetic
contrast sensitivity in chicks. This suggests that uncoupling of gap
junctions between retinal neurons may mediate some actions of DA
and NO.
Commercial Relationships: Michelle Teves, None; Qing Shi, None;
William K. Stell, None; Derek Eng, None
Support: Alberta Innovates - Health Solutions (AIHS) Summer
Student Award (MT); Natural Sciences and Engineering Research
Council (NSERC; Canada) Discovery Grant (WKS) and
Undergraduate Student Research Award (DE); Foundation Fighting
Blindness (FFB; Canada) EYEGEYE Research Training Fund (WKS)
Differential gene expression in tree shrew retina compared with
retinal pigment epithelium (RPE) in response to six hours of
minus-lens wear
Li He, Michael R. Frost, Thomas T. Norton. Department of Vision
Sciences, University of Alabama at Birmingham, Birmingham, AL.
Purpose: Extensive evidence suggests local regulation of ocular
growth. The retina initiates signals that regulate this growth and RPE
is an important transmitter/translator of those signals. We examined
and compared early gene expression patterns in the retina vs. RPE
from eyes of the same experimental animals responding to short-term
minus-lens wear.
Methods: Starting 24 days after normal eye opening, a group of
seven tree shrews wore a monocular −5 D lens for six hours to
initiate increased axial elongation and the development of lensinduced myopia. The untreated contralateral eye served as a control.
Quantitative real-time PCR was used to measure gene expression in
retina and RPE tissues separately. Forty-four genes of interest were
chosen based on the literature and preliminary studies in this lab.
Results: RNA yields were typically around 20 mg from the retina and
2 mg from the RPE. After six hours of minus-lens wear, significant
differential mRNA expression changes (treated vs. control) were
found for four genes in the retina: BMP2, CTGF, and EGR1 were
down-regulated, whilst SST was up-regulated. In the RPE, eleven
genes showed significant differential expression: GJA1, IGF2R,
and LRP2 were up-regulated, whilst APOE, DRD1, NOS1, OPN4,
PENK, SLC18A2, SSTR2, and VIP were down-regulated. No
gene was differentially expressed in both tissues. These relatively
small expression changes, typically <2-fold, are induced prior to
measurable changes in refraction or axial length.
Conclusions: Six hours of minus-lens wear is sufficient to produce
differential gene expression in tree shrew retina and in RPE. The
pattern of gene expression differs between the two, suggesting
that emmetropization-related signaling is transformed as it moves
from retina to RPE. Expression changes in some of these genes
have been reported in chick retina/RPE, analyzed together, after 6
hours of minus-lens wear (Stone et al. IOVS 2011; 52:5765-5777).
Our tree shrew results suggest that some of these changes occur in
retina and some in RPE, suggesting that each ‘compartment’ in the
emmetropization signaling cascade should be analyzed separately.
Commercial Relationships: Li He, None; Michael R. Frost, None;
Thomas T. Norton, None
Support: NIH Grants EY005922 and EY003039 (P30)
C57BL/6 mouse eyes treated by dopamine D1 receptor agonist
and antagonist during form deprivation: an opposite effect on
axial length and refractive development
Xiangtian Zhou, Weiwei Xiong, Furong Huang, JInglei Yang, Jia Qu.
School of Ophthalmology and Optometry, Wenzhou Medical College,
Wenzhou, Zhejiang, China.
Purpose: Dopaminergic activities in the eye have been shown to
modulate development of form deprivation myopia (FDM). However,
the mechanism of dopamine receptors involved in FDM is complex.
In this study, we investigated effects of dopamine D1 receptor
(DRD1) on the development of refraction and axial components of
the eyes treated with form deprivation using selective DRD1 agonist
and antagonist.
Methods: Four-week-old C57BL/6 mice (n=245) raised in
normal visual environments or form deprivation received daily
intraperitoneal injection of either SKF38393 (DRD1 agonist, n=121)
or SCH39166 (DRD1 antagonist, n=124) for four weeks. Each of the
groups was divided into 4 sub-groups including control, vehicle and
drug treatments with two different dosages. The refraction, corneal
radius of curvature and axial components of the eye were measured
in all animals prior to and after the treatments.
Results: After four weeks of treatment, eyes treated with SKF38393
showed a dose dependent inhibition to the development of FDM
with a slower increase in vitreous chamber depth and axial length
compared to the vehicle group. In contrast, SCH39166-treated
animals showed a dose dependent promotion of FDM with a more
rapid increase in vitreous chamber depth and axial length compared
to the vehicle group. No significant differences in corneal radius
of curvature, anterior chamber depth or lens thickness among the
different groups were found during the experimental period. The
refraction and axial components of the eyes raised in normal visual
environments were not affected neither by SKF38393 nor SCH39166.
Conclusions: DRD1 agonist and antagonist exert opposite effects on
refractive development and ocular growth during form deprivation.
An activation of DRD1 can inhibit the development of FDM in
C57BL/6 mice.
Commercial Relationships: Xiangtian Zhou, None; Weiwei Xiong,
None; Furong Huang, None; JInglei Yang, None; Jia Qu, None
Support: 973 program: 2011CB504602; NSFC:81371047
Outdoor Exposure Does Not Inhibit Experimental Myopia
Richard A. Stone1, Yuval Cohen1, Alice M. McGlinn1, Sherrill
Davison2, Susan Casavant2, Jiayan Huang1, Tejvir S. Khurana3,
Machelle T. Pardue4, 5, P M. Iuvone4. 1Ophthalmology, Univ of
Pennsylvania School of Medicine, Philadelphia, PA; 2Laboratory
of Avian Medicine and Pathology, New Bolton Center, Kennett
Square, PA; 3Physiology, Univ of Pennsylvania School of Medicine,
Philadelph1a, PA; 4Ophthalmology, Emory University School of
Medicine, Atlanta, GA; 5Rehab R&D Center of Excellence, Atlanta
VA Medical Center, Atlanta, GA.
Purpose: Rearing chicks and mammals under high-intensity
laboratory lighting inhibits form-deprivation myopia, and it has
been proposed that increasing outdoor exposure of children might
favorably influence clinical myopia. We assessed the effects of “realworld” outdoor exposures on goggle-induced myopia in chicks.
Methods: Two cohorts of white Leghorn chicks, after placement
of a uniocular translucent goggle at 5 or 9 days of age, were reared
outdoors in a rural setting during daylight hours to the extent
permitted by weather; they were not exposed to electric lighting.
Two control cohorts were reared under incandescent lighting (500
lux) without outdoor lighting exposure, with light cycle durations
matched to sunrise-sunset times of the outdoor chicks. Retinal
dopamine/DOPAC (HPLC with electrochemical detection) and ocular
refractions/dimensions (refractometry, ultrasound) were measured
after 10 or 11 days. Analysis used paired t-test or one-way ANOVA.
Results: Non-goggled eyes had some 2.7 diopters (D) of hyperopia
in all cohorts. The cohorts of goggled eyes averaged 12-23 D of
myopia relative to control eyes, with no evidence that outdoor rearing
lessened the myopic response. In fact, the myopic response was
exaggerated in goggled eyes of the older outdoor vs. indoor cohort
(-23 vs. -13 D; P<0.001). Similarly, the vitreous chambers and axial
lengths were elongated in goggled eyes relative to controls, also with
no evidence that outdoor rearing attenuated the myopic response
(P>0.05, all between-cohort comparisons). In the younger cohort,
retinal dopamine or DOPAC (pg/mg protein) in non-goggled eyes
and the reductions in contralateral goggled eyes (dopamine ~15%;
DOPAC ~48%) were similar between indoor and outdoor reared
chicks (P>0.4, all between-cohort comparisons).
Conclusions: Contrary to current hypotheses, outdoor exposure does
not reproduce the anti-myopia effects of increasing artificial lighting
intensity in the lab, at least in chicks. The outdoor environment
has much variability in richness of scene and chromaticity, along
with major shifts in illumination from movement/density of clouds,
individual movement into and out of shady areas, and direction
of gaze. All these features were inherent to our study. While some
element of outdoor and/or lighting exposure may favorably affect
refractive development, our results point to a level of complexity
that is not being adequately considered in laboratory or clinical
investigations.
Commercial Relationships: Richard A. Stone, None; Yuval
Cohen, None; Alice M. McGlinn, None; Sherrill Davison, None;
Susan Casavant, None; Jiayan Huang, None; Tejvir S. Khurana,
None; Machelle T. Pardue, None; P M. Iuvone, None
Support: NIH R01 EY022342 (RAS), NIH P30 EY001583 (Penn),
NIH R01 EY016435 (MTP), NIH R01 EY004864 (PMI), P30
EY006360 (PMI), Mackall Trust (RAS), RPB (RAS, PMI).
Influence of Oral 7-Methylxanthine on Lens-induced and Form
Deprivation Myopia in Chickens
Kai Wang1, 2, Diane Nava2, Klaus Trier3, Christine Wildsoet2.
1
Ophthalmology Department, Peking University People’s Hospital,
Beijing, China; 2Wildsoet Lab, School of Optometry, University
of California, Berkeley, Albany, CA; 3Trier Research Laboratories,
Hellerup, Denmark.
Purpose: 7-methylxanthine (7-MX), a non-selective adenosine
antagonist, is approved as an anti-myopia oral medication in
Denmark. To learn more about its mechanisms, this study examined
its ability to inhibit lens-induced (LIM) and form deprivation myopia
(FDM) in young chickens.
Methods: Forty-five young White-Leghorn chickens were divided
into four groups, groups 1 and 2 being assigned -10 D lenses (n=10
each), and groups 3 and 4, white diffusers (n=12 & 13 resp.). All
optical treatments were monocular, applied to left eyes, starting eight
days after hatching, and extending for 10 days. From the beginning
of the treatment period, groups 2 and 4 received at 9 am and 3 pm
each day, two doses of oral 7-MX (30 mg/kg, samples provided by K
Trier). Sterile distilled water was used as the diluent, with xanthangum added to ensure uniform suspension of 7-MX. Control groups
(1 and 3) received the xanthan-gum solution without 7-MX, with the
dosing schedule as per 7-MX. Baseline refractive errors and axial
ocular dimensions were measured before treatments, using static
retinoscopy and high frequency A-scan ultrasonography respectively,
and measurements were repeated every other day thereafter.
Results: 7-MX had a small inhibitory effect on LIM but no effect
on FDM, as reflected in the refractions recorded at the end (LIM:
-6.43 ± 1.30 D, 7-MX versus control, -8.30 ± 1.53 D; FDM: -10.19
± 7.89 D, 7-MX versus -10.77 ± 7.20 D, control)(Figure 1). For LIM
groups, the interocular difference in optical length was smaller in the
7-MX group. And final intergroup difference (0.12 mm) was mainly
of a product of differences in anterior chamber depth (0.11 mm).
Both groups showing similar interocular differences in other key
ocular parameters (vitreous chamber depth and lens thickness). No
significant intergroup differences in these parameters were noted for
the FDM groups.
Conclusions: For the dose tested, 7-MX showed only a weak
inhibitory effect against LIM and no effect against FDM in the
chicken. This result adds weight to evidence suggesting a scleral
site of action for the anti-myopia effect of 7-MX, because unlike
mammalian and primate eyes, avian eyes have an additional cartilage
layer, which largely determines eye size.
Figure 1. Interocular changes in mean spherical equivalent refractive
error (SER), normalized to baseline values, for the lens-wearing
group (LIM, A) and form deprived groups (FDM, B).
Commercial Relationships: Kai Wang, None; Diane Nava, None;
Klaus Trier, None; Christine Wildsoet, None
Support: NIH/NEI EY12392
Changing Material Properties of the Tree Shrew Sclera during
Minus Lens Compensation and Recovery
Rafael Grytz1, John T. Siegwart2, Thomas T. Norton2. 1Department of
Ophthalmology, University of Alabama at Birmingham, Birmingham,
AL; 2Department of Vision Sciences, University of Alabama at
Birmingham, Birmingham, AL.
Purpose: To estimate two material properties (collagen fibril crimp
angle and elastic modulus) of the remodeling tree shrew sclera during
monocular -5 D lens wear and recovery.
Methods: Tensile tests were performed on 3-mm wide scleral strips
(0-50 g, 30 sec) obtained from juvenile tree shrews exposed to
three different visual conditions: (i) normal development (24, 28,
and 35 days of visual experience; n = 2, 3, and 3, respectively); (ii)
monocular -5 D lens wear to induce axial elongation and myopia (1,
2, 4, and 11 days of lens wear; n = 5 per group); and (iii) recovery
from the myopia (1, 2, 4, and 10 days of recovery with no lens after
11 days of lens wear; n = 3 per group). Collagen fibrils are crimped
in the unloaded sclera and uncrimp as the tissue stiffens under
load. Inverse numerical analyses were performed to estimate the
collagen fibril crimp angle (unloaded) and elastic modulus using a
microstructure-based constitutive model.
Results: The fitted crimp angle was significantly higher in the
treated eye vs. control eye after 2 days and peaked after 4 days of -5
D lens wear (p < 0.05). This difference was reduced but remained
significantly higher after 11 days. In contrast, the difference in crimp
angle rapidly decreased after the lens was removed and was not
significant after 1 day of recovery. A rapid increase in the elastic
modulus (up to 2-3 fold) was seen in both eyes (control and treated)
after starting or stopping the -5 D lens wear. The increase was highly
transient during lens wear, but more sustained during recovery.
Compared to normally developing eyes, this stiffening effect was
significant during the first 2 days of monocular lens wear in both
eyes, while it remained significant up to day 4 and 10 of recovery in
the treated and control eye, respectively.
Conclusions: The estimated change in the crimp angle of scleral
collagen fibrils is temporally associated with the change in axial
elongation rate during myopia development and recovery. This
finding suggests that axial elongation may be controlled by a
remodeling mechanism that modulates the collagen fibril crimp as
well as creep rate (Siegwart and Norton, Vision Res. 1999;39:387407). The binocular changes in scleral stiffness during monocular
lens treatment and recovery are not temporally associated with the
change in axial elongation, indicating that scleral stiffening may not
be causally related to axial elongation in myopia.
Commercial Relationships: Rafael Grytz, None; John T.
Siegwart, None; Thomas T. Norton, None
Support: NIH grants R01 EY005922, EY003909 (P30); EyeSight
Foundation of Alabama; Research to Prevent Blindness
336 Non-melanoma intraocular lesions: Retinoblastoma and
beyond
Tuesday, May 06, 2014 11:00 AM–12:45 PM
Contributing Section(s): Retinal Cell Biology, Retina
Comparative Analysis Of ABCG2+ Stem-Like Retinoblastoma
Cells And Induced Pluripotent Stem Cells As Three-Dimensional
Aggregates
Gail M. Seigel1, 2, Linda Cassidy1, Robert Diaz3, Ruby Y. Tsai3.
1
Center for Hearing and Deafness, University at Buffalo, Buffalo,
NY; 2SUNY Eye Institute, Buffalo, NY; 3Applied StemCell, Inc.,
Menlo Park, CA.
Purpose: Retinoblastoma (RB), an intraocular malignancy of early
childhood, expresses a number of stem cell markers including
ABCG2. In this study, we compared ABCG2+ and ABCG2- RB
cells with induced pluripotent stem cell (iPSC)-derived embryoid
bodies to assess their potential for pluripotency as three-dimensional
aggregates. We tested the hypothesis that both iPSC-derived
embryoid bodies and ABCG2+ cells would both preferentially
exhibit pluripotent stem cell markers and less mature retinal marker
expression as compared with ABCG2- cells.
Methods: Immunomagnetic enrichment of WERI-RB27 and
Y79 retinoblastoma cells created populations that were ABCG2+
or ABCG2-. Enriched ABCG2+ and ABCG2- populations were
examined as aggregates in three-dimensional culture and compared
with embryoid bodies formed by iPSCs. Resulting cell aggregates
were assessed by immunohistochemistry for a variety of stem cell
and mature markers.
Results: ABCG2+ and ABCG2- cells formed aggregates that
differed morphologically from iPSC-derived embryoid bodies.
Immunostaining demonstrated that both ABCG2+ and ABCG2cell aggregates were immunoreactive to tubulin III (ectoderm), but
immunonegative for smooth muscle actin (mesoderm) and alpha-feto
protein (endoderm). However, ABCG2+ aggregates exhibited greater
immunoreactivity to stem cell markers (ABCG2, ALDH1A1 and
CD164), but less immunoreactivity to mature markers (MAP-2 and
S-Antigen) as compared with ABCG2- RB cells. In contrast, iPSCinduced embryoid bodies contained cells that were immunoreactive
for primitive markers including Nestin, PAX6, CD164 and
ALDH1A1.
Conclusions: Aggregate cultures of enriched ABCG2+ RB cells
possess a higher degree of stem-like features as compared with
ABCG2- RB cells. Both populations express Tubulin III, a marker of
embryonic ectoderm, but neither alpha feto-protein (endoderm) nor
smooth muscle actin (mesoderm) as compared with pluripotent iPSCderived embryoid bodies. This suggests a restriction to ectodermal
lineage for both ABCG2+ and ABCG2- RB cells. These results may
have implications for RB tumor development, as well as the potential
to lead to novel therapeutic approaches for tumor eradication in RB.
WERI-RB27 retinoblastoma cell aggregate with immunoreactivity to
ABCG2 (red) and CD164 (green). DAPI (blue) labels nuclei.
Commercial Relationships: Gail M. Seigel, Applied StemCell, Inc.
(R); Linda Cassidy, None; Robert Diaz, Applied StemCell, Inc. (E);
Ruby Y. Tsai, Applied StemCell, Inc. (E)
Support: This work was supported by the Cornell Center on
the Microenvironment & Metastasis through Award Number
U54CA143876 from the National Cancer Institute and NYSTEM
C026412.
Optical Coherence Tomography Enables Imaging of
Retinoblastoma Tumor Initiation in the TAg-RB Mouse Model
Andrea Wenzel1, 2, Mehdi Shadmand1, 2, Timothy W. Corson1, 2.
1
Eugene and Marilyn Glick Eye Institute, Indiana University School
of Medicine, Indianapolis, IN; 2Ophthalmology, Indiana Univ School
of Medicine, Indianapolis, IN.
Purpose: Retinoblastoma is the most common intraocular
malignancy in children. Although significant advances in treatment
have decreased mortality in recent years, there continues to be
a high morbidity associated with these therapies and therefore
a pressing need for new therapeutic options. Transgenic mouse
models are popular for testing new therapeutics as well as studying
the pathophysiology of retinoblastoma. The TAg-RB model has
the closest molecular and histological resemblance to human
retinoblastoma tumors; these mice inactivate pRB by retinal-specific
expression of the Simian Virus 40 T-antigens. Optical coherence
tomography (OCT) has previously been used to characterize TAg-RB
tumors in 10-13 week-old mice. Here, we evaluated whether OCT
imaging could be used to document tumor growth in the TAg-RB
model at the earliest stages of tumor development.
Methods: The Micron III rodent imaging system was used to obtain
fundus photographs and OCT images of both eyes of TAg-RB mice
regularly from 2 to 20 weeks of age to document tumor development.
Tumor morphology was confirmed by histological analysis.
Results: Light-colored, intraretinal tumors, preferentially in the
periphery, were readily seen by funduscopy in animals ≥8 weeks
of age. By OCT, hyperreflective tumor masses arising in the inner
nuclear layer were evident as early as five weeks, even when no
pathology was yet evident by funduscopy. These masses grew into
discrete, discoid tumors that developed more irregular morphology
over time, eventually merging and displacing the inner retinal layers
into the vitreous.
Conclusions: OCT is a novel, non-invasive imaging modality for
tracking early TAg-RB tumor growth in vivo. Using OCT, we were
able to characterize tumor growth as early as 5 weeks, corresponding
to the earliest stages at which tumors are histologically evident, and
before they are evident by funduscopy. Tracking tumor growth from
its very earliest stages will allow better analysis of the efficacy of
novel therapeutics tested in this powerful mouse model.
Commercial Relationships: Andrea Wenzel, None; Mehdi
Shadmand, None; Timothy W. Corson, Phoenix Research Labs (F)
Support: Phoenix Research Labs, ACSIRG, NIH/NCATS TR000163
Lymphocytic microparticles suppress growth of retinoblastoma
Qian Qiu, Chun Yang, Houda Tahiri, Carmen Gagnon, Pierre Hardy.
Research Center of CHU Sainte-Justine, University of Montreal,
Montreal, QC, Canada.
Purpose: Retinoblastoma (Rb) is an aggressive childhood cancer of
the developing retina that is associated with epigenetic deregulation
of several cancer pathways. In addition to the significantly
upregulation of the proto-oncogene spleen tyrosine kinase (SYK),
the angiogenic potential of Rb correlates with invasive growth and
metastasis. Lymphocyte-derived microparticles (LMPs) possess
strong antiangiogenic effect against pathological ocular angiogenesis
and potent inhibitory effect on cell viability. This study is designed to
elucidate the mechanisms underlying the anti-Rb effect of LMPs.
Methods: LMPs were produced from human T cell lymphoblastlike cell line cells (CEM T cells) after 0.5 μg/mL actinomycin D
stimulation and isolated LMPs were characterized with annexin
V staining and gated using 1.0 mM beads. Rb cell line (Y-79) and
primary cultured Rb cells (isolated from primary site intraocular
retinoblastoma of Rb patients) were subjected to WST-1, cellular
senescent, apoptotic assay and FACS cell cycle analysis after treated
with LMPs. Quantitative RT-PCR, immunohistochemistry and
Western blot were performed to detect interest gene expression and
protein levels in LMPs and in Rb cells.
Results: LMPs significantly reduced Rb cell viability in a dosedependent manner. LMPs in a concentration of 10mg/ml significantly
induced Rb cell-cycle arrest at G0/G1 phase with associated increases
of the senescence-associated β-galactosidase activity. LMPs in higher
concentrations (≥20mg/ml) induced cell death (46% apoptotic cells in
LMPs-treated primary cultured Rb cells compared to 14% in control
cells). LMPs treatment has a significant suppression effect on the
expression of SYK in primary Rb cells. Moreover, LMPs contain a
considerable amount of p21(cip1), a cell cycle inhibitor. Inhibition of
p21(cip1) significant induced pro-apoptotic effect of LMPs.
Conclusions: LMPs dose-dependently induced Rb cell senescence or
apoptosis; the mechanism favouring this effect is directly mediated
by p21(cip1) pathway. These data may open unexpected avenues for
the development of novel therapeutic strategies that are particularly
useful and relevant for the treatment of Rb cancer.
Commercial Relationships: Qian Qiu, None; Chun Yang, None;
Houda Tahiri, None; Carmen Gagnon, None; Pierre Hardy, None
Support: FRSQ
Topotecan pharmacokinetics and safety after super-selective
ophthalmic artery infusion concomitant to melphalan in children
with retinoblastoma
Paula J. Taich1, Alejandro Ceciliano2, Emiliano Buitrago1, Francisco
Villasante2, Claudia Sampor1, Gabriel Mato1, Guillermo L.
Chantada1, Paula Schaiquevich1. 1Hospital de Pediatria JP Garrahan,
Buenos Aires, Argentina; 2Maternidad Suizo Argentina, Argentina,
Buenos Aires, Argentina.
Purpose: To characterize topotecan pharmacokinetics and safety after
super-selective ophthalmic artery infusion (SSOAI) in combination
with melphalan in children with retinoblastoma.
Methods: Topotecan SSOAI concomitant to melphalan was offered
to children with retinoblastoma between October 2011 and July
2013. The SSOAI therapy was performed according to published
guidelines. Consenting patients received SSOAI topotecan (0.5-1
mg) concomitant to melphalan (3-7 mg) in unilateral administration.
Blood samples were collected from a peripheral access before
starting and at the end of the infusion of each drug and 0.5, 1, 2 and 3
h after finishing SSOAI. Topotecan was quantified by HPLC and the
pharmacokinetics was characterized using a nonlinear mixed effects
modeling approach. After each chemotherapy cycle, hematological
toxicities were graded according to international criteria. Patients
received both drugs at no predetermined order (sequence effect).
Results: A total of 21 patients received SSOAI topotecan
concomitant to melphalan in 39 cycles. The median (range) age and
weight at the first cycle was 1.6 years (0.75-7.4) and 11.6kg (7.930), respectively. Topotecan pharmacokinetics was best described
by a 2-compartment model with an additive residual error model
according to the limit of quantitation (5ng/ml). Topotecan mean (s.e)
pharmacokinetic parameters calculated included clearance: 0.67 L/h/
kg (0.07); volume of distribution of the central compartment: 0.53 L/
kg (0.09), intercompartmental clearance: 2.86 L/h/kg (0.34); volume
of distribution of the peripheral compartment: 0.72 L/kg (0.07) and
a median (range) systemic exposure corrected by dose of (AUC/D):
95.5 (ng*h/ml)/mg (34.5-237.9). Adverse events included 5 grade
3/4 neutropenia with an incidence of 12.8 % of myelosupression. The
sequence of drug administration was not statistically associated with
topotecan clearance (p>0.05).
Conclusions: The present data are in agreement with topotecan
pharmacokinetics after endovenous administration previously
reported in children. Topotecan systemic exposure was low and is
in correspondence with the low incidence of hematological toxicity
(12.8%).
Commercial Relationships: Paula J. Taich, None; Alejandro
Ceciliano, None; Emiliano Buitrago, None; Francisco Villasante,
None; Claudia Sampor, None; Gabriel Mato, None; Guillermo L.
Chantada, None; Paula Schaiquevich, None
Support: Supported by: Fund for Ophthalmic Knowledge, New
York, New York, US; Consejo Nacional de Investigaciones
Cientificas y Tecnicas, Buenos Aires, Argentina (grant no.:
11220090100343); Agencia Nacional de Promocion CientificaFONCYT, Buenos Aires, Argentina (PICT Bicentenario no.: 20102271); Hospital JP Garrahan, Buenos Aires, Argentina and Fundacion
Natalie D. Flexer de Ayuda al Niño con Cancer, Buenos Aires,
Argentina.
Evaluation of the Efficacy of Topotecan Loaded Au-Tethered
Liposomes and AU-011 for the Treatment of Retinoblastoma in
vitro
Kristen Jijelava1, Shin Kang1, Uday Kompella2, Shelley A. Durazo2,
Elisabet de los Pinos3, John MacDougall3, Hans E. Grossniklaus1.
1
Ophthalmology, Emory University School of Medicine, Atlanta,
GA; 2Pharmaceutical Sciences, University of Colorado, Denver, CO;
3
Aura Biosciences, Cambridge, MA.
Purpose: The purpose of the study is to test our hypothesis that if
WERI-Rb-1 cells are exposed to topotecan, topotecan loaded Autethered DPPC/DPGPTE liposomes treated with near-infrared (NIR)
pulsed laser, or AU-011 treated with a light-emitting diode (LED)
laser, that the result will be cell death.
Methods: WERI-Rb-1 cells were grown to 3-4x10^5 cells/mL and
exposed to different concentrations of topotecan (5-20 mg/50 mL)
and topotecan loaded Au-tethered DPPC/DPGPTE liposomes (10-50
mg/50 mL). After treatment with topotecan loaded Au-tethered DPPC/
DPGPTE liposomes, the cells were exposed to an 810 nm diode laser
(1500 mW, 1000 msec at 50 msec repeat intervals) for 2 minutes.
Similarly, WERI-Rb-1 cells were treated with multiple concentrations
of AU-011 (Human papillomavirus virus-like particles conjugated
with IR700 dye) (0.1-3 nM) and exposed to 16J of light using a LED
lamp. Cell viability was determined at multiple time points using a
trypan blue viability stain and a hemocytometer.
Results: Topotecan at a concentration of 20 mg/50 mL causes 99.7%
cell death (p<0.001) by 4 hours. There was no statistically significant
difference in cell viability rates at any time point between the 20
mg/50 mL of topotecan loaded Au-tethered DPPC/DPGPTE liposome
groups that received NIR pulsed laser therapy as compared to the
same group that did not receive laser therapy. The AU-011 at 0.1 nM,
1 nM, and 3 nM concentrations following exposure to 16J with an
LED lamp all resulted in a statistically significant (p < 0.05) increases
in cell death (83.3-91.7%, 87.5-100%, and 95.0-100% respectively)
compared to the control (1.56-8.75% dead cells) at 1, 4, and 24 hours.
Conclusions: Topotecan at a concentration of 20 mg/50 mL
effectively and rapidly kills WERI-Rb-1 cells in vitro. NIR pulsed
laser therapy applied for a clinically relevant duration does not
cause the necessary rise in temperature of the gold nanoshells to
induce the release of a significant amount of topotecan from the
liposomes. However, AU-011 in combination with LED therapy
presents a promising novel method to effectively induce cell death of
retinoblastoma cells locally without being cytotoxic to cells that do
not receive LED exposure.
Commercial Relationships: Kristen Jijelava, None; Shin Kang,
None; Uday Kompella, None; Shelley A. Durazo, None; Elisabet
de los Pinos, Aura Biosciences (E); John MacDougall, Aura
Biosciences (E); Hans E. Grossniklaus, None
Support: Supported in part by an unrestricted department grant from
Research to Prevent Blindness, Inc
Small molecules that selectively inhibit growth of MYCNARB1+/+
retinoblastoma cells
Kamakshi Sishtla1, 2, Timothy W. Corson1, 2. 1Eugene and Marilyn
Glick Eye Institute, Indianapolis, IN; 2Department of Ophthalmology,
Indiana University School of Medicine, Indianapolis, IN.
Purpose: Retinoblastoma is the most common pediatric ocular
cancer, traditionally thought to be always caused by loss of both
alleles of the RB1 tumor suppressor gene. However, recent work
showed that amplification of the MYCN oncogene can also spur the
development of retinoblastoma in a fraction of unilateral patients
with normal RB1 alleles. Here, our aim was to identify small
molecules from a library of known bioactive compounds that show
selective growth inhibition of a MYCNARB1+/+ retinoblastoma cell
line (RB3823) over a MYCNARB1-/- cell line (Y79). Such molecules
may be leads for therapy of MYCNARB1+/+ retinoblastoma, and
will further elucidate the molecular differences between these two
subtypes of retinoblastoma.
Methods: Compounds from the LOPAC1280 library were tested
to judge their effect at 10 μM on cell proliferation in both Y79 and
RB3823 cell lines using Alamar Blue after a 48 hour incubation in
384 well format. Compounds that reduced cell proliferation by at
least 40% in one or both cell lines were tested twice more in both cell
lines to confirm efficacy. Dose response testing was conducted using
compound concentrations ranging from 100 pM to 1 mM in 384 well
format.
Results: In initial screening, 95 compounds showed cytotoxicity to
one or both cell lines. Secondary screening yielded 9 compounds
that reduced RB3823 proliferation by at least 40% compared to
untreated cells. Confirmatory screening identified 6 of those 9
compounds that reduced RB3823 proliferation by at least twice
that of Y79. In dose-response assays of 5 of these compounds, one
compound showed reproducibly higher efficacy in RB3823 than Y79.
Dimethoxy-naphthoquinone (DMNQ) had a GI50 in RB3823 at least
3 times lower than that for Y79. DMNQ is an oxidizing quinone that
is thought to act by depleting cellular reduction potential leading to
oxidation of proteins and DNA, causing cell death.
Conclusions: Given our findings, potential may exist for selective
treatment options for aggressive, MYCN-driven retinoblastoma.
DMNQ has not been used in humans, but a DMNQ derivative has
previously shown efficacy in a mouse lung carcinoma xenograft.
Further characterization of DMNQ’s mode of action in the
retinoblastoma context is required, but the door is open to the
family of quinones as potential treatment options for MYCNARB1+/+
retinoblastoma.
Commercial Relationships: Kamakshi Sishtla, None; Timothy W.
Corson, None
Support: NIH NCATS TR000163 and TR000006
Establishment of a new retinoblastoma mouse model by
intravitreal injection of human retinoblastoma Y79 cells into
nude mice eyes – Comparison of SLO/OCT vs. histological follow
up
Alexander V. Tschulakow1, Hans-Peter Rodemann2, Ulrich
Schraermeyer1, 3, Sylvie Julien1, 3. 1section of experimental
vitreoretinal surgery, institute for ophtalmic research, Tuebingen,
Germany; 2Department of Radiation Oncology, Tuebingen, Germany;
3
STZ OcuTox, Preclinical Drug Assessment, Hechingen, Germany.
Purpose: Retinoblastoma is the most frequent ocular tumor in
children and if let untreated, can cause death. The aim of this
study was to create a novel xenograft-nude mouse-model, which
closely resembles the situation in the patients and to investigate the
development and spread of the tumor by using SLO/OCT as well as
histology methods.
Methods: Human retinoblastoma Y79 cells were intravitreally
injected in both eyes of immune-deficient nude mice. The mice
were closely monitored for any phenotype changes during the whole
experiment. The frequency of retinoblastoma incidence and growth
velocity were analysed 3, 6, 9 and 12 weeks after cell injection. The
tumor was characterized in vivo by SLO/OCT as well as ex vivo by
electron microscopy and hematoxylin eosin (HE) staining. Moreover,
potentially occurring metastases were investigated via histological
screening of internal organs.
Results: Already three weeks post-injection, animals developed
a retinoblastoma. After five weeks, the eyes began to swell in
individual animals and they showed a similar phenotype to that
of untreated retinoblastoma patients. After 12 weeks, 67.5% of all
analyzed eyes (29 of 42) presented a retinoblastoma. The SLO/OCT
analysis could only be performed in eyes with a tumor at an early
stage (till week three). In all cases in which SLO/OCT- analysis was
possible, the results were in accordance with the histological analysis
(Fig 1a-c). The tumors were found in the vitreous body and in some
cases also within the retina and the subretinal space. In only one
mouse, brain metastases were observed.
Conclusions: Our retinoblastoma mouse model mimics the situation
observed in patients. At early stages, the SLO/OCT analysis
correlated with the histology findings. Therefore SLO/OCT can be
used for the detection of tumors and might be used for monitoring the
success of potential therapy approaches. When the tumors were too
large, only histological investigations were possible.
Figure 1: Three weeks after intravitreal injection of Y79 cells in nude
mice eyes the cells formed a tumor (red arrow), which broke through
the retina (red circle). a) SLO image, b) OCT image and c) the
corresponding HE-stained slide (X100).
Commercial Relationships: Alexander V. Tschulakow, None;
Hans-Peter Rodemann, None; Ulrich Schraermeyer, None; Sylvie
Julien, None
Support: Deutsche Kinderkrebsstiftung 2012.08
Role of HMG protein in Primary Retinoblastoma
Mithalesh Singh1, Seema Kashyap1, Lata Singh1, Neelam Pushker2,
Seema Sen1, Anjana Sharma3, Bhavna Chawla2. 1Ocular Pathology,
Dr R. P. Centre for Ophthalmic Sciences, All India Institute of
Medical sciences, New Delhi, India; 2Ophthalmology, Dr R. P. Centre
for Ophthalmic Sciences, All India Institute of Medical sciences, New
Delhi, India; 3Ocular Microbiology, Dr R. P. Centre for Ophthalmic
Sciences, All India Institute of Medical sciences, New Delhi, India.
Purpose: Retinoblastoma is a malignant tumor composed of
embryonic tumor cells from retinoblasts of neuroepithelial origin.
High mobility group proteins (HMG) are the member of non histone
nuclear factors associated with cell proliferation, differentiation and
neoplastic transformation. High mobility group (HMG) proteins is
a newly recognized protein regulating cancer cell tumorigenesis,
expansion and invasion. However, the role of HMGB1 is still unclear
in retinoblastoma.
Methods: Prospective analysis of 70 primary enucleated
retinoblastoma cases over a period of one year. Expression of
HMGB1 was performed by immunohistochemistry (IHC) in formalin
fixed retinoblastoma specimens and their results were confirmed
by western blotting. mRNA expression was performed by semiquantitative Reverse Transcriptase PCR (RT-PCR).
Results: A total of 70 eyes were taken of which 10(14.28%) eyes
had bilateral involvement. Ages ranged from 7months to 8years. 56
(80%) cases were reported as poorly differentiated tumors whereas
46(65.71%) and 14(20%) cases had calcification and necrosis
respectively. Histopathologically, 16(22.85%) had massive choroid
invasion, 13(18.57%) had optic nerve invasion, 6 cases each had
scleral and ciliary body invasion. Strong expression of HMGB1
were seen in 41/70(58.57%) cases. mRNA expression was seen in 36
cases (51.4%) by RT-PCR. Expression of HMGB1 was statistically
significant with poor differentiation (p=0.0436) and optic nerve
invasion (p=0.0473).
Conclusions: Overexpression of HMGB1 is seen more in poorly
differentiated tumors and those with, histopathological high risk
factors. HMGB1 could serve as a poor prognostic marker in
retinoblastoma. Better understanding of the molecular mechanisms
underlying HMGB1 function could yield novel therapeutic
approaches to anti-cancer strategies.
Commercial Relationships: Mithalesh Singh, None; Seema
Kashyap, None; Lata Singh, None; Neelam Pushker, None; Seema
Sen, None; Anjana Sharma, None; Bhavna Chawla, None
Ubiquitin carboxyl-terminal esterase L1 (UCHL1) expression is
reduced in retinoblastoma tumor samples
Patricia Sanchez-Diaz1, 2, Melanie Kane1, Erik P. Cummings1, Judy C.
Chang2, Gail E. Tomlinson2, 3, Jaclyn Y. Hung2, 3. 1Rosenberg School
of Optometry, University of the Incarnate Word, San Antonio, TX;
2
Greehey Children’s Cancer Research Institute, University of Texas
Health Science Center at San Antonio, San Antonio, TX; 3Pediatrics,
University of Texas Health Science Center at San Antonio, San
Antonio, TX.
Purpose: Ubiquitin carboxyl-terminal esterase L1 (UCHL1) is a
deubiquitinase enzyme within the ubiquitin proteasome system that
seems to either promote or to block cancer progression in a context
dependent manner. UCHL1 is highly expressed in nervous tissue
including retina, but there is no data regarding UCHL1 expression or
function in retinoblastoma. We used tissue arrays to measure UCHL1
protein expression in human retinoblastoma tumor samples and
compared it to normal retina and to other ocular tissues.
Methods: Retinoblastoma tissue arrays were purchased from
USBiomax, Inc. Each tissue contained 12 cores of normal ocular
tissue (including 4 retinal), and 28 retinoblastoma cores. UCHL1
expression was measured by immunohistochemistry using a rabbit
polyclonal antibody against UCHL1 (Abcam), HRP-polymer
(TexGen) and DAB (Sigma-Aldrich) as per manufacturer’s
guidelines. Two independent observers ranked the area of the tissue
stained (S) from 0-100 (0=no staining; 100=all tumor stained) and
the intensity of the staining (I) from 0-3 (0=weak; 3= very strong).
The quick-score method (Q=SxI) was used to compare UCHL1
expression across the tissue array. T-test analyses were used to
determine statistically significant differences in UCHL1 levels
(p<0.05).
Results: Low Q-scores (0-60) were obtained for 75% of the nonretinal ocular tissues and for 53% of the tumor samples. Intermediate
Q-scores (100<Q<200) were obtained for 25% of the non-retinal
ocular tissues and for 32% of the retinoblastoma samples. High
Q-scores (Q>200) were obtained for all retinal samples and for 11%
of the retinoblastomas. The high levels of UCHL1 detected in retinal
samples were statistically significant compared both to non-retinal
ocular tissues (p=0.00043) and to retinoblastomas (p=0.004). The
observed staining pattern in normal retinal was in agreement with
previous reports and concentrated in neuroretinal cells while UCHL1
expression in retinoblastoma seemed to cluster in regions of high
mitotic index known as Flexner–Wintersteiner rosettes.
Conclusions: Our data was consistent with a reduced UCHL1
expression in retinoblastoma compared to normal retina. Ongoing
experiments using retinoblastoma cell lines as model system will
enable us to elucidate potential roles for UCHL1 in retinoblastoma
pathogenesis and may also help us find novel molecular targets for
retinoblastoma.
Commercial Relationships: Patricia Sanchez-Diaz, None; Melanie
Kane, None; Erik P. Cummings, None; Judy C. Chang, None; Gail
E. Tomlinson, None; Jaclyn Y. Hung, None
Analysis of Mitochondrial DNA Mutations and Altered Protein
Expression in Human Retinoblastoma
Lata Singh1, Seema Kashyap1, Neeru Saini2, Neelam Pushker3, Seema
Sen1, Tapas C. Nag4, Anjana Sharma5, Sameer Bakhshi6, Bhavna
Chawla3, Jasbir Kaur7. 1Ocular Pathology, All India Institute of
Medical Sciences, New Delhi, India; 2Functional Genomics Unit,
Institute of Genomics and Integrative Biology, New Delhi, India;
3
Ophthalmology, Dr. R. P. Centre for Ophthalmic Sciences, All
India Institute of Medical Sciences, New Delhi, India; 4Anatomy,
All India Institute of Medical Sciences, New Delhi, India; 5Ocular
Microbiology, Dr. R. P. Centre for Ophthalmic Sciences, All India
Institute of Medical Sciences, New Delhi, India; 6Medical Oncology,
All India Institute of Medical Sciences, New Delhi, India; 7Ocular
Biochemistry, Dr. R. P. Centre for Ophthalmic Sciences, All India
Institute of Medical Sciences, New Delhi, India.
Purpose: Mitochondria are critical for cellular function in cancer
and play an important role in cell differentiation and survival.
Displacement loop (D-loop) is a control site for expression of the
mitochondrial genome. Mutations in D-loop region may alter the rate
of mtDNA transcription and replication. Genetic variability in D-loop
region has been suggested to affect the function of mitochondrial
complexes and could contribute to tumor initia
tion. The purpose of this study was to determine if mutations in the
mitochondrial D-loop region could cause or effect the expression
of mitochondrial complex (s) and the morphological changes of
mitochondria in human primary retinoblastoma tissues.
Methods: In the present study, the entire D-loop region of mtDNA
was amplified in two overlapping polymerase chain reaction
fragments (Nested-PCR) and variations were evaluated in 24
primary retinoblastoma patients by direct DNA sequencing methods.
Morphology of mitochondria was studied by transmission electron
microscopy (TEM). Expression of mitochondrial complex I was
performed in all the 24 cases by immunohistochemistry and then
validated by western blotting on representative cases.
Results: A total of 301 mutations were observed at 170 positions in
the mitochondrial D-Loop region. The most common variations were
73A-G (83.3%), 263A-G (70.8%) and 16223C-T (66.6%) followed
by 30 novel mutations. Loss of mitochondrial complex I was seen in
19/24 (79.16%) cases by immunohistochemistry. Western blotting
was performed to confirm the immunoreactivity results. Electron
microscopy showed numerous degenerated and swollen mitochondria
in tumor cells. On statistical analysis, the expression of mitochondrial
complex I correlated significantly with poorly differentiated
retinoblastoma and tumor invasion.
Conclusions: This is the first study to show a high frequency in
mt D-loop variations and deficiency of mitochondrial complex I in
retinoblastoma tumor. Electron microscopy revealed morphological
changes in mitochondria which may be due to damage in mtDNA
genome. D-loop variations could probably cause alteration in
mitochondrial complex (s) which is still being investigated. Exploring
mtDNA alterations might be helpful for developing biomarkers in the
management of retinoblastoma patients.
Commercial Relationships: Lata Singh, None; Seema Kashyap,
None; Neeru Saini, None; Neelam Pushker, None; Seema Sen,
None; Tapas C. Nag, None; Anjana Sharma, None; Sameer
Bakhshi, None; Bhavna Chawla, None; Jasbir Kaur, None
Review of anatomopathological features and high-risk factors in
45 primary enucleated eyes with retinoblastoma
Alexandre Azevedo, Luiz Teixeira, Juliana Soares, Carla D. Macedo.
UNIFESP, Sao Paulo, Brazil.
Purpose: To evaluate the prevalence of high-risk factors in primary
enucleated eyes with retinoblastoma diagnostic at UNIFESP/GRAAC
from 2007 to 2013
Methods: Observational, retrospective study of consecutive pacients
submbitted to primary enucleation at UNIFESP/GRAAC with
anatomopathological diagnostic of Retiblastoma from 2007 to 2013
. The histopathological features of the enucleated specimen were
reviewed after obtaining approval from the institutional review
board . High-risk histopathologic features for metastatic disease
were defined as the presence of 1 or more of the following : an
área of massive posterior uveal invasion >or = 3 mm, post-laminar
optic nerve invasion, or a combination of any nonmassive choroidal
invasion with any degree of nonretrolaminar optic nerve invasion
.The association between the presence of high-risk histopathological
features and intraocular grouping (ICRB-international classification
of retinoblastoma), laterality, and tumor differentiation was analyzed
with fisher’s exact test .
Results: Of 45 eyes, 5 (11.1%) were classified as group D and
40 as E (ICRB). High-risk retinoblastoma were identified in 26
eyes (57.7%), all in Group E. Sample ages ranged from 0,5 to 85
months, median 24 months . There were choroid massive invasion
in 19 eyes (42.2%), post-laminar invasion in 11(24.4%), and the
combination of any nonmassive choroidal invasion with any degree
of nonretrolaminar optic nerve invasion in 2 (4.4%). The only
statistically significant association was between presence of high-risk
factor and IRCB grouping (P=0.0095). Neither tumor differentiation
or lateralitty showed statistically significant association with highrisk factors .
Conclusions: About half of the primary enucleated patients in our
service needed adjuvant systemic chemotherapy to prevent metastatic
disease.
The ICRB had strong association predicting high-risk histopathologic
features of retinoblastoma.
Neither tumor differentiation or lateralitty showed statistically
significant association with high-risk factors .
Commercial Relationships: Alexandre Azevedo, None; Luiz
Teixeira, None; Juliana Soares, None; Carla D. Macedo, None
Histopathologic Grading of Anaplasia for Retinoblastoma
Pia R. Mendoza1, G B. Hubbard1, Jill R. Wells1, Charles S. Specht2,
Qing Zhang1, Hans E. Grossniklaus1. 1Ophthalmology, Emory
University, Atlanta, GA; 2Pathology, Penn State Milton S. Hershey
Medical Center, Hershey, PA.
Purpose: We hypothesize that there are cytologic characteristics of
retinoblastoma that may be important for prognostication. Cellular
anaplastic change is known to be a significant phenotypic expression
of genetic instability and malignant transformation that can be readily
recognized under a microscope. The objective of this study is to
determine whether the degree of anaplasia correlates with routinely
assessed histologic features and clinical outcomes in a series of
retinoblastoma patients.
Methods: This retrospective study involved the review of
demographic, clinical, and pathologic findings from 92 patients who
underwent primary enucleation for retinoblastoma. Anaplasia was
graded as none, mild, moderate, or severe; defined by increasing
nuclear size, number of mitoses, nuclear hyperchromatism,
pleomorphism and angularity. Pathologic and clinical data were
compared using Kaplan-Meier estimates of event free survival and
overall survival. The Fisher’s exact test was used to analyze the
association between anaplasia grade and other histologic factors.
Results: The distribution of anaplasia grades was as follows: 15
mild (16%), 53 moderate (58%), and 24 severe (26%). Six tumors
had retinoblastoma associated with retinocytoma; all retinocytoma
components had no anaplasia. Increasing grade of anaplasia was
associated with decreased overall survival (p=0.03). There was no
statistically significant correlation between anaplasia and event
free survival. Histopathologic factors statistically associated with
anaplasia were level of differentiation (p<0.001), optic nerve invasion
(p=0.005), and choroidal invasion (p=0.02).
Conclusions: Anaplasia grading may be a useful adjunct to standard
histopathologic criteria in identifying high risk retinoblastoma
patients who may need adjuvant therapy.
Kaplan-Meier curve showing the association of increasing severity of
anaplasia grade with decreased overall survival
Commercial Relationships: Pia R. Mendoza, None; G B.
Hubbard, None; Jill R. Wells, None; Charles S. Specht, None;
Qing Zhang, None; Hans E. Grossniklaus, None
Histopathological Analysis of Cell Division Cycle 25 (CDC25)
Phosphatase Protein in Retinoblastoma
Seema Kashyap1, Lata Singh1, Neelam Pushker2, Seema Sen1, Anjana
Sharma3, Bhavna Chawla2. 1Ocular Pathology, All India Institute
of Medical Sciences, New Delhi, India; 2Ophthalmology, Dr. R.
P. Centre for Ophthalmic Sciences, All India INstitute of Medical
Sciences, New Delhi, India; 3Ocular Microbiology, Dr. R. P. Centre
for Ophthalmic Sciences, All India Institute of Medical Sciences,
New Delhi, India.
Purpose: Retinoblastoma is the most common childhood intraocular
malignant tumor of the developing retina . Cell Division Cycle
25 (CDC25) phosphatase is an essential regulator of the cell
cycle machinery, functioning as a positive regulator by activating
Cyclin-Dependent Kinases (CDK). CDC25A plays a pivotal role in
controlling cell proliferation during development and tumorigenesis.
Overexpression of CDC25A is detected in a number of tumors which
implies dysregulation in malignant transformation. However, the role
of CDC25A in patients with Retinoblastoma is still unknown.
Methods: Prospective analyses of 60 primary enucleated
retinoblastoma cases over a period of one year (Jan 2011Dec 2012). CDC25A protein expression was investigated by
Immunohistochemistry in formalin fixed paraffin embedded sections
and then validated by western blotting. Cytoplasmic staining was
graded as weak/negative (1+), moderate (2+) and strong (3+).
Semi-quantitative analysis for expression of CDC25A mRNA was
performed by the Reverse-Transcriptase PCR (RT-PCR). Expression
of CDC25A was correlated with tumor differentiation and various
histopathological high risk factors.
Results: There were total of 45 poorly differentiated retinoblastomas
and 15 well differentiated retinoblastomas. Necrosis and
calcification was found in 37 (61.6%) and 17 (28.3%) respectively.
Massive choroidal invasion, optic nerve invasion and scleral
invasion was found in 20/60, 17/60 and 7/60 cases respectively.
Immunohistochemistry showed CDC25A expression in total of
38/60 (63.3%) cases. Western blotting was performed to confirm
immunoreactivity results on representative cases. mRNA expression
was seen in 31/60 (51.6%) cases by RT-PCR. Expression of CDC25A
showed statistically significant correlation with poor tumour
differentiation and tumor invasion (p<0.05).
Conclusions: Our results suggest that increased expression
of CDC25A plays an important role in the pathogenesis of
retinoblastoma. CDC25A was associated with invasion of ocular
coats and poor differentiation. CDC25A expression might be a
potential molecular target for novel drug development in tumor
biology.
Commercial Relationships: Seema Kashyap, None; Lata Singh,
None; Neelam Pushker, None; Seema Sen, None; Anjana Sharma,
None; Bhavna Chawla, None
Retinoblastoma (Rb) in Saudi Arabia- Fifteen Year Retrospective
Comparative Review of a Registry: 1983-1997 vs. 1998-2007 at
King Khaled Eye Specialist Hospital
Amir Pirouzian1, 2, Saleh Mesfer2, Hind Al Katan2, Azza M. Maktabi2,
Mohammad Karoui2, Nasira Asghar2, Babar Zaman2, Wafa Ahmed2,
Rajiv Khandekar2, Deepak P. Edward1, 2. 1Wilmer Eye Institute, Johns
Hopkins University, Baltimore, MD; 2Ophthalmology, King Khaled
Eye Specialist Hospital, Riyadh, Saudi Arabia.
Purpose: Published data regarding Rb in Saudi Arabia (KSA) is
sparse. The objective of this study was to compare the clinical and
pathologic features, and evolving patterns of the treatment from
1983-1997 (Early) to 1998-2013 (Late) from an Rb registry; the
registry represents all Rb cases in KSA.
Methods: The Rb registry was queried for the following parameters:
gender, laterality, age, clinical features and stage at presentation;
gender, laterality, regional distribution, history, co-morbid diseases,
pathologic features, surgical treatment modality and treatment
outcome.
Results: In the early group (EG) 343 patients were identified vs.
461 patients in the late group (LG). The gender distribution was
similar in both groups. The median (inter quartile range) age of
presentation was 24 (11, 39) months in EG vs. 18(8, 31) months in
LG (p <0.001). Unilateral Rb was noted in EG vs. LG [198 (58%)
vs. 277 (60%)]. Positive family history was <10% in both groups.
However, in EG, 120 (33%) of parents were first cousins vs. 124
(28%) in the LG (p=0.12). Leukocoria was the most common
presenting symptom in both groups 255 (80%) EG vs. 346 (75%) in
LG (p=0.13). At presentation, tumor confined to the eye was more
common the LG group (67% EG vs. 84% LG). Vitreous seeding was
more common in LG [302(62%) LG vs.156 (45%) in EG]. In the
non-surgical group, photocoagulation rate increased in the LG [106
(23.19%) vs. 13(3.8%) in EG; p<0.001 ]. The rate of chemotherapy
increased from 96 (28%) in EG to 158 (34.64%) in LG (p =0.06).
The rate of external beam radiation decreased [125 (36.98%) EG
vs. 107(23.41%) in LG, (p <0.001)]. The rate of extraocular tumor
extension was similar in both groups (53.7% EG vs. 51.6% LG).
The rate of full thickness choroidal involvement decreased from 40
(14.4%) EG to 40(10.34%) in LG, p =0.11; ON involvement in LG
decreased to 22 (3.4%) compared to 48(9.8%) in EG (p <0.001).
Conclusions: Most Rb clinical parameters remained unchanged over
30 years except an earlier median age of presentation suggesting
earlier tumor detection. However, an increasing number of Rb was
seen in the last fifteen years. The pathologic features in LG were less
advanced suggesting earlier Rb detection. A significant increase in
the non-surgical treatment approaches of Rb was noted in the last 15
years.
Commercial Relationships: Amir Pirouzian, None; Saleh
Mesfer, None; Hind Al Katan, None; Azza M. Maktabi, None;
Mohammad Karoui, None; Nasira Asghar, None; Babar Zaman,
None; Wafa Ahmed, None; Rajiv Khandekar, None; Deepak P.
Edward, None
SPONTANEOUS RESOLUTION OF MACULAR PUCKER IN
TWO PATIENTS WITH RETINOBLASTOMA
Jelena Potic1, 2, Maja Beck Popovic3, Marie-Claire Gaillard1, Aubin
Balmer1, Francis L. Munier1. 1Hopital Ophtalmique Jules-Gonin,
Lausanne, Switzerland; 2University of Belgrade, University Eye
Clinic, Clinical Center of Serbia, Belgrade, Serbia; 3Unité d’hématooncologie pédiatrique CHUV, Lausanne, Switzerland.
Purpose: We report on two patients treated for bilateral sporadic
retinoblastoma (RB), who presented a macular pucker (MP) that
resolved spontaneously.
Methods: Fundus imaging was performed under anesthesia including
RetCam photography, optical coherence tomography (OCT)
(Bioptigen), B-scan ultrasonography (OtiScan), and fluorescein
angiography.
Results: Patient 1 presented with group D and group A disease at
the age of 5 months. Patient 2 was diagnosed group C and group
D disease at 15 months of age. Following neoadjuvant systemic
chemotherapy, secondary enucleation was performed for the group
D eye of both patients in order to prevent optic nerve invasion by
the residual tumors and to avoid salvage treatment by conformal
stereotactic radiotherapy of the posterior pole. The follow eye of
both patients was characterized by a temporally located para-macular
tumor,2.5 papillary diameters away from the fovea. In patient 1,
tumor control (type IV regression) was achieved by combining
transpupillary diode-mediated hyperthermia and argon laser
photocoagulation.Five months after completion of the last treatment,
thickening (970 micrometers) of the foveal area was observed
secondary to the contraction of a MP. This lesion spontaneously
resolved 24 months later, with flattening of the macula area as
documented by OCT. At 6 years of age his visual acuity was 0.8. In
patient 2, the paramacular lesion was treated by chemohyperthermia
and hyperthermia resulting in a flat scar. Five months later, the OCT
showed the formation of a MP creating a retinal fold across the fovea.
Spontaneous resolution of the MP could be observed over the next 18
months,with restoration of the macular anatomy as shown by OCT.
At last visit, visual acuity was 0.63, which is within the normal range
for his age (2 years and 10 months).
Conclusions: Although indirect ophthalmoscopy remains the gold
standard for the management of RB, OCT imaging has improved
the sensitivity of detecting tumour recurrences and complications
especially at the posterior pole. The threshold for macular pucker
formation appears to be low in case of tumors located temporally
to the fovea and aligned with the papillo-macular bundle. To our
knowledge, this is the first time that spontaneous resolution of a
macular pucker is documented in retinoblastoma patients. The
regression occurred over a 18 to 24 months period, stressing the need
to delay the indication to vitrectomy.
Commercial Relationships: Jelena Potic, None; Maja Beck
Popovic, None; Marie-Claire Gaillard, None; Aubin Balmer,
None; Francis L. Munier, None
Retinal vessel architecture in retinoblastoma pre and post
treatment
Clare Wilson1, 2, Karen Wong3, M. S. Sagoo3, M Ashwin Reddy3.
1
Ophthalmology Department, Great Ormond Street Hospital, London,
United Kingdom; 2Department of Vision Science, UCL Institute of
Ophthalmology, London, United Kingdom; 3Ophthalmology, Barts
Health, London, United Kingdom.
Purpose: This study aimed to quantify the vessel parameters
including width and tortuosity in patients with retinoblastoma before
and after treatment.
Methods: 20 RetCam images from 10 children diagnosed with
retinoblastoma at Royal London Hospital were analysed. The
sentinel vessel width and tortuosity values were analyzed with semiautomated software CAIAR (Computer Aided Image Analysis of
Retina) at the time of diagnosis and after effective treatment. Data
were analyzed with two tailed paired-t test.
Results: The mean width of sentinel vessel pre-treatment is 2.59
and post-treatment is 1.60 (p= 0.001; SD= 1.07); the mean tortuosity
value of sentinel vessel pre-treatment is 2.55 and post treatment is
1.19 (p=0.011; SD= 2.02). The sentinel vessel width and tortuosity
value were significantly less after effective treatment.
Conclusions: Prompt treatment is paramount to saving the eye and
preventing death from metastasis in retinoblastoma. Retinal vessel
width and tortuousity decreases with disease regression. Following
further study, the quantification of retinal vessel architecture may be
of use as a valuable screening tool, predictor of disease progression
and assessment of treatment response in retinoblastoma patients.
Commercial Relationships: Clare Wilson, UCL Business (P);
Karen Wong, None; M. S. Sagoo, None; M Ashwin Reddy, None
Support: Childhhood Eye Cancer Trust (CHECT) Project Grant
2013
Primary and Salvage Proton Radiotherapy for Intraocular
Retinoblastoma: 1990 – 2013
Yoshihiro Yonekawa1, Shannon M. MacDonald2, John E.
Munzenrider2, Shizuo Mukai1. 1Ophthalmology, Massachusetts Eye
and Ear Infirmary, Boston, MA; 2Radiation Oncology, Massachusetts
General Hospital, Boston, MA.
Purpose: Proton radiotherapy allows highly focal tumor targeting
with sub-millimeter precision while minimizing exposure of
surrounding tissues. Of note, tissues distal to the target volume
are completely spared. We present one of the first reports of
proton therapy as a primary or salvage treatment modality in the
management of intraocular retinoblastoma.
Methods: This was a consecutive, retrospective, interventional case
series of patients from 1990 to 2013.
Results: Forty-seven subjects were identified, and 11 were excluded,
resulting in 46 eyes from 36 patients for analyses. Median age at
diagnosis was 248.5 days (range 6 days to 2.5 years), and 33 (92%)
had bilateral disease. Eyes were treated using 40-46.8 Gy(RBE)
(Gray, Radiobiological Equivalent), divided into 20-26 fractions.
Median follow-up after proton therapy was 6.8 years (range 1.1-22.1
years). Proton therapy was used as the primary treatment modality
in 24 eyes. Tumor control was achieved in 19 (79%) eyes; 9 of 10
(90%) Group B, 3 of 4 (75%) Group C, 6 of 9 (67%) Group D, 1 of
1 (100%) Group E; 3 of 3 (100%) Reese-Ellsworth Group I, 7 of 7
(100%) Group II, 3 of 4 (75%) Group III, 1 of 4 (25%) Group IV,
and 5 of 6 (83%) Group V. Twenty-two received proton therapy as
salvage therapy; 18 after failed chemoreduction, 3 after failed focal
ablation, and 1 after failed intra-arterial chemotherapy. Tumor control
was achieved in 18 (82%) of eyes; 8 of 9 (89%) Group B, 5 of 5
(100%) Group C, 4 of 7 (57%) Group D, and 1 of 1 (100%) Group
E; 3 of 3 (100%) Reese-Ellsworth Group I, 3 of 3 (100%) Group II,
4 of 4 (100%) Group III, 1 of 1 (100%) Group IV, and 7 of 11 (64%)
Group V. Of eyes with tumor control, 7 (26%) underwent cataract
extraction, 4 (15%) developed radiation retinopathy, and 1 (4%)
developed radiation optic neuropathy. Median final visual acuity was
20/70 (range 20/16-NLP), and 18 (67%) had final visual acuity >
20/200. There were no cases of in-field secondary malignancies, and
all patients survived.
Conclusions: Proton radiotherapy achieved excellent tumor control
both as a primary or salvage treatment modality, without inducing
any cases of in-field secondary malignancies.
Commercial Relationships: Yoshihiro Yonekawa, None; Shannon
M. MacDonald, None; John E. Munzenrider, None; Shizuo
Mukai, None
Support: Mukai Fund, Massachusetts Eye and Ear Infirmary, Boston,
MA
Focal treatment of retinoblastoma tumors with simultaneous
810nm and 532nm lasers
Ashwin Mallipatna, Vandhana Surendranath. Retinoblastoma
Service, Narayana Nethralaya, Bangalore, India.
Purpose: Focal laser therapy is an essential aspect of treatment of
intraocular retinoblastoma, with the utilization of multiple types of
lasers and methods of delivery. The effect of the 810nm laser and
532nm laser are different, and simultaneous use of both lasers on a
single tumor in the same treatment session is not common. This study
describes the results of using this combination of lasers on tumor
response. We describe our rationale and experience in utilizing the
effects of a combination of both lasers in an attempt to achieve a
more rapid reduction in tumor size.
Methods: We retrospectively analyzed the simultaneous use of the
810 and 532nm lasers delivered through an indirect ophthalmoscope
in 14 children with intraocular retinoblastoma.
Results: A total of 32 tumors were individually analyzed in 18
eyes of 14 children with intraocular retinoblastoma. An initial
gentle application of 810nm laser rendered the tumor opaque,
without affecting the tumor vasculature. Once the tumor vasculature
was clearly visible, application of the 532nm laser was able to
photocoagulate this tumor vasculature. With this technique, we
were able to demonstrate rapid reduction in tumor size in specific
instances, along with documentation of the effect of each laser with
ocular coherence tomography. None of the cases experienced laser
related complications after the combination of lasers. Twenty-eight
tumors went on to be inactive. Three eyes required enucleation for
persistent tumor activity.
Conclusions: A combination of 810nm and 532nm lasers used in the
same session might help augment the effect of focal laser therapy.
We would suggest this technique be used in certain situations to help
a rapid reduction in tumor size. Caution must be exercised to ensure
that we do not over apply laser, leading to laser related complications.
Commercial Relationships: Ashwin Mallipatna, None; Vandhana
Surendranath, None
Treatment Patterns and Survival of Patients with
Retinoblastoma: A Surveillance, Epidemiology, and End Results
Dataset Evaluation
Diana A. Tamboli1, Arun D. Singh2, Alan Topham3. 1Ophthalmology,
Loyola University Chicago, Stritch School of Medic, Maywood, IL;
2
Ophthalmology, Cleveland Clinic, Cleveland, OH; 3Coalition of
National Cancer Cooperative Group Inc., Philidelphia, PA.
Purpose: Treatment of Retinoblastoma (Rb) over the years has
shifted from enucleation and radiation towards multimodal therapy
involving chemotherapy and focal therapy. We sought to analyze
the SEER database to determine the treatment patterns of Rb and its
impact on survival from 1975 to 2010.
Methods: The SEER dataset was used to identify cases of Rb using
ICD-03 histology codes. Special permission was granted by the
SEER administration to release chemotherapy information for this
study (information which is not available in the publically available
SEER dataset). Treatment of Rb for patients with locoregional
disease was characterized as surgical therapy, radiation therapy,
chemotherapy or any form thereof across 4 time periods from
1975-2010. Overall survival was performed using the Kaplan-Meier
method and compared using the log rank method.
Results: There were 1452 cases of Rb identified from 1975-2010
with 48% of patients being male and 30% presenting with bilateral
disease. Twelve hundred twenty (84%) patients presented with
localized disease. Table 1 shows grouped treatment patterns over time
with an increase in chemotherapy (+/- any treatment) from 16.5% to
50.2% and a decrease in surgery (+/- any treatment) from 96.2% to
88.5% and decrease in radiation from 15.2% to 4.9% from the 19751979 time period to the 2000-2010 time period. Ten year overall
survival was approximately 97% for the two most contemporary time
periods (1990-1999 and 2000-2010), 94% for 1975-1979 and 92% for
1980-1989.
Conclusions: Treatment trends for Rb show an increase in
chemotherapy utilization with a decrease in surgery and radiation
therapy from 1975-2010. Increased utilization of chemotherapy
corresponds with an improvement in survival. Future studies with
in depth analysis will be required to confirm treatment superiority
of various forms of chemotherapy (intravenous, intra arterial,
intravitreal).
Table 1: Grouped Treatment Patterns over Time (Note the sum of
each column does not sum to 100%; each row in independent of each
other)
= 9.5). Vitreous seeding was associated with anterior chamber angle
invasion (OR = 3.3). Ectropion uvea was associated with ciliary body
invasion (OR = 20.7), massive choroidal invasion (OR = 45.7) and
optic nerve invasion (OR = 36). Massive choroidal invasion could be
predicted by clinical signs of buphthalmos [Adj OR = 0.08 (95% CI
0.007 – 0.9)] and uveal ectropion [Adj OR = 0.05 (95% CI 0.004 –
0.7 )].
Conclusions: This study suggests that certain clinical features in
advanced retinoblastoma can be associated with HRF on pathologic
examination. The presence of buphthalmos and uveal ectropion
at presentation correlated with at least 2 HRF massive choroidal
invasion ..and ciliary body invasion.
Commercial Relationships: Azza M. Maktabi, None; Mohammed
Karaoui, None; Saleh Al-Mesfer, None; Hind Al Katan, None;
Sahar M. Elkhamary, None; Deepak Edward, None
Figure 1: Overall Survival by Time Period (p<0.02)
Commercial Relationships: Diana A. Tamboli, None; Arun D.
Singh, None; Alan Topham, None
Clinical Predictors of high risk pathological features in advanced
enucleated Retinoblastoma.
Azza M. Maktabi1, Mohammed Karaoui5, Saleh Al-Mesfer2, Hind
Al Katan1, Sahar M. Elkhamary3, Deepak Edward4. 1Pathology
and Laboratory department, King Khalid Eye Specialist
Hospital,KKESH, Riyadh, Saudi Arabia; 2Oncology Division, King
Khalid Eye Specialist Hospital, Riyadh, Saudi Arabia; 3Diagnostic
Imaging Department, King Khalid Eye Specialist Hospital,
Riyadh, Saudi Arabia; 4Reasearch Department, King Khalid Eye
specialist Hospital, Riyadh, Saudi Arabia; 5Medicine and Peadiatric
Department, King Khalid Eye Specialist Hospital, Riyadh, Saudi
Arabia.
Purpose: Advanced retinoblastoma can present with a spectrum of
clinical findings that often leads to enucleation. However, a decision
on adjuvant treatment is based on the presence of high risk features
(HRF) determined on pathologic examination. The aim of this study
is to determine whether certain clinical features of advanced disease
could predict pathologic HRF that would merit adjuvant treatment.
Methods: Medical records and histopathology of enucleated
specimens from 69 patients who underwent enucleation prior to
receiving any treatment at King Khaled Eye Specialist Hospital over
were reviewed. Clinical findings were recorded in a database. The
histologic slides were evaluated for HRF including anterior chamber
angle invasion, iris invasion, ciliary body invasion, massive choroidal
invasion, optic nerve invasion beyond lamina cribrosa, intrascleral
infiltration, and extrascleral extension. Univariate analysis was
performed to determine odd ratio
Results: Sixty nine eyes (34 boys and 35 girls; mean age 19 months)
were included in this study . Hyphema was associated with iris
invasion [Odd’s ratio (OR) = 12)] and ciliary body invasion (OR =
12). Buphthalmos was associated with massive choroidal invasion
(OR = 30.5), optic nerve invasion (OR = 10). Increased intraocular
pressure was associated with anterior chamber angle invasion (OR =
3.9), ciliary body invasion (OR = 13)] and optic nerve invasion (OR
Long-term outcomes of Group B eyes in retinoblastoma patients
treated with chemoreduction and low-dose IMRT radiation
therapy as salvage: The Children’s Hospital Los Angeles
Experience
Dagny Zhu2, 1, Jesse L. Berry1, 2, Rima Jubran1, Thomas C. Lee1,
A. Linn Murphree1, 2, Jonathan W. Kim1, 2. 1Children’s Hospital
Los Angeles, Los Angeles, CA; 2Keck School of Medicine at the
University of Southern California, Los Angeles, CA.
Purpose: To evaluate long-term outcomes of Group B eyes of
retinoblastoma patients.
Methods: Retrospective chart review of patients diagnosed with
retinoblastoma and designated Group B in at least one eye from
January 1, 1991 to December 31, 2011 at Children’s Hospital
Los Angeles (CHLA) was done. Overall, 115 Group B eyes of
102 patients were included; 18 had bilateral disease. Primary
chemoreduction (CRD) with vincristine, etoposide, and carboplatin
was administered along with local consolidation. Salvage therapy
if required was administered in the form of low-dose intensitymodulated radiation therapy (IMRT) as salvage for recurrent or
persistent tumor. Primary outcome measure was globe salvage.
Results: Of 115 Group B eyes, 113 were initially treated with CRD
and local consolidation. Three cycles of primary CRD with local
consolidation cured 98 of 113 eyes (87%). Recurrent or persistent
tumors were found in 15 eyes: 8 were treated with additional
cycles of CRD, 1 with proton beam, 4 with IMRT, and 2 with both
additional CRD and IMRT. Of the 7 irradiated eyes, 6 (86%) were
salvaged and 1 (14%) required enucleation. Of the 8 eyes treated with
additional CRD, 1 (12.5%) eye was salvaged and 7 (87.5%) required
enucleation. Final visual acuity ranged from 20/20 to count fingers;
92% had vision better than 20/40. Average follow-up was 2.6 years
(range 0.2 – 19 years).
Conclusions: Three cycles of systemic CRD with local consolidation
for Group B retinoblastoma demonstrated a very high rate of globe
preservation. For tumor recurrences, low dose IMRT therapy offers
an effective salvage treatment.
Commercial Relationships: Dagny Zhu, None; Jesse L. Berry,
None; Rima Jubran, None; Thomas C. Lee, None; A. Linn
Murphree, None; Jonathan W. Kim, None
Long-Term Outcomes of Group C Eyes in Retinoblastoma
Patients Treated With Chemoreduction and Low-Dose IMRT as
Salvage
Lilangi Ediriwickrema1, Jesse L. Berry2, Rima Jubran3, Thomas C.
Lee1, 2, A. Linn Murphree2, Jonathan W. Kim1, 2. 1Ophthalmology,
University of Southern California, Los Angeles, CA; 2The Vision
Center, Children’s Hospital Los Angeles, Los Angeles, CA; 3The
Children’s Center for Cancer and Blood Diseases, Children’s Hospital
of Los Angeles, Los Angeles, CA.
Purpose: To evaluate outcomes of Group C eyes of unilateral and
bilateral retinoblastoma patients treated with primary chemoreduction
and intensity modulated radiotherapy (IMRT) as salvage.
Methods: Retrospective chart review of patients diagnosed with
unilateral and bilateral retinoblastoma and designated Group C in at
least one eye from January 1, 1991 to December 31, 2011. Overall,
36 Group C eyes of 36 patients were included; 26 had bilateral
disease. Primary chemoreduction (CRD) with vincristine, etoposide,
and carboplatin with local consolidation was administered, followed
by external beam radiation in the form of low-dose intensitymodulated radiation therapy (IMRT) as salvage for persistent or
recurrent tumor. Primary outcome measure was globe salvage.
Results: Of the 26 Group C eyes in patients with bilateral
retinoblastoma, none were enucleated primarily; 5 of the 10 unilateral
Group C eyes were enucleated primarily. Thus, CRD and local
therapy was used to treat 31 retinoblastoma eyes. CRD cured 22
of 31 (71%) Group C eyes. Recurrences were found in 9 eyes; five
underwent secondary enucleation due to poor visual prognosis, and
four received low dose IMRT as salvage therapy. Of the 4 irradiated
eyes, 100% were salvaged and none required enucleation (Figure
1). Final visual acuity ranged from 20/20 to no light perception
with 13 eyes having 20/80 or better visual acuity in the bilateral
retinoblastoma patients. Average follow-up was 98 months (range 22
to 211 months). Chemoreduction was well tolerated by all patients.
Conclusions: Estimates of eye survival of Group C eyes in
bilateral patients at 12 months is 91.7%; at 60 months eye
survival is estimated to be 87.5% (Figure 2). Systemic treatment
for retinoblastoma demonstrated an extremely high rate of globe
preservation with acceptable complications and many eyes retaining
functional vision.
Figure 1: Treatment Modality and Percent Recurrence of Group C
Eyes (n=31 eyes)
Figure 2: Kaplan-Meier Estimate of Eye Survival of Group C Eyes in
Bilateral Patients (n=24 eyes)
Commercial Relationships: Lilangi Ediriwickrema, None; Jesse
L. Berry, None; Rima Jubran, None; Thomas C. Lee, None; A.
Linn Murphree, None; Jonathan W. Kim, None
Support: Retinoblastoma International, Inc.; The Institute for
Families, Inc.; Children’s Hospital of Los Angeles
Intraarterial Chemotherapy (Ophthalmic Artery Chemosurgery)
for Group D Retinoblastoma
Anthony Daniels1, Y. Pierre Gobin2, 3, Brian Marr3, Jasmine H.
Francis3, Scott E. Brodie4, 3, David H. Abramson3. 1Divisions of
Ocular Oncology and Retina, Department of Ophthalmology,
Vanderbilt Eye Institute, Nashville, TN; 2Neurosurgery /
Interventional Radiology, Weill Cornell Medical College, New York,
TN; 3Ophthalmic Oncology, Memorial Sloan-Kettering, New York,
NY; 4Ophthalmology, Mount Sinai School of Medicine, New York,
NY.
Purpose: To evaluate intraarterial chemotherapy (OAC) as a
treatment for group D eyes, both those that have failed other
treatments and in the treatment-naïve setting. While group A-C eyes
can often be managed with other treatment modalities, these other
treatments are less successful in group D eyes.
Methods: IRB-approved retrospective review of all Group D eyes
treated with OAC from 5/2006-12/2012 at our institution. Patients
were treated according to our previously-published techniques.
Demographics, prior treatment, OAC agents used, outcomes and
adverse events were recorded.
Results: 100 patients (109 eyes) with group D retinoblastoma
who underwent OAC were included. 45 eyes were treatment-naïve
and 63 eyes had received prior treatments elsewhere. 6 infants (7
eyes) underwent IV carboplatin bridge until old enough to undergo
OAC. Median age at first treatment was 16 months (range 3-252).
Treatment-naïve patients were significantly younger than patients
previously treated elsewhere (19.5 vs. 37.1 months, p=0.0005).
Median number of OAC sessions/eye was 3 (range 1-9), with no
difference between pretreated and naïve groups (p=0.34). 106/109
eyes received IA melphalan, but only 31 eyes received melphalan as
the only agent. 41 eyes received carboplatin, and 76 eyes received
topotecan (never as a single agent). 78/109 eyes received >1 drug
over the course of treatment. 24 eyes (17 pretreated, 7 treatmentnaïve, 0 bridge) failed treatment and required enucleation during the
study period. Eyes that were treatment-naïve prior to us initiating
OAC or bridge-OAC were much less likely to ultimately require
enucleation (13% of treatment-naïve vs. 30% of pretreated eyes;
OR for prior treatment=2.8; p=0.035). OAC-related adverse events
included bronchospasm (44 patients), Grade 3/4 neutropenia (31
patients), periocular edema (16 eyes), forehead hyperemia (14
eyes) or madarosis (10 eyes). 3 patients developed metastases (all
survived), and 1 child developed (and died from) a second nonocular cancer. No patient developed a new intraocular tumor during
treatment or follow-up.
Conclusions: In Group D eyes, primary OAC (or Carboplatin bridge
to OAC in young infants) is able to achieve globe salvage in 87%
of eyes. 70% of eyes that failed prior conventional therapy could
be salvaged. OAC prevented the development of subsequent, new
intraocular tumor foci. These results are notably better than published
series using other methods.
Commercial Relationships: Anthony Daniels, None; Y. Pierre
Gobin, None; Brian Marr, None; Jasmine H. Francis, None; Scott
E. Brodie, None; David H. Abramson, None
Support: Fund for Ophthalmic Knowledge, Inc.
Efficacy of second-course ophthalmic artery chemosurgery for
retinoblastoma
Jasmine H. Francis1, Y. P. Gobin2, Brian Marr1, 2, Irwin Tendler1, 2,
Scott E. Brodie3, 1, David H. Abramson1, 2, Ira J. Dunkel1, 2. 1Memorial
Sloan-Kettering Cancer Center, New York, NY; 2Weill-Cornell
Medical College, New York Presbyterian, New York, NY; 3Mount
Sinai School of Medicine, New York, NY.
Purpose: To evaluate the efficacy of second-course ophthalmic
artery chemosurgery (OAC) following progression of disease after
completing initially successful ophthalmic artery chemosurgery for
retinoblastoma.
Methods: Single-arm, retrospective study of 31 eyes in 32 patients
treated with second-course OAC at Memorial Sloan-Kettering
Cancer Center between May 2006 and July 2013, with a median 34
months follow-up. The study included patients who underwent a
successful course of IA chemotherapy, with a minimum of 2 months
of progression-free follow-up at monthly examinations, but who
subsequently required additional OAC for recurrent tumor. Outcome
measurements included progression free survival and ocular survival.
Kaplan Meier survival estimates were generated and the Mantel-Cox
test was used to compare curves.
Results: Eyes requiring second-line OAC had initially received
a mean of 3.1 OAC infusions. They developed progression of
disease and necessity for second-course OAC at a mean of 8.7 mos
following initial OAC. The 2-year Kaplan-Meier ocular survival and
progression free survival estimates following second-course OAC
were 80.2% (95% confidence interval [CI], 58.5-91.3%) and 47.0%
(95% confidence interval [CI], 27.8-64.0%), respectively. Vitreous
seeds were present in 17 (53%) of eyes requiring second-course
OAC and were significantly associated with progression of disease
following second-course OAC (p=0.01). Neither prior treatment
status, age at initial OAC, addition of new drug, or increasing number
of infusions during second-course OAC was significantly associated
with progression free survival.
Conclusions: Retinoblastoma eyes requiring second-course OAC
following initial OAC treatment have good salvage rates. However,
these eyes often require additional (third or fourth-course) OAC or
other treatment modalities due to progression of disease after secondline OAC, particularly if vitreous seeds are present at the time of
initial OAC failure.
Commercial Relationships: Jasmine H. Francis, None; Y. P.
Gobin, None; Brian Marr, None; Irwin Tendler, None; Scott E.
Brodie, None; David H. Abramson, None; Ira J. Dunkel, None
Support: Fund for Ophthalmic Knowledge
Outcome of 20 eyes with recurrent or refractory retinoblastoma
using selective intra-arterial and/or intravitreal chemotherapy
Luiz Teixeira1, 3, Jose R. Fonseca2, 3, Juliana Soares3, Camila H.
Hashimoto3, Carla D. Macedo3. 1Ophthalmology, Federal University
of São Paulo, Sao Paulo, Brazil; 2Radiology, Federal University of
São Paulo, São Paulo, Brazil; 3Pediatric Oncology Institute, Federal
University of São Paulo, São Paulo, Brazil.
Purpose: To evaluate the outcome of 20 eyes, with recurrent or
refractory retinoblastoma after intravenous chemotherapy, using
selective intra-arterial chemotherapy (SIAC) and/or intravitreal
chemotherapy (IViC).
Methods: Retrospective study, approved by the institutional
review board, of 20 eyes of 17 patients. SIAC was used as first
option in cases with retinal or subretinal disease with or without
vitreous seeding. IViC was used for isolated vitreous disease or
for complementary treatment in eyes with partial vitreous seeding
response to SIAC. Focal therapy was used as needed to consolidate
treatment.
Results: Twenty eyes of 17 patients were treated. 15 (75%) eyes
were treated with SIAC, 2 (10%) eyes with IViC and 3 (15%) eyes
with both therapies. SIAC was used in 18 eyes. (melphalan plus
topotecan and carboplatin in 15 eyes; melphalan plus topotecan in
1 eye and melphalan in 2 eyes). The median infusions per eye were
2 (range 1-3). Dose ranges were 3-5 mg for melphalan, 0,3-0,4 mg
for topotecan and 30-40 mg for carboplatin. Five eyes received IViC
(melphalan 30 μg in 0,05 ml – every 7 days). The median injectios
were 5 (range 2-7). At a median follow up of 11 months (range 3-30
months) all patients are alive with no metastatic disease. 17 of 20
eyes (85%) were preserved.
Conclusions: The use of both therapies SIAC and IViC as
isolated modality or in combination to treat recurrent or refractory
retinoblastoma showed successfully results in globe preservation.
Commercial Relationships: Luiz Teixeira, None; Jose R. Fonseca,
None; Juliana Soares, None; Camila H. Hashimoto, None; Carla
D. Macedo, None
Indocyanine green-mediated Photothrombosis in the treatment of
Vasoproliferative Retinal Tumors: a case series
Enrico Bertelli, Michael Simonazzi. Divisione Oculistica, Ospedale
Centrale Bolzano, Bolzano, Italy.
Purpose: To assess the efficacy of indocyanine green-mediated
photothrombosis (ICGMP) in the treatment of Vasoproliferative
Retinal Tumors (VPRT) with various size and clinical expression.
Lesions were all peripheral and symptomatic, due to associated
manifestations at the level of retina and/or vitreous.
Methods: Retrospective, consecutive case series.
4 consecutive patients, 2 males, 2 females, aged 39 to 62 years,
presenting unilateral, single VPRT lesions of variable size (1,5 to 7
mm maximum diameter) underwent ICGMP at our institution. Late
pooling or staining of ICG dye within the lesion was considered an
essential prerequisite for the treatment.
Thirty minutes after the administration of a loading dose of 1 mg/kg
of intravenous ICG multiple, confluent, 2 mm wide, infrared 810 nm
diode laser spots were applied on the surface of the tumors. The final
number of spots applied were related to the tumor size. Duration of
each spot was up to 60 sec., with power between 400 and 1200 mW.
Pain perception by the patient during treatment was used as limit for
the single laser exposure. Diode laser power was increased until pain
threshold was reached, starting at 300 mW.
Results: No change in tumor aspect was observed during and
immediately after treatment. ICG late images were taken after
treatment, showing no hyperfluorescence of the lesion in all cases.
In 2 cases 2 treatments were sufficient to obliterate the lesion. In the
remaining 2 cases 3 and 4 treatments respectively were necessary, in
order to achieve a complete destruction of the lesion.
In 2 cases an associated vitreoretinal interface disorder (stage 3
macular hole) underwent successful vitreoretinal surgery 2 to 3
months after treatment of the VPRT lesion.
Visual acuity improved in 3 cases and remained stable in 1 case, in
which faint vitreous hemorrhage was present at first observation, with
patient retaining full vision.
All lesion regressed to fibrotic and partly pigmented scars.
Conclusions: In the reported case series (4 eyes of 4 patients)
ICGMP achieved complete obliteration of single VPRT lesions of
different size (1,5 to 7 mm). Significant improvement or stabilization
of function was achieved in all cases.
ICGMP is less invasive than cryotherapy and less expensive than
Photodynamic Therapy.
It may deserve further attention in the treatment of selected retinal
vascular tumors characterized by late ICG pooling or staining.
Commercial Relationships: Enrico Bertelli, None; Michael
Simonazzi, None
Natural History of Circumscribed Choroidal Hemangiomas
Angelica G. Ortiz1, Marco A. Gonzalez2, J William Harbour2,
Bernadete Ayres2. 1University of Miami, Miami, FL; 2Bascom Palmer
Eye Institute, Miami, FL.
Purpose: To evaluate the natural course of the clinical and
ultrasonographic characteristics of circumscribed choroidal
hemangiomas.
Methods: This is a non-comparative consecutive case series
from January 2000 through July 2013. Patients presenting with
circumscribed choroidal hemangiomas at the Bascom Palmer Eye
Institute were identified. Data was collected on demographics,
clinical features, treatments, and visual outcomes.
Results: A total of 78 eyes in 78 patients were identified. The mean
age at presentation was 54.9 years (range: 19 to 88). The mean follow
up after presentation was 42.8 months. Presenting visual acuity was
equal or better than 20/50 in 37 of 87 patients (43%). 42 patients had
an associated serous retinal detachment. The mean height on initial
ultrasonography was 2.4 mm (range 1 to 5 mm). Treatment included
photodynamic therapy in 40 eyes. Final visual acuity was equal or
better than 20/50 in 38 of 87 patients (44%).
Conclusions: Circumscribed choroidal hemangiomas are rare,
vascular hamartomas. In spite of the benign nature of this tumor,
many patients in the present study developed sight-threatening
complications requiring treatment.
An understanding of the natural history of circumscribed uveal
hemangioma is important for predicting disease trends and
determining the timing of treatment.
Commercial Relationships: Angelica G. Ortiz, None; Marco A.
Gonzalez, None; J William Harbour, None; Bernadete Ayres,
None
Utility of wide-field autofluorescence imaging in screening for
new retinal capillary hemangiomas associated with von Hippel
Lindau disease
Ilya Leskov, Shizuo Mukai. Massachusetts Eye and Ear Infirmary,
Boston, MA.
Purpose: Fluorescein angiography (FA) is used for the identification
of retinal capillary hemangiomas in patients with von Hippel Lindau
disease (VHL). We show that new retinal hemangiomas can also be
identified using wide-field autofluorescence imaging, which may
obviate the need for fluorescein administration in some cases.
Methods: A consecutive, retrospective case series of patients with
von Hippel Lindau disease that were seen between 2011 and 2013.
Results: Thirty-one patients with previously identified mutations
of the VHL gene were seen between 2011 and 2013. During this
time period, they underwent 102 wide-field retinal imaging studies
using the Optos system, which included autofluorescence as well
as FA. Seventeen patients (55%, median age 34 years) were found
to have retinal capillary hemangiomas, of whom 11 developed a
total of 17 new hemangiomas during the study period. Fourteen
patients (45%, median age 14.5 years) remained hemangioma-free.
Of the newly identified hemangiomas, 9 were located within the
autofluorescence image field. Notably, all 9 of these lesions (100%)
were detectable using autofluorescence. All new hemangiomas that
were not visualized with autofluorescence were located outside
of the autofluorescence image field. Hemangiomas that remained
active despite treatment did not appear significantly different on
autofluorescence from ones that had been treated successfully.
Conclusions: Wide-field autofluorescence imaging is as effective as
FA for detecting new retinal capillary hemangiomas associated with
von Hippel Lindau disease. While FA is superior for surveillance
of previously treated hemangiomas, obtaining autofluorescence
images from retinal periphery may obviate the need for fluorescein
administration in screening younger VHL patients for new
hemangiomas.
Commercial Relationships: Ilya Leskov, None; Shizuo Mukai,
None
Results of Single spot limited PDT in patients with choroidal
haemangioma
Marilette Stehouwer1, Jan E. Keunen2, Reinier O. Schlingemann1,
Frank D. Verbraak1. 1Ophthalmology, AMC, Amsterdam,
Netherlands; 2Ophthalmology, Radboudumc, Nijmegen, Netherlands.
Purpose: To study the long term outcome of patients with a
circumscribed choroidal hemangioma (CCH) treated with a single
spot limited photodynamic therapy (PDT) with Visudyne.
Methods: Twenty patients with a CCH were treated between 2001
and 2010 with a single spot limited PDT for CCH. The single spot
covered only the most prominent part of the tumour, using a standard
diode laser (692 nm), with a radiance exposure of 50 J/cm2, starting 6
min after a 1-min infusion with Visudyne (6 mg/m2 BSA).
In a retrospective design the long term outcome was evaluated.
Outcome parameters were: change in visual acuity (VA) pre,- and
post PDT, presence of regression of the tumour on ultrasound (USG),
presence and change of leakage on OCT.
Results: Regression of the tumour was seen in all patients with
USG during follow up (mean follow-up 17.7 months, range 3 to
36 months). In 12 patients, the tumour became undetectable on
ultrasound examination. The VA pre-PDT varied from 20/400 to
20/20, and was less than 20/40 in 10 patients. After treatment in
only 5 patients VA remained below 20/40. Overall, Improvement of
VA to 20/25 or higher was seen in 14 patients. In all patients, retinal
leakage on OCT disappeared following PDT. Three patients had a
recurrence of the CCH with retinal leakage, 4.5, 3 and 9.3 years after
PDT treatment. In all of them the tumour was still detectable after the
first PDT. After retreatment with limited PDT, 2 patients recovered
with a VA of 20/20, the other patient showed regression of the tumour
but without improvement of VA (20/800). In the 17 patients without
recurrence of leakage all parameters remained stable during followup.
Conclusions: Circumscribed choroidal hemangioma is effectively
treated with a single spot limited photodynamic therapy with
improvement of VA in the majority of cases. A recurrence can be seen
especially when the tumour is not completely regressed after initial
PDT.
Commercial Relationships: Marilette Stehouwer, None; Jan
E. Keunen, None; Reinier O. Schlingemann, None; Frank D.
Verbraak, None
Bevacizumab injections in patients with a choroidal neovascular
membrane secondary to choroidal osteoma
Maria Pefkianaki1, Vasilios Papastefanou1, 2, Richard Andrews1,
Victoria Cohen1, 2, Mandeep S. Sagoo1, 2. 1Moorfields Eye Hospital,
London, United Kingdom; 2St Bartholomew`s Hospital, London,
United Kingdom.
Purpose: To present the outcomes of a series of patients with
choroidal neovascular membrane (CNV) secondary to a choroidal
osteoma.
Methods: In this retrospective series patients underwent complete
clinical and imaging assessment (fundus photo, fluorescein
angiography and optical coherence tomography) and were managed
with intravitreal anti-VEGF injections. Visual acuity and central
retinal thickness were recorded pre treatment and at the end of follow
up period.
Results: Six patients were included in this study. Of this, 4/6 had
predominantly classic or classic and 2/6 patients had minimally
classic or occult CNV. Treatment with intravitreal anti-VEGF was
applied in all cases (1-3 injections of bevacizumab). Median follow
up was 6 months (6-10 months). Visual acuity improved in all
patients by 2-6 Snellen lines. CNV completely regressed in 4 cases
and partially regressed in 2 cases. Central retinal thickness change
ranged from -222 μm to +60 μm.
Conclusions: Intravitreal bevacizumab is an effective treatment
modality in the management of vision threatening choroidal
neovascular membrane secondary to choroidal osteoma
Commercial Relationships: Maria Pefkianaki, None; Vasilios
Papastefanou, None; Richard Andrews, None; Victoria Cohen,
None; Mandeep S. Sagoo, None
En face OCT imaging of retinal and choroidal tumors
Cinzia Mazzini, Daniela Bacherini, Andrea Giorni, Giulia Pieretti,
Ugo Menchini. Department of Surgery and Translational Medicine,
Eye Clinic, University of Florence, Florence, Italy.
Purpose: To describe optical coherence tomography (OCT) features
of retinal and choroidal tumors using the en face technique.
Methods: Prospective observational case series. We included in our
study 66 eyes of 65 patients with retinal or choroidal tumors. Patients
underwent ophthalmoscopic examination, fundus photography, A
and B-scan ultrasonography, SD-OCT and en face OCT. All the OCT
images were obtained by CIRRUS OCT (Carl Zeiss, Meditec) with
the 512x128 macular cube acquisition. For each retinal and choroidal
lesion qualitative characteristics (tumor outline, reflectivity and/or
shadowing of choroidal layers) were assessed.
Results: In all cases, en face OCT was able to identify the tumor
from the surrounding normal choroid, allowing to determine
the tumor area. En face OCT scan is able to detect structural
and morphological details. In our cases, en face OCT imaging
of choroidal nevi shows a well demarcated, sub-retinal pigment
epithelium (RPE) hyporeflectivity area, while images of choroidal
melanomas show a sub-RPE hyporeflective area with irregular and
hyper-reflective edge due to vascular tissue. Choroidal hemangioma
shows a circumscribed sub-RPE hyperreflective area surrounded
by a hyporeflective ring. Choroidal metastasis often show an
irregular hyporeflective area, but in some cases we observed a
well-demarcated round shape. Choroidal osteoma is well visualized
as an irregular hyporeflective area. We evaluated en face OCT
morphological features of retinal astrocytoma and RPE adenoma.
Even small choroidal lesions nondetectable by ultrasonography can
be objectively identified and measured by this new technique.
Conclusions: En face OCT could be a useful diagnostic modality
for imaging the retina and RPE overlying a choroidal lesion. This
diagnostic approach may provide some information about peculiar
details of choroidal and retinal lesions which cannot be imaged with
other techniques and it will probably represent an useful tool to
monitor the changes during follow-up.
Commercial Relationships: Cinzia Mazzini, None; Daniela
Bacherini, None; Andrea Giorni, None; Giulia Pieretti, None; Ugo
Menchini, None
Ciliary Body Melanocytomas: Ultrasonographic Characteristics
and Natural Course in 10 Cases
Phillip Tenzel, Marco A. Gonzalez, J. Antonio Bermudez Magner, J
William Harbour. Bascom Palmer Eye Institute, Miami, FL.
Purpose: To evaluate the natural course of the clinical and
ultrasonographic characteristics of ciliary body melanocytomas.
Methods: This is a non-comparative consecutive case series of
patients with biopsy proven ciliary body melanocytomas. Data was
collected on demographics, clinical features, treatments, and visual
outcomes.
Results: A total of 10 eyes in 10 patients were identified. The mean
age at presentationwas 65.1years (range of 55 to 75). The mean
follow up after presentation was 35 months. Mean initial ultrasound
height was 2.19 mm (range of 0.7 to 5.5 mm). Clinical factors
associated with ciliary body melanocytomas were dark brown color,
pigment deposition into trabecular meshwork, stability of size on
ultrasonography, and shaggy contour on ultrasonography.
Conclusions: Ciliary body melanocytomas are rare, benign
melanocytic tumors that can be difficult to distinguish from malignant
melanoma. Certain clinical and ultrasonographic characteristics are
unique to melanocytomas and may be used as diagnostic tools to help
identify these uveal tumors.
Commercial Relationships: Phillip Tenzel, None; Marco A.
Gonzalez, None; J. Antonio Bermudez Magner, None; J William
Harbour, None
Autofluorescence Pattern of Optic Disc Melanocytoma
Panagiotis Salvanos1, 2, Tor P. Utheim3, Morten Carstens
Moe1, 2, Ragnheidur Bragadottir1, 2, Nils Eide1. 1Department of
Ophthalmology, Oslo University Hospital, Oslo, Norway; 2University
of Oslo, Oslo, Norway; 3Department of Medical Biochemistry, Oslo
University Hospital, Oslo, Norway.
Purpose: To evaluate the autofluorescence changes caused by optic
disc melanocytoma (ODM).
Methods: Three eyes from 3 patients with ODM were included.
Ultra-widefield autofluorescent images (UW-FAF) were obtained
with the 532nm laser (Optomap P200Tx).
Results: Mean patient age was 44 years. Mean follow-up time
from the time of first observation was 16.5 years. Best-corrected
visual acuity was 20/25. Clinical examination revealed a domeshaped, darkly pigmented tumor on or adjacent to the optic disc in
all patients, with a mean tumor basal dimension 1.3 mm and mean
tumor thickness by ultrasonography 1 mm. UW-FAF revealed a
totally hypofluorescent mass with sharply demarcated, feathery edges
on or adjacent to the optic disc. No hyperfluorescent changes due to
orange pigment or subretinal fluid in the tumor area or the periphery
were seen. There was no major discrepancy in the size of the tumor
between autofluorescent images and ophthalmoscopy.
Conclusions: UW-FAF imaging is a novel, noninvasive adjuvant
tool in the differential diagnosis of pigmented fundus lesions and
might offer more insight in the pathophysiology of optic disc
melanocytoma.
Commercial Relationships: Panagiotis Salvanos, None; Tor
P. Utheim, None; Morten Carstens Moe, None; Ragnheidur
Bragadottir, None; Nils Eide, None
Clinical characteristics of intraocular lymphoma in southern
Japan
Wakako Yoshinaga, Kumiko Nakao, Taiji Sakamoto. Ophthalmology,
Kagoshima University, Kagoshima City, Japan.
Purpose: Because of the epidemic of Human T cell leukemia/
lymphotropic virus type 1 (HTLV-1) in southern Japan (antibody
prevalence 6%), the clinical characteristics of intraocular lymphoma
in southern Japan differ from those in other areas. The purpose of
this study was to find the clinical features of patients diagnosed with
intraocular lymphoma in an academic referral hospital in southern
Japan.
Methods: The Kagoshima University Hospital is located in
Kagoshima, the southernmost prefecture of main-islands of Japan,
which covers 1.7 million of populations. We reviewed the medical
records of patients with intraocular lymphoma diagnosed and treated
at the Kagoshima University Hospital from 2000 to 2011.
Results: During the study period, 26 cases, 10 men and 16
women, were diagnosed with intraocular lymphoma. The mean
age at presentation was 64.2 years. Follow-up period ranged from
1 month to 10 years with a median of 19 months. Eight patients
had lymphoma in an unilateral eye and 18 had bilateral eyes final
examination at the time. The most frequent symptom was visual
disturbance or blurred vision. On ophthalmological check-ups, the
most common sign was vitreous opacity. Ocular disease concurrent
with central nervous system (CNS) lymphoma was observed in
73.0%, while the localized ocular disease was found in 7.7% of the
patients. Intraocular lymphoma occurred as the primary lymphoma in
57.7%, and as ocular relapse of the primary CNS lymphoma in 15.4%
the cases. The 5-year survival rate was 60.2%. A positive serological
test for HTLV-1 was found only in 2 cases.
Conclusions: The bilateral onset, elderly female and vitreous
infiltration were common characteristics of intraocular lymphoma in
southern Japan. Intraocular lymphoma with no systemic involvement
was rare. Despite its high epidemic, HTLV-1 was associated with
uveitis, but not intraocular lymphoma.
Commercial Relationships: Wakako Yoshinaga, None; Kumiko
Nakao, None; Taiji Sakamoto, None
Life Expectancy of Patients with Intraocular Lymphoma after
Peripheral Blood Stem Cell Transplantation
Manfred Zierhut, Tarek Bayyoud, Bianka Sobolewska, Christoph
M. Deuter. Centre for Ophthalmology, University of Tuebingen,
Tuebingen, Germany.
Purpose: Primary intraocular lymphoma (PIOL) is the ocular
disorder with the shortest 5-year survival time. It is mostly a diffuse
large B-cell non-Hodgkin lymphoma. Up to 80% are associated
with or will develop CNS-lymphoma. This is the reason for treating
even isolated ocular disease systemically. Current treatment involves
mainly intravenous high dose methotrexate (MTX) for PIOL.
Peripheral blood stem cell transplantation (PBSCT) has been shown
to be very effective for primary CNS lymphoma.
Methods: Retrospective observational study of 5 patients with
intraocular lymphoma without (n=2) or with CNS involvement
(n=3). All patients had standardized clinical and ophthalmological
assessments. All patients received PBSCT in addition to high dose
intravenous MTX. Three of these patients received additional
chemotherapeutics and 1 patient radiation therapy.
Results: Five patients (3 female and 2 male) with intraocular
lymphoma were included in this study. The mean age at the initial
presentation was 55.2 years (range: 40-74 years). The mean time of
survival, starting with the initial symptoms, was 77.2 months (range:
25-136 months). All patients showed complete response of their
intraocular and CNS-lymphoma. Today all patients are alive without
any signs of relapses.
Conclusions: Treatment of intraocular lymphoma with PBSCT in
addition to chemotherapeutics resulted in all patients in complete
remission and long survival time.
Commercial Relationships: Manfred Zierhut, None; Tarek
Bayyoud, None; Bianka Sobolewska, None; Christoph M. Deuter,
None
374 Myopia
Tuesday, May 06, 2014 3:45 PM–5:30 PM
Relationship between axial length and 64 cells retinal thickness or
optic disc-to-fovea angle in young healthy eyes
Takehiro Yamashita, Taiji Sakamoto, Yuya Kii, Minoru Tanaka,
Kumiko Nakao. Ophthalmology, Kagoshima University, Kagoshima,
Japan.
Purpose: To determine relationship between axial length and 64 cells
retinal thickness with posterior pole asymmetry scan (PPA) or optic
disc-to-fovea angle (OFA) in young healthy eyes.
Methods: A prospective study of 64 healthy right eyes was
performed. All participants (mean age 25.6 ± 3.5) underwent
comprehensive ophthalmologic examination, including axial length,
color fundus photographs and optical coherence tomography
(OCT). The 64 cells retinal thicknesses were measured by the PPA
of Spectralis SD-OCT. The OFA was calculated using the fundus
photographs. The relationships between the retinal thickness of the 64
cells or the OFA and the axial length were investigated using linear
regression analysis.
Results: Retinal thicknesses of central 4 cells were not significantly
correlated with the axial length (R=-0.07 to 0.01, p >0.05). Retinal
thicknesses of almost other cells (54 cells) were significantly
negatively correlated with the axial length (R=-0.25 to -0.56, p
<0.05). The OFA was not significantly correlated with the axial length
(R=-0.17, p=0.19).
Conclusions: The retinal thicknesses of the central 4 cells and OFA
were constant regardless of the axial length in young healthy eyes.
Commercial Relationships: Takehiro Yamashita, None; Taiji
Sakamoto, None; Yuya Kii, None; Minoru Tanaka, None; Kumiko
Nakao, None
Clinical Trial: UMIN000006040
Genome-wide scleral micro- and messenger-RNA profiling in the
mouse myopia model
Ravi Metlapally1, Hanna Park2, Kevin Wang1, Christopher C. Tan2,
Jacob G. Light2, Machelle T. Pardue2, 3, Christine Wildsoet1, 4.
1
Optometry, UC Berkeley, Berkeley, CA; 2Ophthalmology, Emory
University, Atlanta, GA; 3Rehab R&D Center of Excellence, Atlanta
VA Medical Center, Atlanta, GA; 4Vision Science Graduate Group,
UC Berkeley, Berkeley, CA.
Purpose: miRNAs (micro-RNAs) play critical roles in normal
physiological as well as disease processes. Recent studies implicated
miRNAs in scleral remodeling and normal ocular growth. Through
the modulation of scleral extracellular matrix genes, miRNAs have
potential to be therapeutic scleral targets for myopia prevention/
retardation strategies. As a first step, genome-wide miRNA and
mRNA (messenger-RNA) expression profiles in myopic and control
eyes of mice were studied using microarray analyses.
Methods: C57BL/6J mice (n=7, P28), reared under a 12 hr lightdark cycle, were form-deprived (FD) unilaterally for 2 weeks.
Refractive error and axial length measurements were made using
photorefraction and 1310nm SD-OCT. Scleral RNA samples from
FD and control eyes were processed for mirBASE version 19 miRNA
(1279 miRNAs) and Affymetrix GeneChip Gene 2.0 ST (>28000
mRNAs) microarrays. Statistical analyses were performed using
the NIA (National Institute on Aging) Array Analysis Tool. Briefly,
raw data were background subtracted, log-transformed, normalized
and average intensities as well as fold differences calculated. Group
comparisons were made using ANOVA. Functional biological
pathways were identified using WebGestalt software.
Results: FD eyes showed myopic shifts in refractive error (mean
interocular difference: -2.90 +/- 0.86D, p<0.05). Comparison of
scleral RNA profiles for FD and control eyes revealed a total of 54
miRNAs and 261 mRNAs differentially expressed (fold change >1.25
fold in either direction, max FC=1.63 for miRNA, 2.7 for mRNA)
(p<0.05, min p=0.0001). miRNAs of the let-7 family, previously
implicated in matrix remodeling in other tissues, were upregulated
in eyes developing axial myopia. Notable statistically significant
(p<0.05) pathways showing over-representation of genes included
intermediate filament organization, scaffold protein binding, detection
of stimuli, calcium ion, G-protein, and photo-transduction pathways.
Conclusions: A number of scleral mi- and m-RNAs showed
differential expression linked to myopia in the mouse model, further
supporting the involvement of miRNAs in scleral remodeling. The
observed general trend of relatively small fold changes is perhaps
indicative of a tightly controlled regulatory mechanism for scleral
gene expression. Further validation and molecular studies aimed at
understanding specific mechanisms related to myopia are planned.
Commercial Relationships: Ravi Metlapally, None; Hanna Park,
None; Kevin Wang, None; Christopher C. Tan, None; Jacob G.
Light, None; Machelle T. Pardue, None; Christine Wildsoet, None
Support: NIH-NEI K08EY022670 to RM, NIH-NEI R01016435 to
MTP, NIH-NEI R01 EY012392 to CFW
Spatial and temporal mapping of retinal sublayer and choroidal
thicknesses in the chick using 3-dimensional segmentation with
optical coherence tomography imaging
Diane Nava1, 4, Akhila Raman1, Claudia Nieuwenhuis2, Anwar
Nunez-Elizalde3, Christine Wildsoet1, 4. 1Vision Science Group,
UC Berkeley, Berkeley, CA; 2Department of Computer Science,
Technische Universität München, Garching, Germany; 3Helen Wills
Neuroscience Institute, UC Berkeley, Berkeley, CA; 4Center for Eye
Disease and Development, UC Berkeley, Berkeley, CA.
Purpose: Interest in changes in thickness of the retina (RT) and
choroid (CT) has grown in myopia research, yet studies using
animal models typically use A-scan ultrasonography (US), which is
limited to on-axis measurements, and blockface photography, which
precludes longitudinal tracking. This study sought to exploit in vivo
spectral domain optical coherence tomography (SD-OCT) imaging to
cover the latter deficiencies.
Methods: Young chicks were fitted with either monocular +5 D
single vision (SV) lenses or multifocal (MF) lenses with a +5 D
peripheral defocus. For each eye, several overlapping rectangular
and radial SD-OCT scans covering central and peripheral retina and
choroid were collected (14x14 mm FOV, 100 b-scans, Bioptigen,
NC). Scans were segmented to extract RT and CT using a custom
automated algorithm or a manual segmentation software that uses
defined boundaries from points projected from an 11 by 11 grid. The
data were then reconstructed in 3D and smoothed using a pythonbased algorithm. 3D maps of overlapping scans were merged, after
which the absolute nasal, temporal, superior and inferior fields were
redefined using the angle of the pectin and the location of the area
centralis (AC). Interocular and temporal thickness difference maps
were obtained through registration. Data were compared to RT and
CT from US, photographs of embedded blocks and 10 um vertical
sections.
Results: Peripheral choroidal thickening was detected in the MF
group. RT and CT data obtained with our custom method were
comparable to values obtained from manual calipers, US, blockface
photography, and histological sections, with improved interobserver reliability and resolution. The retinal ganglion cell layer
thickness maps show a peak superior and nasal to the tip of the
pectin, consistent with histological data defining the AC, and served
as a useful reference for validating segmentation algorithms and
quantifying lens-induced regional CT changes.
Conclusions: Our new methods for the visualization and
quantification of chick SD-OCT data allows for fast and accurate
characterization of point-to-point regional and temporal differences
in RT and CT in vivo, providing valuable new insight into the effects
of novel optical defocus manipulations, as well as ex vivo, providing
a replacement for photography applied to blockfaces and histological
sections.
Commercial Relationships: Diane Nava, None; Akhila Raman,
None; Claudia Nieuwenhuis, None; Anwar Nunez-Elizalde, None;
Christine Wildsoet, None
Support: NIH Grant EY12392, NIGMS Grant R25GM090110
Chick Retinal Pigment Epithelium Responds to Imposed Defocus
in Minutes
Yan Zhang, Albert Truong, Feng Zhao, Christine Wildsoet. Center
for Eye Disease & Development, School of Optometry, Univ of
California, Berkeley, Berkeley, CA.
Purpose: We previously reported that gene expression of BMP2, 4,
and 7 in chick RPE show differential regulation by optical defocus
in as little as 2 hours, which suggest their involvement of RPE in
the early stage of eye growth regulation. This study was conducted
to further characterize the temporal profile of these defocus-induced
BMP gene expression changes in the chick RPE
Methods: White-Leghorn chicks wore monocular -10 or +10 D
lenses from 14 days of age for 5, 15, 30 or 60 minutes. At the end of
the lens treatment periods, chicks were sacrificed, eyes enucleated,
RPE isolated and RNA extract. RNA was subjected to cDNA
synthesis and then qPCR. Expression levels for lens-treated eyes
were compared to those of their fellow eyes
Results: As reported previously, differences in BMP gene expression
were detected in chick RPE samples from eyes subjected to positive
(+) versus negative (-) lens treatments. With positive lenses, 5 and
30 min of wear was sufficient to up-regulate the gene expression of
BMP2 and BMP4 in treated eyes respectively, with up-regulation
also detected at the other 3 time points for BMP2 and 60 minutes
treatment for BMP4. For 5, 15, 30 and 60 min of +lens treatment,
BMP2 was up-regulated 2.0-fold (p < 0.05, n = 5), 3.4-fold (p < 0.05,
n = 7), 11.3-fold (p < 0.01, n = 7), and 5.0-fold (p < 0.001, n = 7),
respectively, while BMP4 was up-regulated 7.8-fold (p < 0.05, n =
7) and 2.9-fold (p < 0.05, n = 7) after 30 and 60 minutes of treatment
respectively. In contrast, BMP7 did not show differential expression
with these short 5-60 min +lens treatments, and nor was differential
expression detected with 5, 30, and 60 min of -lens treatment for
these three BMPs. A trend of down-regulation of BMP2 was observed
after 60 minutes of -lens treatment (6 out of 8 birds), but curiously,
this gene showed significant up-regulation at 15 minutes (2.5-fold, p
< 0.05, n = 8)
Conclusions: This study provides further evidence for the
involvement of the RPE in eye growth regulation signaling cascades.
The differences in the temporal profiles of gene expression changes
induced by positive versus negative lenses, i.e., positive lenses
inducing very rapid changes, suggest different pathways are involved.
Finally, differences in the temporal profiles of the three genes studied
- BMP2, 4 and 7, suggest that they are regulated differently. These
proteins may interact synergistically across time during eye growth
regulation
Commercial Relationships: Yan Zhang, None; Albert Truong,
None; Feng Zhao, None; Christine Wildsoet, None
Support: NIH grants R01EY012392 (CFW), K08EY023609 (YZ),
K12EY017269 (YZ)
Quantitative analysis of tessellated fundus and its association
with choroidal thickness in healthy eyes
Naoya Yoshihara, Takehiro Yamashita, Yuya Kii, Minoru Tanaka,
Kumiko Nakao, Taiji Sakamoto. Ophthalmology, Kagoshima
University, Kagoshima, Japan.
Purpose: To investigate the relationship between the tessellated
fundus index (TFI) and choroidal thickness (CT) by the objective
method.
Methods: A prospective observational cross-sectional study
comprised 100 right eyes of 100 healthy young Japanese participants.
All participants (mean age 25.8 ± 3.9) underwent comprehensive
ophthalmologic examination, including axial length, color fundus
photograph. The subfoveal choroidal thickness (SFCT) and the
nasal choroidal thickness (NCT) being located 750 μm nasal
from the fovea were measured using enhanced depth imaging of
Spectralis OCT. Fundus color between the fovea and the optic disc
was quantified on digital color fundus photographs as a surrogate
of the degree of the tessellated fundus using Image J. The TFIs was
calculated as the ratio of mean red-green-blue (RGB) intensity. We
used following three formula; TFI1: (R-G)/R, TFI2: R/(R+G+B),
TFI3: (R-G)/(R+G+B). The relationship between the TFIs and the
choroidal thickness and the axial length was investigated using linear
regression analysis.
Results: The mean axial length was 25.3 ± 1.4 mm. The all TFIs
were significantly associated with the SFCT or NCT (R=-0.20 to
-0.24, p<0.05). The correlation coefficients between the TFI2 and
SFCT or NCT were higher than the correlation coefficients between
the TFI1 or TFI3 and SFCT or NCT. However, all the TFIs were not
significantly associated with the axial length.
Conclusions: The degree of tessellated fundus was increased as the
choroidal thickness decreased in young healthy eyes.
Commercial Relationships: Naoya Yoshihara, None; Takehiro
Yamashita, None; Yuya Kii, None; Minoru Tanaka, None;
Kumiko Nakao, None; Taiji Sakamoto, None
Clinical Trial: UMIN000006040
Is Retinal Shape different in Asians and Caucasians?
Estimation from Peripheral Refraction and Peripheral Eye
Length Methods
Pavan K. Verkicharla1, David A. Atchison1, Marwan Suheimat1,
Katrina L. Schmid1, Ankit Mathur1, Edward A. Mallen2, Xin Wei3,
Noel A. Brennan3. 1School of Optometry & Vision Science, Institute
of Health & Biomedical Innovation, Queensland University of
Technology, Brisbane, QLD, Australia; 2School of Optometry and
Vision Science, University of Bradford, Bradford, United Kingdom;
3
Johnson & Johnson Vision Care, Inc., Jacksonville, FL.
Purpose: Race appears to be associated with myopiogenesis,
with East Asians showing high myopia prevalence. Considering
structural variations in the eye, it is possible that retinal shapes are
different between races. The purpose of this study was to quantify
and compare retinal shapes between racial groups using peripheral
refraction (PR) and peripheral eye lengths (PEL).
Methods: A Shin-Nippon SRW5000 autorefractor and a Haag-Streit
Lenstar LS900 biometer measured PR and PEL, respectively, along
horizontal (H) and vertical (V) fields out to ±35° in 5° steps in 29
Caucasian (CA), 16 South Asian (SA) and 23 East Asian (EA) young
adults (spherical equivalent range +0.75D to −5.00D in all groups).
Retinal vertex curvature Rv and asphericity Q were determined from
two methods:
a) PR (Dunne): The Gullstrand-Emsley eye was modified according
to participant’s intraocular lengths and anterior cornea curvature.
Ray-tracing was performed at each angle through the stop, altering
cornea asphericity until peripheral astigmatism matched experimental
measurements. Retinal curvature and hence retinal co-ordinate
intersection with the chief ray were altered until sagittal refraction
matched its measurement.
b) PEL: Ray-tracing was performed at each angle through the anterior
corneal centre of curvature of the Gullstrand-Emsley eye. Ignoring
lens refraction, retinal co-ordinates relative to the fovea were
determined from PEL and trigonometry.
From sets of retinal co-ordinates, conic retinal shapes were fitted in
terms of Rv and Q. Repeated-measures ANOVA were conducted on Rv
and Q, and post hoc t-tests with Bonferroni correction were used to
compare races.
Results: In all racial groups both methods showed greater Rv for the
horizontal than for the vertical meridian and greater Rv for myopes
than emmetropes. Rv was greater in EA than in CA (P=0.02), with Rv
for SA being intermediate and not significantly different from CA and
EA. The PEL method provided larger Rv than the PR method: PEL:
EA vs CA 87±13 vs 83±11 m-1 (H), 79±13 vs 72±14 m-1 (V); PR: EA
vs CA 79±10 vs 67±10 m-1 (H), 71±17 vs 66±12 m-1 (V). Q did not
vary significantly with race.
Conclusions: Estimates of Rv, but not of Q, varied significantly with
race. The greater Rv found in EA than in CA and the comparatively
high prevalence rate of myopia in many Asian countries may be
related.
Commercial Relationships: Pavan K. Verkicharla, Johnson
& Johnson Vision Care, Inc. (F); David A. Atchison, Johnson &
Johnson Vision Care, Inc. (F); Marwan Suheimat, Johnson &
Johnson Vision Care, Inc. (F); Katrina L. Schmid, Johnson &
Johnson Vision Care, Inc. (F); Ankit Mathur, Johnson & Johnson
Vision Care, Inc. (F); Edward A. Mallen, Johnson & Johnson Vision
Care, Inc. (F); Xin Wei, Johnson & Johnson Vision Care, Inc. (E);
Noel A. Brennan, Johnson & Johnson Vision Care, Inc. (E)
Support: Johnson & Johnson Vision Care, Inc.
Identification of integrin receptor subunits in the guinea pig
sclera
Kevin Wang1, Ravi Metlapally1, Christine Wildsoet1, 2. 1School of
Optometry, University of California Berkeley, Berkeley, CA; 2Vision
Science Graduate Group, University of California Berkeley, Berkeley,
CA.
Purpose: Treatment for myopia, the result of excessive elongation
of the globe, has been directed mostly at the symptoms and not
at the core issue of elongation. The ocular dimensional changes
reflect increased scleral remodeling, which is also associated with
biomechanical weakening of the sclera. As integrins have been linked
to scleral remodeling previously, they represent potential targets for
myopia treatments aiming to slow ocular elongation. As a first step,
this study aimed to characterize the integrin subunits in the guinea
pig sclera, a common myopia model.
Methods: Primers for α and β integrin subunits were designed using
NCBI entries from a genome sequencing project, Primer3, and UCSC
Genome Browser Bioinformatics. Total RNA was extracted from
both intact scleral samples and fibroblasts cultured from guinea pig
sclera. Reverse transcription was performed to produce cDNA and
PCR used to amplify products of a predetermined size. Finally, PCR
products were sequenced to confirm their identity.
Results: PCR analysis of intact guinea pig scleral samples revealed
twenty-four of the twenty-six known integrin subunits, corresponding
to a possible twenty-two combinations of integrins, while only
twenty-one of the twenty-six known subunits were detected in
cultured scleral fibroblasts, allowing for nineteen potential integrin
combinations. Specifically, scleral tissue expressed all known integrin
alpha subunits except integrin αD and αE, which were also not
expressed in scleral fibroblasts although expression of integrin αD
and αE was confirmed in guinea pig liver. Integrins αL, αM, and
αX were also not expressed in scleral fibroblasts. Both guinea pig
scleral tissue and scleral fibroblasts expressed all known integrin beta
subunits. All results were verified through sequencing.
Conclusions: While the above results are similar to those of a
previous study in the tree shrew, another popular myopia model,
guinea pig sclera expressed all known mammalian beta integrin
subunits while tree shrew sclera did not express β2-, β3-, β6-, and
β7-integrin subunits. However, alpha integrin subunits found in tree
shrew sclera were also found in guinea pig sclera. Data provided from
this study will help guide future studies directed at understanding the
relationship between scleral integrins and myopia.
Commercial Relationships: Kevin Wang, None; Ravi Metlapally,
None; Christine Wildsoet, None
Support: T35EY007139 to KW, K08EY022670 to RM,
R01EY012392 to CFW
Changes in mRNA expression of Endothelial Growth Factor A,
C, D and Vascular Endothelial Growth Factor Receptor 1, 2, 3
during induction of deprivation myopia in chickens
Marita P. Feldkaemper, Frank Schaeffel. Centre for Ophthalmology,
Institute for Ophthalmic Research, Tuebingen, Germany.
Purpose: It was previously reported that the vascular epithelial
growth factor inhibitor Bevacizumab can slow the development of
deprivation myopia in chickens and suppress choroidal thickening
that normally occurs during recovery from deprivation myopia
(Mathis and Schaeffel, ARVO 2013). Furthermore, Sheng, Zhu
and Wallman (ARVO 2012) found that VEGF isoform V165 can
transiently thin the choroid in chickens. We have further studied the
role of VEGF in myopia by measuring the choroidal expression of
different VEGF isoforms and their receptors, both after long-term
deprivation and during recovery from myopia.
Methods: 7 days old White Leghorn chicks were treated with (a)
diffusers for 8 days on both eyes but diffusers were removed on
one eye one hour before choroidal tissue analysis, (b) no treatment
except for that one eye was occluded 1 hour before tissue analysis,
(c) unilateral diffuser treatment for 16 days, (d) unilateral diffuser
treatment for 15 days followed by removal of a diffuser for one day
(n= 6 chicks in groups a-d). Semi-quantitative real-time PCR was
used to quantify mRNA levels of VEGFA (transcript variant 1 and 2),
VEGFC, VEGFD, VEGFR1, VEGFR2 and VEGFR3. Beta-actin and
HPRT served as reference genes.
Results: Most mRNA levels of the VEGF isoforms were not
changed during deprivation myopia. Only VEGFD mRNA was
significnatly up-regulated after 16 days of diffuser wear with a 46%
increase in normalized mRNA expression (paired t-test, p=0.02).
Neither recovery for 1 hour or of 1 day had any effect of the mRNA
expression of the various VEGF genes.
Conclusions: Changes in choroidal mRNA expression levels (VEGF
D) were evident only after long-term treatment of the chickens with
diffusers. This growth factor might therefore either play a role in the
maintainance of the myopic state or its induction is related to early
degenerative processes in high myopia.
Commercial Relationships: Marita P. Feldkaemper, None; Frank
Schaeffel, None
The association between initial choroid thickness and subsequent
ocular growth rate in young chicks: Evidence for different
choroidal mechanisms in growth inhibition vs stimulation
Kristen Totonelly, Xiaoying Zhu, Pearl Thai, Rinita Zanzerkia,
Debora L. Nickla. New England College of Optometry, Boston, MA.
Purpose: Thick choroids are associated with ocular growth inhibition
and thin choroids with growth stimulation. Whether this is relevant to
the mechanisms underlying the signal cascade that mediates scleral
growth in response to visual or pharmacological stimuli is unknown.
These studies asked whether choroid thickness in young chicks
predicts subsequent ocular growth rates under various conditions.
Methods: We determined the correlation between choroid thickness
and subsequent ocular growth rate (scleral GAG synthesis for group
6) under the following conditions. (1) Plus lens-wear for 4 days (d)
(n=14). (2) Minus lens-wear for 5 d (n=16). (3) Diffuser-wear for 5
d (n=16). (4) Fellow untreated eyes (n=53). (5) Daily injections of
quinpirole (n=12), apomorphine (n=17), atropine (n=11), pirenzepine
(n=10), or a single injection of oxotremorine (n=27). (6) Eyecups of
RPE, choroid and sclera cultured for 24 hrs (n=9). Some of these data
are retrospective.
Results: There was a negative correlation between initial choroid
thickness and subsequent ocular growth rate in visual conditions in
which eye growth was inhibited (plus lens: r=-0.662, p<0.01) and
in untreated eyes (r=-0.393, p<0.005), but not when growth was
stimulated (minus lens: r=0.08, p=0.72; diffusers: r=0.036, p=0.9).
In untreated eyes and in eyes wearing diffusers or minus lenses
there was also a correlation between initial choroid thickness and
subsequent thinning (p<0.05); there was no such correlation for plus
lenses. In eyecups, there was a negative correlation between choroid
thickness and scleral GAG synthesis (r=-0.79, p<0.01). Drugs: There
was a negative correlation between choroid thickness and growth rate
in eyes injected with quinpirole (r=-0.72, p=0.008) but the data for
apomorphine did not reach significance. There was no correlation for
eyes injected with muscarinic agents.
Conclusions: That initial choroidal thickness does not predict
ocular growth rate under all conditions implies that thickness is
not correlated with the efficacy of a mechanical barrier to a signal
molecule. This is also supported by the negative correlation between
thickness and quinpirole-mediated growth inhibition. Finally, the
results support different roles for the choroid in growth stimulation vs
inhibition.
Commercial Relationships: Kristen Totonelly, None; Xiaoying
Zhu, None; Pearl Thai, None; Rinita Zanzerkia, None; Debora L.
Nickla, None
Support: NIH EY013636
Gaze-Induced Axial Length Changes in Highly Myopic Eyes as
Gauged by Magnetic Resonance Imaging
Quan V. Hoang1, Jonathan Tang1, Julie Goldman1, Jane Y. Pan2, 1,
Stanley Chang1. 1Ophthalmology, Harkness Eye Institute, Columbia
University, New York, NY; 2Methodist Girls’ School, Singapore,
Singapore.
Purpose: To determine if axial length changes occur in highly
myopic eyes undergoing the stress and strain of normal eye
movement.
Methods: A prospective imaging study was performed on highly
myopic patients (> 26 mm of axial length) with a clinical diagnosis
of staphyloma. 3-D MRI scans were acquired while subjects gazed in
5 directions (primary, nasal 15°, temporal 15°, superior 10°, inferior
10°). Volume renderings were manually reoriented so that the plane
of the limbus (the cornea-sclera interface) was normal to the plane of
the screen. Four axial length measurements were taken at 90-degree
rotations around the central axis and averaged for each eye in every
gaze. Eye axial lengths at each eccentric gaze were compared to the
axial length in primary gaze using a fixed effects regression allowing
for person-specific and eye-specific effects (n = 40).
Results: Axial lengths were unchanged in temporal gaze when
compared to primary gaze (p = 0.89). Axial lengths shortened in nasal
gaze (-0.07 mm, p = 0.04, 95% CI -0.148 to 0.008) and in superior
gaze (-0.08 mm, p = 0.03, 95% CI -0.163 to 0.003). Axial lengths
increased by +0.12 mm when changing from primary to inferior
gaze (p = 0.001, 95% CI [0.043 to 0.196]). Linear regressions for
individual patients demonstrated that the F-test of the axial length
measured in the four gazes were jointly different from the axial
length measured in primary gaze. This difference was significant at
the p < 0.05 level in 33 out of 40 eyes (82.5%).
Conclusions: Eye lengthening appears to occur only in inferior
gaze, which is of interest given past clinical studies suggesting an
association between excessive near work and myopia development
and progression.
Commercial Relationships: Quan V. Hoang, None; Jonathan
Tang, None; Julie Goldman, None; Jane Y. Pan, None; Stanley
Chang, Alcon (F)
Support: NIH 5 KL2 TR 81-8 (QVH) and an unrestricted grant from
Research to Prevent Blindness (New York, New York)
Lack of oblique peripheral astigmatism in the chicken eye,
comparison to the human eye and possible consequences for
emmetropization
Felix Maier1, Arne Ohlendorf2, Siegfried Wahl2, Frank Schaeffel1.
1
Institute for Ophthalmic Research, Section for Neurobiology of
the Eye, Tuebingen, Germany; 2Institute for Ophthalmic Research,
ZEISS Vision Science Lab, Tuebingen, Germany.
Purpose: Primates display considerable off-axis astigmatism. In
humans, its magnitude can be described by a parabolic function:
astigmatism relative to the pupil axis = 3.28*10-3 * angle2
(Howland, 13th IMC in Tuebingen 2010). Howland also proposed
that peripheral astigmatism may represent a cue for emmetropization,
since either its tangential or radial axis are in better focus, depending
on the spherical refractive error. It is known that the chicken eye
emmetropizes independently in the center and the periphery but not
whether it uses peripheral astigmatism.
Methods: Infrared photorefraction was used to map out the
refractions over the horizontal visual field, both in the vertical and
horizontal meridians, in three 43 day old chicks (6 eyes) and three
near emmetropic male human subjects (6 eyes, age 34.7 ± 6.8 years).
Chicks were trained to accept that the operator turned their heads
as desired by holding their beak. Videos were recorded and eye
orientation and refractions were determined from the position of the
first Purkinje image relative to the pupil center and the brightness
slopes in the pupil. Human subjects were measured with a scanning
photorefractor (Tabernero et al. 2009).
Results: There was no significant difference in the amount of
astigmatism in chicks and humans in the center of the visual field
(0 deg: chicks -0.35±0.79 D, humans 0.65±0.60 D, p = 0.30).
Similar to what was found by Howland, astigmatism increased in
our subjects’ eyes to the periphery with the function: astigmatism =
2.21*10^-3*angle^2-0.0245*angle+1.589. Strikingly, in the chick
eye astigmatism did not increase in the periphery. Highly significant
differences were found in the peripheral astigmatism in humans and
chicks (at 40 deg in the temporal visual field: humans 4.21±2.39 D,
chicks -0.63±0.80 D, p < 0.001 (unpaired t-test) and at 20 deg in
the nasal visual field: humans 3.69±1.16 D, chicks 0.74±0.44 D, p
< 0.001; the trend is similar at 20 deg in the temporal visual field:
humans 1.68±1.40 D, chicks 0.30±0.29 D, p = 0.07 and in the nasal
visual field at 40 deg humans 5.17±3.71 D, chicks 0.78±0.11 D, p =
0.12).
Conclusions: The chick eye is perhaps the first vertebrate eye
without oblique peripheral astigmatism. While the optical design of
the crystalline lens in the chick eye must be fascinating, the lack of
peripheral astigmatism suggests that emmetropization cannot rely on
it.
Commercial Relationships: Felix Maier, None; Arne Ohlendorf,
Carl Zeiss Vision International GmbH (E); Siegfried Wahl, Carl
Zeiss Vision International GmbH (E); Frank Schaeffel, None
Support: Werner Reichardt Centrum für Integrative
Neurowissenschaften Pool Projekt 2012-12
Blue light protects against temporal frequency dependent
refractive changes
Stephanie Britton, Stephan Hanowsky, Frances J. Rucker. Biomedical
Science and Disease, New England College of Optom., Boston, MA.
Purpose: A previous experiment indicated that chicks exposed to
luminance flicker without blue light showed a hyperopic shift at
higher temporal frequencies and a myopic shift at lower frequencies.
In this experiment we test the hypothesis that blue light is important
in emmetropization by comparing the response of chicks to
luminance flicker with and without blue light over a range of
temporal frequencies.
Methods: 4-5 day old White Leghorn chicks were exposed daily
for three days (9am to 5pm) to sinusoidal luminance modulation
at 80% contrast, at one of six temporal frequencies: 0, 0.2, 1, 2, 5,
10 Hz. Luminance flicker “without blue” light was created with
in-phase modulation of red and green, while “with blue” flicker was
created with in-phase modulation of red, green and blue light. Mean
illumination was 680 lux. Chicks were maintained in a dark chamber
overnight. Changes in ocular components were measured before and
after the experiment with a non-contact ocular biometer (Lenstar LS
900), and refractive error was measured with a Hartinger Coincidence
Refractometer. Photokeratometry was used to calculate corneal
astigmatism and images were analyzed with Image J.
Results: Refraction, eye length, and choroidal changes were
dependent on temporal frequency (p<0.03 all) and on the interaction
between frequency and blue light (p<0.03 all). Without blue,
refractions were more hyperopic with higher temporal frequencies,
and more myopic at lower temporal frequencies. There was a
hyperopic shift (> 1 D) at 5 and 10 Hz, and a myopic shift (> -0.6 D)
at ≤2 Hz, and an increase in astigmatism along J45 (p=0.04). With
blue light, refraction remained constant (mean change -0.24 D).
Without blue light, there was less eye growth at high frequencies
and more at low frequencies. Eyes were 145 mm shorter at 10 Hz
than they were at 0.2 Hz (p<0.003), while with blue, they were
only 77 mm shorter. Without blue light, anterior chamber depths
were deeper (p=0.006), while choroids were thinner at low and
intermediate temporal frequencies, contributing to the myopic shift at
low frequencies. At 5 Hz, choroids thinned 46 mm more without blue
light, than with blue light (p=0.03).
Conclusions: Emmetropization shows a temporal, blue light
sensitivity. Blue light protects against temporal frequency dependent
refractive changes through modification of eye length, choroidal
thickness, anterior chamber depth and corneal curvature.
Commercial Relationships: Stephanie Britton, None; Stephan
Hanowsky, None; Frances J. Rucker, None
Support: New England College of Optometry Internal Research
Award
The effect of near additions and prismatic lenses on
accommodative micro-fluctuations in Chinese myopic children
Huiling Lin1, 3, Drobe Björn2, 3, Lin Meng1, 3, Jin Wanqing1, 3, Chen
Yunyun1, 3. 1School of Optometry and Ophthalmology, Wenzhou
Medical University, Wenzhou, China; 2R&D Optics Asia, Essilor
International, Wenzhou, China; 3WEIRC (WMU-Essilor International
Research Centre), Wenzhou, China.
Purpose: To investigate the effects of near additions and prismatic
lenses on accommodative micro-fluctuations and horizontal
heterophoria in Chinese myopic children.
Methods: 14 myopic children (SE: -0.75D to -3.75D) aged from 9
to 13 participated in the study. Accommodative micro-fluctuations
and horizontal heterophoria were measured through multiple lenses
by means of an open-field infrared autorefractor (WAM-5500,
Grand Seiko Co., Ltd., Hiroshima, Japan) and modified Thorington
technique. Tested lenses were additions (-1.00, 0, +1.00, +2.00 and
+3.00D on each eye) and horizontal prisms (3ΔBI, 2ΔBI, 1ΔBI, 0,
1ΔBO and 2ΔBO on each eye). Lens order was randomized. During
accommodation measurements, children were looking at RSVP (rapid
serial verbal presentations) of Chinese characters at 25cm.
Results: RMS (root mean square) of accommodative response
increased significantly in the condition of -1.0D near addition
(0.28±0.11D) compared to plano lens (0.18±0.06D, p<0.001). No
significant difference in RMS was found between plano and positive
addition lenses (p > 0.05), even though smaller RMS were found for
+1.0D and +2.0D lenses (respectively 0.14±0.05D, 0.13±0.15D). No
significant difference in RMS was found between prismatic lenses
(p=0.22). Heterophoria decreased (more exophoric) with plus-power
addition lenses (p<0.001) and increased with base-in prismatic lenses
(p=0.038). RMS was found to be correlated with the phoria state (r =
0.345, p < 0.001).
Conclusions: Over-correction resulted in higher accommodative
micro-fluctuations while plus-power addition lenses and prismatic
lenses had no influence on accommodative micro-fluctuations. Phoria
decreased with plus-power addition lenses and increased with base-in
prismatic lenses.
Commercial Relationships: Huiling Lin, Essilor International
(F); Drobe Björn, Essilor International (E); Lin Meng, Essilor
International (F); Jin Wanqing, Essilor International (F); Chen
Yunyun, Essilor International (F)
Transient changes in choroidal thickness to different levels of
imposed myopic defocus
Jinhua Bao1, 3, Bjorn Drobe2, 3, Ke Chen1, 3, Hao Chen1, 3. 1School
of Ophthalmology and Optometry, Wenzhou Medical University,
Wenzhou, China; 2R&D Optics Asia, Essilor International, Wenzhou,
China; 3WEIRC, WMU-Essilor International Research Centre,
Wenzhou Medical University, Wenzhou, China.
Purpose: To investigate short-term changes in choroidal thickness
and other ocular biometrics to imposed myopic defocus in young
adults.
Methods: Ocular biometrics of thirteen myopic young adult subjects
was measured before and after 60 minutes of exposure to monocular
defocus (right eye) while watching movies on a 5.5m distant screen.
Choroidal and retinal thicknesses were measured by means of Lenstar
SD-OCT (Heidelberg Spectralis OCT; Heidelberg Engineering,
Heidelberg, Germany) and axial length by Lenstar LS 900 optical
biometer (Lenstar LS 900; Haag Streit AG, Koeniz, Switzerland).
Four different monocular defocus conditions were tested, each on
a separate day: control (no defocus) and myopic defocus (+1D,
+3D and +5D defocus). The fellow eye was optimally corrected for
distance (no defocus).
Results: After 60 minutes, choroidal thickness of the right eye
exhibited a significant increase only for +3D and +5D defocus
conditions (mean change, +1D: +7.53±7.80μm, p>0.05; +3D:
+12.51±11.23μm, p=0.002; +5D: +11.74±8.45μm, p<0.001). Retinal
thickness exhibited no significant change with any myopic defocus
conditions (mean change, +1D: -1.17±2.52μm, +3D: -1.93±1.99μm,
+5D: -1.77±3.49 μm; p>0.1). Optical axial length decreased
significantly only after +5D defocus condition (mean change,
+1D: +2.13±9.67μm, p>0.1; +3D: -1.93±9.05μm, p>0.1; +5D:
-6.56±9.14μm, p=0.024). Choroidal thickness, retinal thickness and
axial length of the left eye showed no significant change (p>0.05).
Conclusions: Significant increase in choroidal thickness occurred in
human subjects after 60 minutes of myopic defocus of 3D or more
but not in axial length. Moreover, there is no evidence to support
the notion that choroidal thickness elongation is proportional to the
degree of myopic defocus.
Commercial Relationships: Jinhua Bao, Essilor International
(F); Bjorn Drobe, Essilor International (F); Ke Chen, Essilor
International (F); Hao Chen, Essilor International (F)
Comprehensive lesions of the retina surrounding the optic nerve
enhance elongation and cause myopia in the guinea pig eye
Sally A. McFadden, Callan Medcalf, Guang Zeng, John Holdsworth.
Faculty of Science and IT, University of Newcastle, Callaghan, NSW,
Australia.
Purpose: Disruption to detailed vision using form deprivation
(FD) induces excessive eye elongation and myopia. The underlying
mechanisms are local to the eye since FD myopia induces excessive
growth after optic nerve section.1 Surprisingly, foveal photoablation
does not inhibit FD myopia in young monkeys.2 However, the region
around the optic nerve (the peripapillary zone or PPZ) expands early
during myopia development and the overlying retina may carry the
pertinent signals.3 We studied the effect of retinal photocoagulation
lesions of the PPZ on both normal and myopic growth.
Methods: Guinea pigs received FD alone (n = 16) or comprehensive
PPZ lesions using multiple spots (laser strength of 100 mW for 50
ms, n = 9) the day prior to FD. FD involved wearing a diffuser over
one eye from 6 to 13 days of age. PPZ lesions were also administered
at the same age in a group not form deprived (n = 7). Refractive error
(using a Nidek autorefractor after cycloplegia) and eye shape (based
on retinal hemisections4) were assessed at the end of the rearing
period. Results are presented as the relative differences between the
two eyes.
Results: FD alone resulted in –3.1D of relative myopia and 100 mm
of relative eye elongation in the PPZ area with relative eye shrinkage
(–47 mm) in the temporal retina. Greater myopia was observed after
PPZ lesions combined with FD (–7.35D, 150 mm) and unlike normal
FD, excessive growth also occurred in the mid periphery, particularly
in nasal retina. PPZ lesions also induced relative myopia in animals
not form deprived (–3.8D, p < 0.001).
Conclusions: Retinal signals from the PPZ may be critical for normal
refractive development and when eliminated, cause exaggeration of
the central elongation so characteristic of myopic eyes. Without these
signals, the eye also grows excessively in the periphery suggesting
they are necessary for relative growth inhibition.
1. Smith EL et al. IOVS (2007), 48(9), 3914-3922.
2. Wildsoet CF, McFadden SA. IOVS (2010) 51 ARVO E-Abstract
1737.
3. Zeng G, McFadden SA. IOVS (2011) 52 ARVO E-Abstract 3923.
4. Zeng G et al. Vision Res. (2013), 76, 77-88.
Commercial Relationships: Sally A. McFadden, None; Callan
Medcalf, None; Guang Zeng, None; John Holdsworth, None
Support: Port Waratah Coal Services, HMRI 13-23, UN NM
G1300470, Ellex Medical Pty. Ltd.
Longitudinal changes in corneal power and axial length in the
Correction of Myopia Evaluation Trial (COMET) Cohort
Mitchell Scheiman1, Li Deng3, Jane E. Gwiazda3, Qinghua
Zhang2, Ruth E. Manny4, Karen D. Fern4, Eric Weissberg3. 1Coll
of Optometry, Salus University, Elkins Park, PA; 2Department of
Preventive Medicine, Stony Brook University Medical Center, Stony
Brook, NY; 331 New England College of Optometry, Boston, MA;
4
College of Optometry, University of Houston, Houston, TX.
Purpose: To describe changes in corneal power and axial length
(AL) in the COMET cohort followed for 14 years, and explore the
relationship between AL and corneal radius (CR) over this time
period.
Methods: 469 ethnically diverse, 6-11 year old children with -1.25
to -4.50 D of myopia were enrolled in COMET. Children wore
either single vision (SVLs) or progressive addition lenses (PALs)
for 5 years and were followed for an additional 9 years wearing
PALs, SVLs, or contact lenses. Additionally, 206 non-myopic young
adults matched by gender, ethnicity, and age with COMET myopes
were recruited at the 12-year visit. Refractive error (cycloplegic
autorefraction), corneal curvature (CC, auto-keratometry), and ocular
component dimensions (A-scan ultrasound) were measured annually
for COMET children, and once for the non-myopic, matched youngadult subjects. Linear mixed model was used to evaluate longitudinal
changes based on all available records adjusting for covariates
(gender, ethnicity, lens type, baseline age and baseline refraction).
Unpaired t-test was used to compare myopes and non-myopes at the
12-year visit. The Pearson correlation coefficient (PCC) between
AL and CC was computed at each visit. The comparison of PCCs
between myopes and non-myopes at the 12-year visit was conducted
using Fisher’s transformation.
Results: Longitudinally, COMET girls had significantly steeper CC
than boys (p<0.0001). Caucasians had the steepest CC, and Hispanics
the flattest (p=0.001). The correlation between AL and CC was -0.70
(p<0.0001) at baseline and decreased to -0.53 (p<0.0001) at the 14year visit. The average AL to CR ratio (AL/CR) was 3.15 at baseline
and increased to 3.31 at the 14-year visit. In the cross-sectional
analysis, the correlation between AL and CC for the COMET myopes
at the 12-year visit was lower than for the matched non-myopes (r
=-0.57 vs.-0.76; p<0.0001). In addition, the AL/CR in myopes was
significantly higher than in non-myopes (3.30 vs. 3.00; p<0.0001).
Conclusions: These data demonstrate significant gender and ethnicity
differences in average corneal curvature as myopia progresses.
In addition, our findings suggest that as axial length continues to
increase in myopic children the cornea may be incapable of further
flattening, contributing to myopia progression.
Commercial Relationships: Mitchell Scheiman, None; Li Deng,
None; Jane E. Gwiazda, None; Qinghua Zhang, None; Ruth E.
Manny, None; Karen D. Fern, None; Eric Weissberg, None
Support: EY11756, EY11754, EY11805, EY11752, EY11740, and
EY11755
Clinical Trial: NCT00000113
The effects of the relative strength of simultaneous competing
defocus signals on emmetropization in infant rhesus monkeys
Baskar Arumugam1, 2, Li-Fang Hung1, 2, Chi-ho To3, Earl L. Smith1, 2.
1
College of Optometry, University of Houston, Houston, TX; 2Vision
CRC, Sydney, NSW, Australia; 3Center for Myopia Research School
of Optometry, Hong Kong Polytechnic University, Hung Hom, Hong
Kong.
Purpose: The aim of this study was to investigate the effect of dual
focus lenses with unequal areas devoted to competing defocus signals
on emmetropization in infant monkeys.
Methods: The subjects were infant rhesus monkeys that, beginning
at 3 weeks of age, were reared with Fresnel lenses that had unequal
areas devoted to the two power zones. The treatment lenses had
central 2 mm zones of zero power and concentric annular zones that
had 33:66 area ratios for alternating powers of +3.0 D and 0 D (n=6;
+3D/pl) or 66:33 ratios for powers of -3.0 D and 0 D (n=6; -3D/pl).
The monkeys wore the treatment lenses over both eyes continuously
until 153±3.4 days. Comparison data were obtained from monkeys
reared with either dual focus lenses with approximately equal power
areas (50:50 area ratios) or full field single vision lenses over both
eyes (FF+3 D, n=6; FF-3 D, n=6). Refractive status, corneal power
and axial dimensions were assessed every 2 weeks throughout the
lens rearing period. Control data were obtained from 33 monkeys
reared with unrestricted vision.
Results: At the end of the treatment period, the median refractive
error and average vitreous chamber depth for the +3D/pl lensreared monkeys were similar to those for animals reared with FF+3
D lenses (OD: +5.19 D vs +4.63 D, p=0.23 and 9.44±0.60 mm vs
9.58±0.32 mm, p=0.65) or similarly powered Fresnel lenses that
had equal areas devoted to each power (OD: +5.25 D, p=1.0), but
axially more hyperopic than those for control monkeys (OD: +2.50
D, p=0.0002; VC: 9.82±0.30 mm, p=0.03). On the other hand, for the
-3D/pl monkeys, refractive development was dominated by the zeropowered portions of the lenses. The median refractive error for the
-3D/pl monkeys was more hyperopic than that found in the FF-3D
monkeys (OD: +2.94 D vs -1.19 D, p=0.02), but similar to that for
monkeys reared with Fresnel lenses that had equal areas devoted to
each power (OD: +3.13 D, p=0.94) and that observed in the control
animals (p=0.24).
Conclusions: The results demonstrate that even though the least
hyperopic/most myopic power zones made up only about 1/3 of the
surface area of the treatment lenses, their associated image planes
dominated refractive development. Overall, the results indicate that
imposing relative myopic defocus over a large part of the retina
provides a strong signal for slowing eye growth.
Commercial Relationships: Baskar Arumugam, None; Li-Fang
Hung, None; Chi-ho To, Inventor (P); Earl L. Smith, Zeiss (P)
Support: NIH Grants EY03611 and EY07551; Vision CRC
Both Lens Induced Hyperopia and Recovery Increase the
Amplitude of Diurnal Rhythm in Refractive Error
Melanie C. Campbell1, 2, Kaitlin Bunghardt1, Marsha L. Kisilak1, 2,
Elizabeth L. Irving2. 1Physics & Astronomy, University of Waterloo,
Waterloo, ON, Canada; 2School of Optometry and Vision Science,
University of Waterloo, Waterloo, ON, Canada.
Purpose: We have reported a larger amplitude of circadian variation
in mean ocular refraction (MOR) in the previously goggled eye
compared with the control eye on removal of positive or negative
lenses. Here we compare the circadian rhythm present during lens
induction of hyperopia in the goggled and control eyes with that
following goggle removal.
Methods: Short term changes in MOR were measured immediately
before and after goggle removal following 6 days of emmetropization
to lens induced hyperopia. Eleven birds, unilaterally treated with
a +10D goggle on the day of hatching, were raised on a 14h/10h
light dark cycle. On day 6, beginning at 8:30 am, Hartmann-Shack
refractive error and A scan ultrasound axial length (to the retina)
were measured every 4 hours, ending on day 9. The goggle was
permanently removed after the 8:30am measurement on day 7.
MOR was analyzed for the largest common pupil. Linear variations
were subtracted and residual sinusoidal variations were fitted before
and after goggle removal. Comparisons were made to previously
presented results. Paired t tests were used, p≤0.05 for significance and
if normalcy or equal variance tests failed, a Wilcoxon Signed Rank
Test was performed.
Results: On day 6 after partial emmetropization (+5.5D), sinusoidal
amplitudes in goggled eyes vs control eyes were significantly larger
for MOR and smaller for length. This implies a difference in the
amplitude of power variation between goggled and control eyes.
Control eyes were not different from eyes of control birds on day 6.
Day 7 diurnal amplitudes for length and MOR after goggle removal
were significantly larger than before removal. The eyes’ amplitudes
no longer differed for length but MOR amplitude was larger in
previously goggled eyes than in control eyes. Acrophases on days 6
and 7 for MOR of goggled eyes were significantly clustered, while
those for control eyes were not (Rayleigh test).
Conclusions: Unlike in previously reported emmetropization to -15D
lenses, after 6 days, following incomplete emmetropization to +10D
lenses, the amplitude of circadian rhythms differed in the control
and goggled eyes. Nonetheless, following goggle removal, as for the
-15D lenses, the amplitude of oscillation of mean ocular refraction
in the treated eye increased. Circadian rhythms could provide
enhanced signals to the direction of defocus both during goggling and
following goggle removal.
Commercial Relationships: Melanie C. Campbell, None; Kaitlin
Bunghardt, None; Marsha L. Kisilak, None; Elizabeth L. Irving,
Visioneering (F)
Support: NSERC Canada
Macrophage Activation Pattern in Human Myopic Choroidal
Neovascular Membranes
Khaled Nassar, El Shaymaa El Far, Julia Luke, Matthias Luke,
Swaantje Grisanti, Salvatore Grisanti. Ophthalmology, Luebeck
University, Luebeck, Schleswig-Holstein, Germany.
Purpose: A little is known about the role of macrophages in myopic
choroidal neovascular membrane (CNV) pathogenesis. The present
study aimed to characterize the pattern of macrophage activation in
CNV caused by degenerative myopia compared to that caused by age
related macular degeneration (AMD).
Methods: 20 surgically excised CNV derived from 15 AMD (5
classic and 10 occult CNV) patient and 5 patient with degenerative
myopia were stained using CD68, CCR7 and CD163 antibodies
staining for pan macrophages, classically activated macrophage
(M1 macrophages) and alternatively activated macrophage (M2
macrophages); respectively. The immunopositive cell density was
calculated per mm2 in 400x images.
Results: All CNVs were untreated and subfoveally located.
Macrophage infiltration was higher in the myopic CNV with a cell
density of M: 268.4±117.78; M1: 137.8 ±189.5; M2: 453.6±313
cell/mm2. In classic AMD related CNV, the cell density of M
macrophages were 251.6±72.8 cell /mm2, M1 macrophages: 131.4±
94.38, and M2 macrophages: 122.8±64.10 cell/mm2. In occult AMD
related CNV, the cell density of M macrophages were 301± 159 cell
/mm2, M1 macrophages: 167 ± 90 cell/mm2 and M2:167 ± 89 cell/
mm2. Alternatively activated macrophages were the dominant type
in myopic CNV (p value: 0.043) and differ significantly from both
classic and occult AMD CNV, (p value: 0.008, 0.01) respectively.
Conclusions: Alternatively activated macrophages might play an
important role in the myopic CNV pathogenesis. The identification of
different activation patterns may be important for distinct therapeutic
strategies.
Fig. 1 Macrophage polarized activation.
A simplified view of immune and tissue-derived signals inducing
classical (M1) and alternative (M2) macrophage polarized activation.
The main functional effects on macrophage functions, molecular
markers and effector molecules are schematically represented in both
cases.
Fig. 2 Immunohistochemical detection of macrophage phenotypes
(M1 and M2).
A) Photomicrographs of myopic CNV depicting CD 163 (M2)
positive macrophages (black arrow). Vascular channels are seen
within the fibrous part of the membrane. B) Photomicrographs of
occult CNV depicting CCR7 (M1) positive macrophages (black
arrow),in close relation to RPE cells, scale bare 20mm.
Commercial Relationships: Khaled Nassar, None; El Shaymaa
El Far, None; Julia Luke, None; Matthias Luke, None; Swaantje
Grisanti, None; Salvatore Grisanti, None
Support: This study was supported in part by the “Jung-Stiftung Für
Wissenschaft und Forschung” Foundation, Hamburg; Germany. The
authors indicate no financial conflict of interest.
Eye Dimensions during Lens Induced Myopia (LIM) and
Recovery in the Chick
Zheng Shao1, 2, Kaitlin Bunghardt1, Marsha L. Kisilak1, 2, Melanie
C. Campbell1, 2. 1Physics and Astronomy, University of Waterloo,
Waterloo, ON, Canada; 2School of Optometry and Vision Science,
University of Waterloo, Waterloo, ON, Canada.
Purpose: The chick eye refocuses out of focus images, primarily
through changes in ocular growth rates. We reported changes in
eye power during recovery from lens induced myopia (LIM) in the
chick eye. Here we analyze changes in eye dimensions in LIM and
recovery.
Methods: Nine Ross Ross chicks were unilaterally goggled
(-15D) on the day of hatching. The goggle was removed on day
7. Retinoscopy and ultrasound were performed; measurements
continued up to day 10 at 10 time points. Mean ocular refraction
(MOR), axial length, the distance from the cornea to the back of the
lens (CBL), lens thickness, and vitreous chamber depth (VCD) were
measured; power changes were calculated. All data were fitted with
linear functions of age, and paired t tests were performed.
Results: During LIM, MOR of the goggled eye partially
emmetropized (-11.3 D) to the goggle by day 7, primarily due to
faster increases in VCD. The lengths and MORs predicted a -3.2 D
power difference between treated and control eyes on day 7. The lens
and CBL were significantly thicker in treated versus control eyes.
After goggle removal, axial length, CBL, and lens thickness for the
treated eye didn’t change significantly between days 7 and 8, while
VCD decreased. Dimensions continued to increase in the control
eye. Lens thickness was still significantly larger in the treated eye
on day 8. Recovery from LIM was complete by day 9 and resulting
dimensions did not differ significantly from the control eye for both
days 9 and 10. Power decreased significantly between days 7 and 8 in
the treated eye and relative to the control eye. After day 8, power of
the treated eye didn’t change significantly while power of the control
eye decreased, and they were not significantly different by day 9.
Conclusions: By day 7 in LIM, the differences in axial length and
MOR between eyes predict a slightly lower power in the goggled eye.
The thicker lens and CBL are consistent with a small power decrease.
During recovery, lens thickness and eye power, as well as length,
are significantly different between treated and control eyes. Days 7
to 9, these properties do not change significantly in the treated eye
but continue to change in the control eye. Differences between the
treated and control eyes, in power and in dimensions, in addition to
length, are present during LIM and recovery. After emmetropization
is complete in recovery, none of the properties considered differ
between the two eyes.
Commercial Relationships: Zheng Shao, None; Kaitlin
Bunghardt, None; Marsha L. Kisilak, None; Melanie C.
Campbell, None
Support: NSERC Canada
Phase-dependent effects of brief periods of myopic defocus on the
rhythms in axial length and choroid thickness in chicks
Debora L. Nickla, Rinita Zanzerkia, Pearl Thai, Kristen Totonelly.
Biosciences, New England College of Optometry, Boston, MA.
Purpose: We have shown that the eye growth inhibition caused by
2-hr periods of myopic defocus is more effective when given during
the mid-day than during the mid-night (Nickla & Totonelly, ARVO
2013). In this study we examined the effects of myopic defocus given
at 4 times of day on the diurnal rhythms in eye length and choroid
thickness.
Methods: 12-d-old chicks wore monocular +10 D lenses for 2 hr
periods at 5:30 am (“dawn”; n=11), 12:00 pm (“day”; n=8), 7:30 pm
(“dusk”; n=11) or 12:00 am (“night”; n=6) for 5 d. Lights were on
from 7:30 am-7:30 pm. Eyes were measured using ultrasound at the
start, and at 6-hr intervals over 24 hrs on the last day (12pm, 6pm,
12am, 6am, 12pm). Refractions (RE) were measured on a Hartinger’s
refractometer. To determine phase and amplitude, data from each
eye was fit with a sine wave. Circular statistics were used for
comparisons of phase. ANOVAs and Bonferroni post-hoc tests were
used to determine between-group significance for growth changes.
Results: Defocus during the day was more effective at inhibiting eye
growth than at dawn or night (change/5d, X-C: ANOVA p=0.014;
-174 mm vs -84 mm and -82 mm; p=0.042, p=0.075 respectively; RE:
3.8 D vs 1.1 and 2.8 D; p<0.05). This was associated with a phasedelay in the rhythm in axial length compared to normal (6:30 pm
vs 2:45 pm; p<0.05; 1-tailed t-test). By contrast, defocus at night
caused a phase-delay in the rhythm in choroid thickness (compared
to dusk and day: 3:15 am vs 12:45 am (p=0.05) and 1:00 am (p<0.05,
1-tailed t-test)). It also abolished the diurnal rhythm in axial length
due to an “acute” growth stimulation from 12 am to 6 am (night vs
day, dawn and dusk, respectively: 119 mm vs -18 mm, -15 mm, 10
mm; ANOVA p<0.001; p<0.01). This effect did not occur in eyes
receiving “normal” vision at night (27 vs 119 mm; p=0.005), in which
growth was inhibited (629 vs 774 mm/7d; p<0.0001). In exp. eyes,
the amplitude of the choroid rhythm was larger than controls in 3
groups (day: 110 vs 51 mm; night: 91 vs 46 mm; dawn: 131 vs 69 mm;
p<0.05).
Conclusions: The greater efficacy of myopic defocus at mid-day is
associated with a phase shift in the axial length rhythm, moving it
towards the choroid rhythm. The lesser efficacy of defocus at night
results from an acute stimulation of eye growth. These results have
implications for future behavioral therapies involving light exposure
to prevent myopia.
Commercial Relationships: Debora L. Nickla, None; Rinita
Zanzerkia, None; Pearl Thai, None; Kristen Totonelly, None
Support: NIH-EY013636
Predicting refractive error from ocular biometrics using
structural equation modeling
Christopher A. Clark, Ann E. Elsner, Benjamin J. Konynenbelt.
School of Optometry, University of Indiana, Bloomington, IN.
Purpose: Previous work has shown that retinal differences may exist
due to refractive error. As an example, total retinal thickness has been
shown to be relatively thicker centrally and thinner peripherally for
myopes compared to emmetropes. These differences may be due
to effects from axial elongation or potential variables influencing
refractive development. If these changes are correct, they should be
able to predict refractive error in subjects.
Methods: Eighty subjects had a battery of tests performed including
axial length, corneal topography, anterior chamber depth, peripheral
refraction, peripheral partial coherence interferometry, and SD OCT
for retinal thickness. The group was randomly split into two groups
of forty subjects, one for model development and the other for model
testing. Two designs were developed to predict central refractive
error. The first group was the complete model using all available
data. The second model was completed using only retinal thickness
changes including thicknesses from the total retina (TRT), outer
nuclear layer (ONL), outer plexiform, inner nuclear layer (INL),
and the inner plexiform layer/ganglion cell layer. The second model
had no data from axial length, corneal topography, etc. Structured
equation modeling was done through SPSS (IBM, Endicott, NY.)
Results: Structural equation modeling using retinal layer thickness
only to predict refractive error had an R2 = 0.273, P = 0.008. Layers
contributing significantly to the model included the TRT, INL and
ONL both centrally and peripherally. Using the full model, including
the axial length, the model improved R2 = 0.698, P = 0.001. As
expected, axial length was the primary contributor to the full model.
Conclusions: Differences in retinal thickness can be used to predict
refractive error. This suggests that these differences are associated
with refractive error and are real changes being detected. It appears
like both central and peripheral retinal thickness differences may be
important. Longitudinal work is needed to determine whether these
differences are due to changes in refraction or if they may directly be
influencing refractive development.
Commercial Relationships: Christopher A. Clark, None; Ann E.
Elsner, None; Benjamin J. Konynenbelt, None
Support: EY022064
Quality of life of patients suffering from pathological myopia:
overview of their social and emotional environment
David Gaucher1, 2, Claire Chartier3, Michel Weber4, Francois
Malecaze5, Salomon Y. Cohen6, Eric H. Souied7, Nicolas Leveziel8.
1
NHC, University Hospital, Strasbourg, France; 2Strasbourg
university, Strasbourg, France; 3Novartis Pharma, Rueil Malmaison,
France; 4Ophthamic department, University hospital of Nantes,
Nantes, France; 5Toulouse University, Toulouse, France; 6CIL, Paris,
France; 7Paris Est University, Creteil, France; 8Poitiers Université,
Poitiers, France.
Purpose: Pathological myopia may affect 2 to 4% of the French adult
population.
The aim of this survey is to better understand patients suffering
from pathological myopia through their experiences, the impact of
the condition on their daily lives, their attitudes and beliefs. This
exploration will enable us to better meet the needs and expectations
of those suffering from this debilitating condition, which we assume
has a significant impact on quality of life and social integration.
Methods: This qualitative survey was conducted in France from
November to December 2013. Twenty patients suffering from
pathological myopia, i.e. patients with myopia over -8 dioptres
were interviewed. The sample is representatively split by sociodemographic criteria such as gender, age and region. A quantitative
survey in 75 patients is planned in January 2014 to assess the
impact of complications in the personal and professional life. Semistructured interviews lasting approximately one hour allowed patients
to speak freely about their experiences. A discussion guide has been
devised, covering the daily lives of patients as well as their care and
the history of their condition.
Results: The first inference is that pathological myopia is a “nonvisible” disease with professional and personal impact, which is
underestimated in circles. The second is the difficult everyday reality
faced by myopic patients, owing to a lack of understanding on the
part of the public and the depreciation of the skills and abilities of
those suffering from pathological myopia. Such are the inferences
drawn from the first interviews conducted. Consequently, patients
are using strategies to conceal the disease in professional and
personal circles, so as to avoid isolation, and rejection and stigma
exist. Possible adaptation strategies are emerging as a result of the
initial interviews, such as research by key resource persons and
development of other senses, such as touch.
Conclusions: This is the first in-depth study into the daily lives
of people suffering from pathological myopia. It aimed to analyse
their social and emotional environment. The results allow health
professionals and general public to be alerted to the emotional and
social difficulties experienced by these patients, which are perhaps
underestimated by ophthalmologists.
Commercial Relationships: David Gaucher, NOVARTIS (C);
Claire Chartier, Novartis Pharma (E); Michel Weber, Novartis (C);
Francois Malecaze, Novartis (C); Salomon Y. Cohen, Allergan (C),
Bausch and Lomb (C), Bayer (C), Novartis (C), Th√©a (C); Eric
H. Souied, Novartis (C); Nicolas Leveziel, Bayer (C), Novartis (C),
Th√©a (C)
Right eyes are longer than left eyes: axial length findings from a
large cataract cohort with consistent refractive findings from a
large twin cohort
Omar A. Mahroo1, 2, Pirro G. Hysi3, Obeda Kailani4, Juliet
Thompson4, Christopher J. Hammond1, 3. 1Ophthalmology,
King’s College London, London, United Kingdom; 2Physiology,
Development and Neuroscience, University of Cambridge,
Cambridge, United Kingdom; 3Twin Research and Genetic
Epidemiology, King’s College London, London, United Kingdom;
4
West Kent Eye Centre, Princess Royal University Hospital, London,
United Kingdom.
Purpose: Small inter-ocular differences exist in the incidence of
certain conditions; for example, retinal detachments affect right eyes
slightly more frequently. This cross-sectional study explored, in two
large cohorts, whether right and left eyes differ in terms of axial
length or myopia (both linked risk factors for retinal detachment).
Methods: For the cataract cohort, axial lengths previously measured
for 12,766 eyes of 8,195 consecutive patients over a 6.5 year period
were analysed. Right and left eyes were compared (t test: paired
for patients with measurements for both eyes; unpaired for those
with measurements available for one eye). For the twin cohort,
refractive error was compared between right and left eyes (paired t
test) for 5,755 twin subjects from 3,199 families from the TwinsUK
database. To adjust for relatedness within families, re-sampling
was performed with one random member of each family (running
10,000 permutations). For 1,186 twins, eye dominance data were also
available.
Results: For 4,571 patients for whom axial lengths were available
for both eyes, mean (SD) axial lengths were 23.55 (1.40) mm and
23.50 (1.40) mm for right and left eyes respectively (p=4.7x10-20).
For 3,624 patients undergoing unilateral surgery, mean axial lengths
were 23.56 (1.28 mm) and 23.47 (1.23) mm respectively (p=0.04).
For twin subjects, right eyes were significantly more myopic than left
eyes (p = 0.04). 65% of twin subjects for whom eye dominance data
were also available were right-eye dominant. Although right eyes
were on average more myopic for right eye dominant subjects, the
laterality was reversed for left eye dominant subjects, but differences
did not reach significance.
Conclusions: Right eyes appear to be, on average, slightly, but
significantly, longer than left eyes. Longer eyes tend to be more
myopic and our refractive data were consistent with this. This
represents a novel finding and, in terms of clinical significance,
may explain the slight laterality imbalance in retinal detachments.
An understanding of mechanisms underlying small differences
in development of the two eyes would shed important light on
the development of myopia; our finding that differences might
be reversed for left eye dominant subjects suggests an interesting
interaction with mechanisms driving eye dominance.
Commercial Relationships: Omar A. Mahroo, None; Pirro G.
Hysi, None; Obeda Kailani, None; Juliet Thompson, None;
Christopher J. Hammond, None
Support: Fight for Sight UK grants (OAM, PH); Wellcome Trust
(CJH). TwinsUK also receives support from the National Institute for
Health Research (NIHR) BioResource Clinical Research Facility and
Biomedical Research Centre based at Guy’s and St Thomas’ NHS
Foundation Trust and King’s College London.
Replication analysis of myopia-associated genes in Japanese
cohort and in highly myopic patients using genome-wide
association study
Munemitsu Yoshikawa1, Kenji Yamashiro1, Masahiro Miyake1, 2, Maho
Oishi1, Yugo Kimura1, Kyoko Kumagai1, Yumiko Akagi-Kurashige1,
2
, Hideo Nakanishi1, 2, Norimoto Gotoh1, 2, Nagahisa Yoshimura1.
1
Department of Ophthalmology and Visual Sciences, Kyoto
University Graduate School of Medicine, Kyoto, Japan; 2Center for
Genomic Medicine/Inserm U.852, Kyoto University Graduate School
of Medicine, Kyoto, Japan.
Purpose: Recently, two large consortium for myopia, Consortium for
Refractive Error and Myopia (CREAM) and 23andMe, reported the
results of genome-wide association analysis (GWAS) on spherical
equivalent (SE) refractive error and myopia age of onset and
found that 29 SNPs and 35 SNPs were associated with myopia in
Caucasians, respectively. In this study, we analyzed the associations
of these myopia-associated genetic loci on axial length (AL) and SE
in Japanese.
Methods: We included 3,248 Japanese healthy volunteers from
the Nagahama Study and 500 Japanese unrelated highly myopic
(AL^26mm) patients from Kyoto University Hospital. From
the results of above two GWAS, 50 genes were seemed to have
associations with myopia. To investigate and replicate these
associations in Japanese, we conducted 2 quantitative trait locus
(QTL) analyses using the Nagahama cohort and 1 GWAS using both
cohorts. The QTL analyses were conducted on AL and SE and GWAS
was conducted on the existence of high myipia. We genotyped
3,248 healthy volunteers using either Illumina OmniExpress2.5M or
HumanHap610K, and GWAS was conducted on the existence of high
myopia using either HumanHap550K or HumanHap660K. For each
analyses, VEGAS (Versatile Gene-based Association Study) program
were applied to perform gene-based association tests on myopiarelated 50 genes.
Results: In our 2 QTL analyses and 1 GWAS, only 4 genes
(RASGRF1, BMP4, GJD2, and CACNA1D) showed statistical
significance in all three analyses and 7 genes (B4GALNT2, SH3GL2,
SETMAR, ADAMTSL1, BICC1, SFRP1, and TOX) showed
statistical significance in two of the three analyses, whereas 27 genes
did not show significance in any of our study. In GWAS on high
myopia, 17 genes showed statistical significance. However, only 6
genes showed statistical significance in QTL analyses on AL and SE
among these 17 genes, respectively. On the other hand, QTL analyses
on AL and SE showed statistically significant 11 genes each, and as
many as 8 genes had statistical significance among these 11 genes in
common.
Conclusions: For previously reported myopia-related genes, we
showed the results of gene-based tests on AL, SE, and existence of
high myopia in our study cohort in Japan. Our data suggests that the
genetic background of Caucasians and that of Japanese for myopia,
and that of myopia and high myopia, could be different.
Commercial Relationships: Munemitsu Yoshikawa, None; Kenji
Yamashiro, None; Masahiro Miyake, None; Maho Oishi, None;
Yugo Kimura, None; Kyoko Kumagai, None; Yumiko AkagiKurashige, None; Hideo Nakanishi, None; Norimoto Gotoh, None;
Nagahisa Yoshimura, None
Guinea pig optic nerve head
Lisa A. Ostrin1, Christine Wildsoet2. 1College of Optometry,
University of Houston, Houston, TX; 2School of Optometry,
University of California Berkeley, Berkeley, CA.
Purpose: The guinea pig is becoming an increasingly popular model
of human myopia. The goal of this study was to characterize and
compare the optic nerve head (ONH) and sclera of the guinea pig
with other animal models of human myopia and glaucoma. Myopia
carries an increased risk of glaucoma. The lamina cribrosa (LC) of
the ONH has been implicated as a site of axon damage in glaucoma
and in many species, is continuous with the sclera. It is plausible that
it is also abnormal in myopia.
Methods: Pigmented and albino guinea pig eyes (n=18, ages
2-3 months) were enucleated and the ONH and surrounding
sclera dissected. After formalin fixation, four eyes were paraffin
embedded for hematoxylin and eosin (H&E) staining. Four eyes
were cryoprotected and frozen for immunostaining. Primary
antibodies included collagen types I-V, elastin, fibronectin and glial
fibrillary acidic protein (GFAP). Remaining eyes were fixed in 2%
gluteraldehyde. Two ONHs were stained with osmium tetroxide
(OsO4) and embedded in resin for transmission electron microscopy
(TEM). Eight ONHs underwent alkali maceration with 10% NaOH
to remove cellular tissue, before staining with OsO4 and dehydration
for scanning electron microscopy (SEM) to evaluate the fiber
organization.
Results: H&E-stained sections showed retinal ganglion cell axons
organizing into fascicles in the prelaminar and laminar region.
Immunostained sections revealed collagen types I, III, IV and V in
the ONH, as well as elastin, GFAP and fibronectin. SEM revealed
the scleral canal to have a well-defined LC with radially-oriented
collagen beams, with spaces between presumably occupied by
ganglion cell axons and other support cells in the intact ONH. TEM
images confirmed the latter, revealing collagen fibrils surrounding
non-myelinated nerve bundles in the LC region, with myelination
and decreased collagen posterior to the LC. Adjacent sclera was
composed of crimped collagen fibers in a crisscross arrangement. The
sclera and LC were qualitatively similar in structure in pigmented and
albino guinea pigs.
Conclusions: Unlike mice, another rodent model for myopia and
glaucoma, the ONH of guinea pig includes a radially-organized,
collagen-based LC, similar to that of the tree shrew, but different
from primates in which organization is a porous collagenous
meshwork. Nonetheless, the overall structural similarity of the LC
suggests that the guinea pig is a suitable model for investigating the
relationship between myopia and glaucoma.
Commercial Relationships: Lisa A. Ostrin, None; Christine
Wildsoet, None
Support: NIH 5K08 EY022696, NIH R01 EY12392
Spectral-domain optical coherence tomography imaging
of the retinal pigment epithelium in myopic choroidal
neovascularization
Laura Dell’Arti1, 2, Diego Vezzola1, Giulio Barteselli1, 3, Chiara
Mapelli2, Eleonora Benatti1, 2, Francesco Viola1, 2, Roberto Ratiglia1, 2.
1
University of Milan, Milan, Italy; 2Ophthalmology, clinical sciences
and community health, Fondazione IRCCS Ca Granda Ospedale
Maggiore Policlinico, Milan, Italy; 3Ophthalmology, Shiley Eye
Center UCSD, San Diego, CA.
Purpose: To analyze the retinal pigment epithelium (RPE)
appearance of inactive myopic choroidal neovascularization (CNV)
with spectral-domain optical coherence tomography (SD-OCT) and
to evaluate its association with the lesion size and the treatment
duration.
Methods: We retrospective reviewed imaging studies of eyes with
inactive myopic CNV after successful treatment. Imaging included
infrared reflectance (IR), fundus autofluorescence (FAF), fluorescein
angiography (FA), indocyanine green angiography, and spectraldomain optical coherence tomography (SD-OCT). Patients were
divided into two groups. Group 1 showed uniformity of the RPE
(defined as continuous and highly reflective layer on SD-OCT)
over the CNV, while group 2 showed an irregular RPE (defined as
disrupted and poorly reflective layer on SD-OCT). The presence
of perilesional hyper-reflective ring (PHR) in IR imaging was
determined; the major diameter (MajD), the minor diameter (MinD)
and the area of the lesion (A2) in early FA images were measured
using the built-in caliper of the SD-OCT device. The number of
treatments performed before CNV stabilization was assessed for
every patient.
Results: Eighty-one eyes of 72 patients were included. Group 1 and
group 2 included 27 and 54 eyes, respectively. MajD, MinD and
A2 of the CNVs were significantly lower in group 1 than in group 2
(p<0.001). Group 2 patients needed greater number of treatments than
group 1 patients (p<0.01). The presence of a PHR was detected more
frequently in group 1 than in group 2 (p<0.01). Binary regression
analysis showed that the only predictor for the presence of a uniform
RPE after successful treatment was the size of the CNV. Conditional
selection of variables showed that the best regression model for the
presence of a regular RPE after successful treatment included size of
the lesion and presence of the PHR.
Conclusions: The uniformity of RPE and the presence of PHR turned
out to be good prognostic factors for myopic CNVs. Size of myopic
CNV influences the development of a uniform RPE over the lesion
and, consequently, can condition the prognosis of the disease.
Commercial Relationships: Laura Dell’Arti, None; Diego Vezzola,
None; Giulio Barteselli, None; Chiara Mapelli, None; Eleonora
Benatti, None; Francesco Viola, None; Roberto Ratiglia, None
Contribution of body length on axial length during normal
eye development in C57BL/6J and 129S1/SvJ wild-type mouse
strains.
Ranjay Chakraborty1, 2, Hanna Park1, 2, Christopher C. Tan1, 2, Megan
Prunty1, 2, Machelle T. Pardue2, 1. 1Ophthalmology, Emory University
School of Medicine, Atlanta, GA; 2Rehab R&D Center of Excellence,
Atlanta VA Medical Center, Atlanta, GA.
Purpose: To determine the influence of body length on eye length
in two different wild-type (WT) mouse strains during normal eye
development.
Methods: Measurements of body length, axial length, and refraction
were retrospectively analyzed for two different WT mouse strains:
129S1/SvJ (n=6) and C57BL/6J (n=8) from 4 to 16 weeks of age.
Body length, from the tip of the nose to the base of the tail were taken
from digital images using image analysis software (ImageJ). Axial
length (using an average refractive index of 1.433 for the entire eye)
and refractions were measured using a 1310 nm spectral domain
OCT (Bioptigen, Inc.) and an infrared photorefractor, respectively.
To elucidate the effect of body length on eye length between strains,
axial length was divided by the body length (eye/body length ratio)
for all animals.
Results: Body length increased significantly with age for both
strains (p<0.001). C57BL/6J mice had significantly larger body
length (average at 10 weeks, 8.60 ± 0.21 cm) compared to 129S1/
SvJ (8.31± 0.13 cm) mice (p=0.011). Axial length also increased
significantly during the development period (p<0.001). However, it
was not found to be significantly different between the two strains
across age (average at 10 weeks, 3.24 ± 0.07 and 3.26 ± 0.05 mm for
C57BL/6J and 129S1/SvJ respectively). After normalizing to body
length, 129S1/SvJ exhibited a significantly larger eye/body length
ratio (average at 10 weeks, 0.039 ± 0.001) compared to C57BL/6J
(average at 10 weeks, 0.037 ± 0.001) at all ages (p=0.025). Similar
to body length, crystalline lens thickness normalized to body length
significantly changed across both strains (129S1/SvJ > C57BL/6J,
p<0.05). Four week old 129S1/SvJ (-4.92 ± 2.37 D) mice had
significantly greater myopic refractions than C57BL/6J mice (+3.80
± 1.45 D; p<0.001), but both strains reached similar hyperopic
refractions by 14 weeks of age. A significant negative association
was observed between the eye/body length ratio and refraction for all
mice across both strains (slope = -0.0001, r 2 = 0.16, p<0.001).
Conclusions: Body length significantly influences axial length in
different mouse strains during development. The ratio of eye length
to body length is important to refractive development, but not the
only predictor of refractive error in mice.
Commercial Relationships: Ranjay Chakraborty, None; Hanna
Park, None; Christopher C. Tan, None; Megan Prunty, None;
Machelle T. Pardue, None
Support: NIH EY016435 (MTP), NIH P30 EY006360, Research to
Prevent Blindness, and the Department of Veterans Affairs
423 Anatomy Development
Wednesday, May 07, 2014 8:30 AM–10:15 AM
Program #/Board # Range: 4439–4472/D0099–D0132
Contributing Section(s): Retinal Cell Biology, Retina, Visual
Psychophysics / Physiological Optics
Program Number: 4439 Poster Board Number: D0099
Retinoic Acid Regulates Neural Crest Migration and
Proliferation in Eye Development
Bahaar Chawla, Brenda L. Bohnsack. University of Michigan, Ann
Arbor, MI.
Purpose: Retinoic acid (RA) is an essential morphogen that is
required for eye development. We previously demonstrated that RA
regulates formation of neural crest (NC)-derived ocular structures in
zebrafish. In the current studies, we further elucidated the role of RA
in regulating NC migration and proliferation.
Methods: Morpholino oligonucleotides (MO) were used in
combination with exogenous regulators of retinoic acid (RA).
Time-lapse imaging of transgenic zebrafish expressing GFP in NC
assessed migration. Embryos were harvested for in situ hybridization,
immunostaining, TUNEL assay, and histology.
Results: Genetic disruption of RA synthesis (Raldh2 MO) or
degradation (Cyp26c1 MO) inhibited early migration from 12-24
hours post fertilization (hpf) of the ventral wave of NC which gives
rise to the jaw and periocular mesenchyme. NC passage through the
ocular fissures into the developing eye (24-60hpf) was also disrupted
in both Raldh2 and Cyp26c1 knockdown embryos. To determine if
this later stage of NC migration was inherently dependent on RA
or if the lack of early migration disrupted further NC development,
pharmacologic treatment with all-trans RA or the aldehdehyde
dehydrogenase inhibitor (DEAB) was initiated after completion
of early NC migration (24hpf). Neither increased (all-trans RA)
nor decreased (DEAB) RA levels inhibited NC migration through
the ocular fissures demonstrating that RA was not intrinsically
required for this later stage of migration. On the other hand, tight
control of RA was necessary for NC organization around the lens
and iris stromal formation at 48-60hpf. Since RA was not essential
for migration from 24-48hpf, additional studies determined if RA
regulated proliferation and survival. All-trans RA significantly
decreased NC and retinal proliferation compared to controls while
DEAB significantly increased the percent of cells in S-phase. In
addition, increased or decreased RA levels resulted in increased NC
and retinal apoptosis in the developing eye.
Conclusions: Tight control of RA was essential for specific cellular
functions during different stages of eye development including early
NC migration, later NC and retinal proliferation and survival, and
finally NC organization and differentiation.
Commercial Relationships: Bahaar Chawla, None; Brenda L.
Bohnsack, None
Support: K08EY022912-01, Research to Prevent Blindness Career
Development Award
Mitochondrial Dysfunctions in Extraocular Muscles and Optic
Nerves of Moebius Syndrome
Mones S. Abu-Asab, Yujuan Wang, Chi-Chao Chan. Lab of Immunol/
Sect of Immunopath, National Eye Institute, Bethesda, MD.
Purpose: Moebius syndrome is an extremely rare congenital
neurological disorder of unknown etiology that primarily affects the
6th (abducens) and 7th (facial) cranial nerves causing motionless
face and lateral immobility of the eyes. We have undertaken an
ultrastructural approach to examine a case of Moebius syndrome in
order to uncover the affected cellular organelles within the muscles
and nerves as well as to infer their related biochemical pathways.
Methods: A postmortem ultrastructural examination was performed
on 12 extraocular muscles, two optic nerves, and a superior oblique
nerve of a Moebius patient. The patient was a 64 year old Caucasian
woman with bilateral facial nerve palsy and partial abducens palsy.
During the last 6 years of her life, she had bilateral ectropion, mapdot-fingerprint dystrophy, cataract, keratitis, lagophthalmos, and
poor vision. The tissues were prepared for transmission electron
microscopy.
Results: All muscle fibers were punctuated with lipid droplets
of various sizes. Mitochondria were abnormal in all specimens
showing various degrees of edema, enlargement, dislocation,
and disintegration of internal membrane and cristae; some
mitochondria had internal lipid accumulation. Three different types
of mitochondrial dysfunctions were identified in the extraocular
muscles and nerves. Mitochondria with internal lipid accumulation
pointed out to a dysfunctional beta-oxidation pathway, the main fat
breakdown process of the cell. There were giant mitochondria that
have been transformed into large aqueous storage vesicle, which was
indicative of a dysfunctionality of a tricarboxylic acid (TCA) cycle
enzyme. The third type of dysfunction is related to the second and
was inferred from the numerous fat droplets within the muscle fibers.
Accumulation of lipid droplets within the cytoplasm results from the
excessive production of acetyl-Co-A in the mitochondria and their
inability to metabolize it through the TCA cycle. Excess of acetylCo-A exits the mitochondria and is utilized in the cytoplasm for fatty
acid synthesis.
Conclusions: The extraocular muscles as well as optic and lateral
nerves of the Moebius patient exhibited three types of mitochondrial
dysfunctions that occurred within the TCA cycle and beta-oxidation
pathway. Such biochemical aberrations within the mitochondria
suggest the possibility that Moebius syndrome etiology may be
mitochondrial in origin.
Commercial Relationships: Mones S. Abu-Asab, None; Yujuan
Wang, None; Chi-Chao Chan, None
Support: N/A
Clinical Trial: N/A
Rat Eyelids, Meibomian Glands, and Glands of Zeis Have
Gastrin Receptors
Mortimer Lorber. Pharmacology & Physiology, Georgetown Univ
School of Medicine, Bethesda, MD.
Purpose: In ARVO 2013, we showed that rat exorbital lacrimal
glands have gastrin receptors. Might this also be true for the contents
of their eyelids?
Methods: Upper llids of 3 male and 2 female rats were excised and
formalin-fixed. Immunohistochemistry employed a rabbit polyclonal
antibody from Alamone Labs in Jerusalem against the gastrin
receptor, CCKBR. This antibody reacted with rat tissues. A 1:500
dilution in buffered saline, pH 7.4, was used. The positive control
was the rat stomach whose parietal cells were stained. The negative
control omitted the primary antibody.
Results: All major eyelid tissues stain. The nuclei of the entire
palpebral conjunctiva stain intensely; their cytoplasm slightly
to moderately. Goblet cell nuclei stain intensely but their mucus
is unstained. Nuclei of the eyelid epidermis stain from slight to
moderately and their cytoplasm moderately. Nuclei of the meibomian
glands generally do not stain, but some are dark. Their cytoplasm
stains slightly to moderately. The Glands of Zeis are near the
eyelash follicles whose nuclei stain intensely, as do the basal and
lower stratum granulosum cells of the epidermis whose cytoplasm
stains prominently. The nuclei of the Glands of Zeis stain slightly or
moderately; their cytoplasm slightly. Myoepithelial cell nuclei stain
less than their cytoplasm. Nuclei of CT fibroblasts stain intensely, but
their cytoplasm only slightly.
Conclusions: Immunoreactive gastrin is abundant in the rat eyelid.
Its presence in rat lacrimal glands which provide aqueous fluid,
and in meibomian glands and glands of Zeis which provide lipids
indicates that gastrin is needed for both major components of tears.
However, gastrin’s action or mechanism of action may differ among
these glands. Gastrin is present throughout the duct system of the
exorbital lacrimal gland, but is in the acini of the eyelid glands whose
duct systems appear shorter than that of the rat exorbital lacrimal
gland.
Commercial Relationships: Mortimer Lorber, None
Observation of Whole-mount Meibomian Glands from Cadaveric
Eyelids using a Fructose-based Optical Clearing Agent
Masataka Ito1, Rika Shirakawa2, Reiko Arita4, Yoko Karasawa3,
Junko Imaki1, Shiro Amano2, Ysushi Kobayashi1, Masaru Takeuchi3.
1
Developmental Anatomy, National Defense Medical College,
Tokorozawa, Japan; 2Ophthalmology, Tokyo University School of
Medicine, Tokyo, Japan; 3Ophthalmology, National Defense Medical
College, Tokorozawa, Japan; 4Ophthalmology, Itoh Clinic, Saitama,
Japan.
Purpose: The chronic dysfunction of meibomian glands known as
meibomian glands dysfunction (MGD) is one of the causes of dry eye
syndrome and is known to be related to aging. Meibomian glands are
clinically observed using meibography, however, those in formalinfixed cadavers are difficult to be observed in whole-mount because of
the stiffness and decreased transparency of the eyelids.
A recently-developed method using fructose-based optical clearing
agent (SeeDB method) enabled observation of the deep structures
of the brain in formalin-fixed experimental animals. We applied this
method to human meibomian glands. The purpose of this study was
to establish the methods to observe the morphology of whole-mount
meibomian glands of the formalin-fixed cadaveric eyelids.
Methods: All cadavers were perfused by 3.3% formalin in 67%
ethanol within 4 days after death and stored in the same fixative more
than 6 months. 13 upper eyelids were dissected from 9 cadavers (69to 98-year-old, male and female). After skin and subcutaneous tissues
were manually removed, the samples were serially incubated in 30%,
60% and 80% (wt/vol) fructose each for 12–24h at room temperature.
Samples were then incubated in SeeDB (80.2% wt/wt fructose) for
24h at 37°C.
Between each step, samples were observed by a dissecting
microscope under transmitted light. Photographs of the glands were
taken by a digital camera and the contrast between the glands and
surrounding connective tissues were compared. In some cases,
observation with meibography was performed.
Results: In cadaveric eyelids, tarsal plates were best cleared by
overnight incubation with 80% fructose solution, but 24h incubation
in SeeDB decreased the contrast of the glands. Observation with
meibography could be done after clearing process with fructose, and
the degeneration of the gland could be scored (meiboscore) in all
cases tested.
Conclusions: The fructose-based optical clearing agent successfully
cleared the palpebral tissues and enhanced the contrast of meibomian
glands in cadavers.
This method could be applied for the evaluation of the morphology of
meibomian glands in formalin-fixed human eyelids.
Commercial Relationships: Masataka Ito, None; Rika Shirakawa,
None; Reiko Arita, None; Yoko Karasawa, None; Junko Imaki,
None; Shiro Amano, None; Ysushi Kobayashi, None; Masaru
Takeuchi, None
Support: NDMC Grant
Comparative Anatomy of Avian Ciliary Muscles in Owls
Charles S. Schobert, Richard R. Dubielzig. Dept of Pathobiological
Sciences, UW-Madison School of Vet Med, Madison, WI.
Purpose: Compare the anatomy of avian ciliary muscles with
particular attention to the order Strigiformes and the Tawny
Frogmouth of the order Podargiformes and speculate how the
anatomy might reflect the environmental and behavioral niche.
Methods: Cases were selected from the archive of the Comparative
Ocular Pathology Laboratory of Wisconsin (COPLOW). Five-micron
H & E sections were examined. Crampton’s muscle and Brücke’s
muscle were identified. Length and area of the muscles were
measured. We could not reliably identify Müller’s muscle.
Results: In the majority of birds examined both muscles were present
and no clear pattern of relative size could be detected. We measured
length and area of Crampton’s and Brücke’s in a variety of species
and discovered that even within the same order there was wide
variation.
In four species of owls (Strigiformes, family Strigidae) examined, a
Crampton’s muscle is evident but no Brücke’s muscle. In contrast, a
fifth owl species examined was the Barn owl (Tyta alba), which has a
vestigial Brücke’s muscle in addition to Crampton’s. Barn owls are in
the family Tytonidae, while all other “typical” owls are in the family
Strigidae.
We also examined the Tawny Frogmouth (Podargus
strigioides), a nocturnal raptor, of the order Podargiformes. It has a
Crampton’s muscle as well as a vestigial Brücke’s.
Conclusions: Owls do not have a Brücke’s muscle and, therefore
lack lens accommodation, relying on corneal accommodation.
Corneal accommodation may be accomplished by contraction
of Crampton’s muscle, resulting in an increased curvature of the
axial cornea. We speculate that the lack of Brücke’s might be a
consequence of the relatively round lens, relatively large globes, or a
relatively large scleral ossicle. The Barn Owl and Frogmouth have a
vestigial Brücke’s. These birds are closely related to the true owls, are
also nocturnal predators and also have a relatively round lens and a
robust ossicle.
Commercial Relationships: Charles S. Schobert, None; Richard
R. Dubielzig, None
Comparative Anatomy of Avian Ciliary Muscles Among
Underwater Sight Hunters
Richard R. Dubielzig, Kari Musgrave, Charles S. Schobert. Pathobiol
Sciences, Univ of Wisconsin-Madison, Madison, WI.
Purpose: Compare the anatomy of avian ciliary muscles in birds with
attention to birds that use their eyes for underwater hunting and how
that ocular anatomy reflects the environmental and behavioral niche.
Methods: Cases were selected from the archive of the Comparative
Ocular Pathology Laboratory of Wisconsin (COPLOW). Five micron
H & E sections were examined. Crampton’s muscle and Brücke’s
muscle were identified. Length and area of the muscles were
measured. We could not reliably identify Müller’s muscle.
Results: Crampton’s muscle was absent in 5 species of penguins.
(Sphenisciformes), cormorants (Pelecaniformes), loons
(Gaviiformes), and puffins (Charadriiformes). However, a robust
Brücke’s muscle was present in all these underwater sight hunters.
In the majority of birds examined both muscles were present and no
clear pattern of relative size could be detected.
Conclusions: These results suggest that birds that hunt underwater
do not have corneal accommodation. These birds also have relatively
flat corneas making the ocular optics nearly the same above and
below water. We speculate the flat corneal is suspended from limbal
sclera that is uniquely rigid and these two features make corneal
accommodation impossible, hence Crampton’s muscle is not needed.
These underwater sight hunters are not closely related species. Some
of these birds have close relatives with a terrestrial life-style that have
a robust Crampton’s. This supports the conclusion that absence of
Crampton’s in these birds is an example of convergent evolution.
Commercial Relationships: Richard R. Dubielzig, None; Kari
Musgrave, None; Charles S. Schobert, None
Anatomical Manifestations of Primary Blast Ocular Trauma
Observed in an Ex Vivo Porcine Model
William E. Sponsel1, 2, Matthew A. Reilly1, Brian J. Lund3, Walter
Gray4, Richard Watson1, 5, Sylvia L. Groth6, Randolph D. Glickman7,
1
, Kimberly Thoe8. 1Biomedical Engineering, UTSA, San Antonio,
TX; 2Visual Science, Rosenberg School of Optometry; UIW, San
Antonio, TX; 3U.S. Army Institute of Surgical Research, JBSA Fort
Sam Houston, San Antonio, TX; 4Geological Sciences, UTSA, San
Antonio, TX; 5Biodynamic Research Corporation, San Antonio,
TX; 6Ophthalmology, Presence St. Francis Hospital, Evanston, IL;
7
Ophthalmology, UTHSCSA, San Antonio, TX; 8Glaucoma Service,
WESMDPA, San Antonio, TX.
Purpose: To qualitatively describe the anatomic features of primary
ocular blast injury observed using an in vitro porcine eye model.
Porcine eyes were exposed to various levels of blast energy to
determine the optimal conditions for future testing.
Methods: Fifty-three (53) enucleated porcine eyes were studied;
13 control eyes and 40 test eyes exposed to a range of blast
overpressure levels. Eyes were pre-assessed with B-scan and UBM
ultrasonography, photographed, mounted in gelatin within acrylic
orbits, and monitored with high-speed videography during blasttube impulse exposure. Post-impact photography, ultrasonography,
and histopathology were performed and ocular damage was
assessed. Injury scoring in each zone followed a new clinically
relevant Composite Injury Scale addressing the practical needs of
those engaged in the treatment of ocular injury or development of
protective eyewear. Injury scores were ascribed on the basis of a
stepwise algorithm to integrate the structural damage in each of the
Zones 1-3 as defined by Pieramici et al (AJO 1997), where Zone 1 is
the external ocular surface, Zone 2 the anterior chamber, and Zone 3
the internal posterior segment.
Results: Strong evidence for primary blast injury was obtained.
Common findings included angle recession, internal scleral
delamination, cyclodialysis, peripheral chorioretinal detachments,
and radial peripapillary retinal detachments. No full-thickness
openings of the eyewall were observed in any of the eyes tested.
Scleral damage demonstrated the strongest associative tendency for
increasing likelihood of injury with increased overpressure.
Table 1 shows the association between primary blast peak
overpressure levels and the tendency for damage to structures in
anatomic Zones 1-3. Note that a level 2 injury as referred to in the
table is one that would require surgery to repair and would result in
chronic pathology.
Conclusions: These data provide convincing evidence that primary
blast can produce clinically significant ocular damage in the absence
of particle impact. We also present a new Cumulative Injury Score
indicating the clinical relevance of observed injuries.
Commercial Relationships: William E. Sponsel, None; Matthew
A. Reilly, None; Brian J. Lund, None; Walter Gray, None;
Richard Watson, None; Sylvia L. Groth, None; Randolph D.
Glickman, None; Kimberly Thoe, None
Support: Department of Defense Vision Research 342 Program,
Award Number W81XWH-12-2-0055
Mapping the entire nerve architecture and sensory neuropeptide
distribution of rabbit iris
Jiucheng He, Haydee E P. Bazan. Ophthalmology & Neuroscience
Ctr, LSU Health Sciences Center, New Orleans, LA.
Purpose: To disclose the entire architecture and sensory neuropeptide
content of rabbit iridal innervation.
Methods: Ten New Zealand albino rabbits were euthanatized and the
whole irises were excised and fixed. The tissues were stained with
antibodies against a neuronal-class β-tubulin III, calcitonin generelated peptide (CGRP), substance P (SP), and vasoactive intestinal
polypeptide (VIP) and whole-mount images were acquired to build
a whole view of the iridal nerve architecture. To obtain the relative
contents of the neuropeptides in the iris, after neuropeptide staining,
the specimens were double stained with β-tubulin III. Relative nerve
fiber densities for each fiber population were assessed quantitatively
on the basis of whole mount view of the entire nerve architecture by
computer-assisted analysis.
Results: The iris nerves are extensions of the ciliary nerves. The
thick nerves run in the iris stroma close to the anterior epithelia,
forming 4-5 stromal nerve rings from the iris periphery to the
pupillary margin. In the anterior surface, fine divisions derivate
from the stromal nerves constitute a nerve network-like structure to
innervate the epithelial cells, with the pupillary margin having the
densest innervation. In the posterior side, the nerve bundles run along
with the pupil dilator muscles in a radial pattern around the pupil. The
morphology of iris nerves in both sides changes with the pupil sizes.
Double staining showed that in the anterior epithelia, CGRP-positive
nerve fibers constitute about 60%, while SP-positive nerves constitute
about 30% of the total nerve contents. In the posterior side, CGRPpositive nerve fibers are about 75% of total nerve contents, while SP
take up only 20%. In addition, there is a very small amount of VIPpositive nerve fibers (less than 1%).
Conclusions: This is the first study to show a three dimensional
map of the entire iris nerve architecture. Considering the anatomical
location, the high expression of CGRP and SP implies that these
neuropeptides may play important role in the pathogenesis of anterior
uveitis, glaucoma, cataracts and chronic ocular pain.
The images show the different patterns of nerve architectures in the
anterior and posterior surface of iris.
The images show the expression of CGRP-positive nerves in the
pupillary margin.
Commercial Relationships: Jiucheng He, None; Haydee E P.
Bazan, None
Support: NIH/NIGMS1P30GM103340-01A1(Bazan, N.G.); NIH/
NEI R01 EY019465-04
Ultra-structural study of vitreo-macular attachments related to
macular ridges in Shaken Baby Syndrome
Ann E. Barker-Griffith1, 2, Mark P. Breazzano3, Hengsheng Fang3,
Susan S. Lee5, Michael R. Robinson5, Jerrold L. Abraham4. 1Ophthal
& Pathol, SUNY Upstate Med Univ-Syracuse, Syracuse, NY;
2
Ophthalmology & Pathology, SUNY Eye Institute, Syracuse, NY;
3
Ophthalmology, SUNY Upstate Medical University, Syracuse,
NY; 4Pathology, SUNY Upstate Medical University, Syracuse, NY;
5
Allergan Sales, Inc., Irvine, CA.
Purpose: If macular ridges seen in Shaken Baby Syndrome relate
to the vitreo-macular attachment anatomy at the area centralis, then
strong vitreous fibers should be visible in young human and monkey
eyes by scanning electron microscopy (SEM).
Methods: Monkey and more than 15 human donor globes, formalinfixed, from infants and young adults, were dehydrated by ethanol,
critical-point dried by CO2, and imaged by SEM. Selected tissues
were processed for histology.
Results: Monkey and young human (<39 years) eyes demonstrate
strong, thick, vitreous fibril attachments at the area centralis that
radiate in a circumferentially oriented pattern. This feature is absent
in older humans (>60 years). All eyes reveal clearly visible retinal
ganglion cells at the macula and area centralis, with associative fibers
that span the pre-retinal surface. Monkey histology demonstrates
vitreous fibers attaching to internal limiting membrane (ILM) at the
area centralis.
Conclusions: Vitreal-retinal ring attachments have previously been
described at the ILM in the retina1 and shown by us to be present
at the macula in young human eyes2. We again demonstrate the
characteristically strong attachments at the area centralis that also
shows a partial canopy of vitreous fibers over this ring complex
in additional young human eyes, as well as monkey eyes. Our
anatomical observation may correlate with macular ridges found
clinically and histopathologically in Shaken Baby Syndrome,
specifically where vitreous fibers insert at the area centralis.
1.
Hogan, Michael J: The vitreous, its structure, and relation
to the ciliary body and retina, Investigative Ophthalmology, 2(5):
418-445, 1963.
2.
Robinson, Michael R; Streeten, BW. Vitreoretinal ring
attachments at the macula in normal young eyes: a scanning electron
microscopic study. Invest. Ophthalmol. Vis. Sci. 28 (Suppl): 119,
1987.
Commercial Relationships: Ann E. Barker-Griffith, Allergan
Sales, Inc. (F); Mark P. Breazzano, None; Hengsheng Fang, None;
Susan S. Lee, Allergan Sales, Inc. (E); Michael R. Robinson,
Allergan Sales, Inc. (E); Jerrold L. Abraham, None
Support: Allergan Sales, Inc., Research to Prevent Blindness, Lions
20-Y1
Modeling the Developmental Growth of the Eye in Subjects Born
at Term and Subjects with Retinopathy of Prematurity (ROP)
Robert J. Munro1, James D. Akula1, 3, Toco Y. Chui4, Ronald M.
Hansen1, 3, Tara L. Favazza1, Anne Moskowitz1, 3, Sanjay P. Prabhu2,
Anne Fulton1, 3. 1Ophthalmology, Boston Children’s Hospital,
Boston, MA; 2Radiology, Boston Children’s Hospital, Boston, MA;
3
Ophthalmology, Harvard Medical School, Boston, MA; 4Indiana
University, New York, NY.
Purpose: Influenced by development and visual experience, oblate
infant eyes become relatively prolate. It is unknown specifically
where and when the eye grows to transform from its neonatal form
to its adult form. We modeled the development of the eye to evaluate
growth as a function of age, and challenged our model against the
abnormal ROP eye.
Methods: We reviewed extant MRIs from term-born and pretermborn ROP patients, aged 0-20 y, for images suitable for generation of
high-resolution, transverse, pupil-optic-nerve sections. Subjects were
categorized as ‘Term’ if they were ≥37 wk postmenstrual age (PMA)
at birth (n=77) or ‘ROP’ if they were preterm and had ROP (n=29).
Using custom software (Chui et al., Ophthalmology 2012; Akula et
al., ARVO 2013, 3058), we segmented the inner surface of the globe.
For every subject, we measured the length of rays from the centroid
to the surface of the eye at 5° intervals. We described the length of
each ray (Lray) as
Lray(x) = b × xn / (xn + kn),
where x is PMA at test, b is asymptotic length of the ray, k is the
age at which the ray reaches length b/2, and n is related to the slope.
We determined the rate of ray elongation, Eray(x) from Lray dy/dx.
The scleral growth that accounts for Eray is 2π×Eray. We note that
scleral growth at point θ, G(x,θ), and at point θ±180°, contribute
nothing to elongation in Eray; conversely, half of the scleral growth
at θ±90° contributes and growth at intermediate angles contributes
intermediate amounts. We solved for growth G(x,θ) at every age and
position.
Results: G was highest at young ages and rapidly declined thereafter.
Relative to Term eyes, growth in ROP eyes was low at young ages
but high at older ages. In Term eyes, G was highest at the equator
early and smoothly shifted to the axial poles with age; in ROP eyes,
G was highest near the axial poles early and abruptly shifted to the
periphery.
Conclusions: Eyes become prolate because, at young ages when
scleral growth is fast, it is predominantly peripheral, and later, when
scleral growth is slow, it is predominantly at the axial poles. The
loci of maximum growth is offset by ~90° at term in ROP eyes. Our
model predicts that the small ROP eye becomes normal (or even
supranormal) in size in adulthood because it is characterized by a
delay in growth followed by prolonged expansion.
Commercial Relationships: Robert J. Munro, None; James D.
Akula, None; Toco Y. Chui, None; Ronald M. Hansen, None; Tara
L. Favazza, None; Anne Moskowitz, None; Sanjay P. Prabhu,
None; Anne Fulton, None
Support: Children’s Hospital Ophthalmology Foundation
Prevalence of HPV in conjunctiva of healthy subjects in Mexican
population
Hector J. Perez-Cano1, Ezequiel G. Díaz-Benítez2, Miriam G. RojasTejeda2. 1Centro de Investigación Biomédica, Hospital Foundation
“Nuestra Señora de la Luz”, Mexico City, Mexico; 2Orbita, Párpados
y Vías Lagrimales, Hospital Foundation “Nuestra Señora de la Luz”,
Mexico City, Mexico.
Purpose: Human papillomavirus (HPV) and their genotypes are
widely distributed in the world. It has no seasonal incidence, it
is one of the sexually transmitted infections more frequent and
16,18,31,33,35,39, 45, 51,52,56,58,59 and 66 genotypes, have been
implicated in the development of cervical cancer. HPV has been
linked in ocular diseases such as the development of pterygium and
ocular surface squamous neoplasia. In this study we determine the
presence of HPV in conjunctiva of healthy subjects.
Methods: We studied a group of 50 healthy subjects of both sexes
over 18 years of age and without ocular pathology. 50 Samples of
scraping conjunctival were studied. We perform DNA extraction. The
presence of HPV by the technique of the polymerase chain reaction
was determined using universal oligonucleotides MY09/MY11.
The positive samples were genotyped by terminators fluorescent
nucleotide sequencing.
Results: We obtained. 50 samples (27 men and 23 women) aged
between 20 and 51 years. The samples analyzed by PCR for the
detection of HPV, 2 were positive; it corresponds to 2% of the
population. The results grouped by gender corresponding to 3% of
the male population and 4% of the female population
Conclusions: The prevalence of HPV found is similar to those
reported in other countries. This study highlights the importance of
focused preventive measurements to diseases caused by this virus,
without forgetting the eye diseases.
Commercial Relationships: Hector J. Perez-Cano, None; Ezequiel
G. Díaz-Benítez, None; Miriam G. Rojas-Tejeda, None
Orbital Complications of Trans-Foramen Ovale Surgical
Manipulation
Christopher D. Weaver, Steven A. Newman. Ophthalmology,
University of Virginia, Charlottesville, VA.
Purpose: Approach through the foramen ovale for pathology
affecting the Gasserian rhizototomy ganglion has been standard
practice for neurosurgery for more than 75 years. Procedures include
glycerol rhizotomy and electro cautery disruption of the trigeminal
nerve. Complications have been uncommon. It is, however, possible
to enter the orbit through the inferior orbital fissure.
Methods: Two cases of patients seen following attempt at
glycerol injection of the Gasserian ganglion developed intraorbital
complications.
Results: In one case an intraorbital hematoma led to transient
diplopia with complete resolution without additional complications.
In another case, the needle lacerated the ophthalmic artery, resulting
in a pseudo-aneurysm which required neuro-interventional coiling.
The motility improved, but the patient was left with a complete optic
neuropathy.
Conclusions: Root cause analysis of these cases indicates that
placement of the needle too far laterally in the cheek permits access
to the inferior orbital fissure, which is otherwise protected by the
lateral wall of the maxilla. Attention to anatomic detail with the
possible use of neuro navigation should make these complications
less frequent in the future.
Commercial Relationships: Christopher D. Weaver, None; Steven
A. Newman, None
Genetic influence of optic disc structure: The Minnesota Twins
Reared Apart Study (MISTRA)
Elena Bitrian1, Karen Armbrust1, Martha M. Wright1, Alana
Grajewski1, 2, Joseph Caprioli3, Erik J. van Kuijk1, Nancy Segal4,
Thomas Bouchard1. 1University of Minnesota, Minneapolis, MN;
2
Bascom Palmer Eye Institute, Miami, FL; 3Jules Stein Eye Institute,
Los Angeles, CA; 4California State University Fullerton, Fullerton,
CA.
Purpose: To assess the influence of genetic and environmental
factors on optic structure, comparing correlations between
monozygotic (MZA) and dizygotic (DZA) twins separated at birth.
Methods: This is a retrospective review of twin pairs from the
Minnesota Study of Twins Reared Apart (MISTRA). Medical
records and optic disc stereo photographs from twins that had been
separated at birth were reviewed. Only patients without glaucoma
or optic nerve diseases were included. Vertical disc diameter (VD),
vertical cup (VC), vertical cup to disc ratio (VCDR), superior rim
(SR), inferior rim (IR), horizontal diameter (HD), horizontal cup
(HC), nasal rim (NR), temporal rim (TR) and horizontal cup to disc
ratio (HCDR) were measured with computer software by the same
masked experienced ophthalmologist and values were correlated in
monozygotic (MZ) and dizygotic (DZ) twins.
Results: A total of 280 eyes from 70 twin pairs (44 MZA and 26
DZA pairs) were included. Mean MZA age was 38.45 ± 12.93
and mean DZA age was 42.38 ± 12.78 years old (p=0.014). Visual
acuity was 1.08 ± 0.24 in MZA and 1.13 ± 0.21 in DZA twin pairs
(p=0.147) and cup to disc ratio, as documented in the clinical
chart, was 0.26 ± 0.16 mm in MZA and 0.23 ± 0.18 in DZA pairs
(p=0.129). There were no statistically significant differences between
digital optic nerve measurements in the MZA and DZA twins. The
measures for MZA and DZA twin pairs, respectively, were 1.82 ±
0.21 mm and 1.80 ± 0.17 mm (p=0.364), VC 0.49 ± 0.31 and 0.45 ±
0.32 (p=0.363), VCDR 0.26 ± 0.18 and 0.25 ± 0.16 (p=0.542), SR
0.64 ±0.12 and 0.64 ± 0.14 (p=0.753), IR 0.71 ± 0.12 and 0.72 ± 0.13
(p=0.472), HD 1.60 ± 0.2 and 1.59 ±0.21 (p=0.730), HC 0.42 ± 0.29
and 0.42 ± 0.31 (p=0.813), NR 0.58 ± 0.11 and 0.57 ±0.08 (p=0.241),
TR 0.52 ± 0.13 and 0.55 ± 0.17 (p=0.069) and HCDR 0.25 ±0.17 and
0.25 ±0.17 (p=0.845).
MZA twin pairs had statistically significant correlations for all
measured disc parameters: VD 0.844, VC 0.790, VCDR 0.750, SR
0.639, IR 0.599, HD 0.746, HC 0.824, NR 0.511, TR 0.647 and
HCDR 0.733. In contrast, DZS twins had statistically significant
correlations for the following measures VD, HD and TR (correlation
coefficients: VD 0.673, VC 0.222, VCDR 0.227, SR 0.164, IR 0.366,
HD 0.570, HC 0.315, NR 0.153, TR 0.683 and HCDR 0.337).
Conclusions: There is a high correlation between optic disc structural
parameters in twins reared apart that it is stronger in MZA than DZA
twin pairs.
Commercial Relationships: Elena Bitrian, None; Karen
Armbrust, None; Martha M. Wright, None; Alana Grajewski,
Alcon Laboratories Inc (F); Joseph Caprioli, Alcon Laboratories Inc
(F), Allergan (C), Allergan (F), New World Medical Inc (F), NIHNEI (F), RPB (F); Erik J. van Kuijk, None; Nancy Segal, None;
Thomas Bouchard, None
Support: Research to Prevent Blindness
Localization of scleral stem/progenitor cells in murine sclera
Pei-Chang Wu1, Chia-Ling Tsai2. 1Ophthalmology, Chang Gung
Memorial Hospital - Kaohsiung, Kaohsiung, Taiwan; 2Dentistry,
Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan.
Purpose: In 2011 we reported the discovery of a group of multipotent
mesenchymal stem cells at the sclera, which we named the scleral
stem/progenitor cells (SSPCs). The purpose of this study was further
to explore the localization of SSPCs in the murine sclera.
Methods: We performed BrdU pulse–chase experiments in
C57BL6/J mice. The mice were administered daily intraperitoneal
injections of a nucleotide dye, bromodeoxyuridine (Brdu), over
7 consecutive days. The BrdU labeled cells were detected by
the immunochemical study in the sclera. As the ABCG and
Sca-1 are good markers for stem cells. We also performed the
immunofluorescence study to localize the SSPCs.
Results: There were significant more BrdU-positive cells in the
anterior sclera in comparison to the posterior sclera. The ABCG and
Sca-1 positive cells were also present in the anterior sclera mostly.
Conclusions: The findings are consistent with the presence of a small
population of scleral stem/progenitor cells localized along the murine
sclera. It may help further studies of scleral diseases such as scleritis
and myopia.
Commercial Relationships: Pei-Chang Wu, None; Chia-Ling Tsai,
None
Support: CMRPG8A0891
Computational Modeling of Internal Eye Injury due to Primary
Blast
Richard Watson1, 5, Walter Gray4, Randolph D. Glickman6, 1, Brian
J. Lund3, William E. Sponsel1, 2, Matthew A. Reilly1. 1Biomedical
Engineering, The University of Texas at San Antonio, San Antonio,
TX; 2Visual Science, Rosenberg School of Optometry; UIW, San
Antonio, TX; 3U.S. Army Institute of Surgical Research, JBSA
Fort Sam Houston, San Antonio, TX; 4Geological Sciences, The
University of Texas at San Antonio, San Antonio, TX; 5Biodynamic
Research Corporation, San Antonio, TX; 6Ophthalmology,
UTHSCSA, San Antonio, TX.
Purpose: Ocular trauma has increased from 0.6% to 6% of battlefield
injuries over the last 140 years. Recent physical experiments have
demonstrated that primary blast can produce significant ocular injury
at low levels of overpressure. In most cases, the mechanisms of
internal injury are obscured due to the difficulty of imaging inside
the eye during the blast event. Alternatively, computational modeling
can provide invaluable insight as to the internal dynamics and forces
occurring inside the eye under primary blast conditions.
Methods: Numerical simulations of primary blast to porcine eyes
were used to support a series of in vitro shock tube experiments. Subglobe rupture levels of blast (overpressures from 50 - 200 kPa) were
used in experiments and modeling. Three dimensional Lagrangian
Finite Element Analysis (FEA) models were created using material
properties from the relevant literature. Internal structures of the
eye were modeled in detail to allow visualization of internal
dynamics under blast conditions. Experimental and hypothetical
blast waveforms with purely positive and purely negative pressure
components were applied to give insights as to the relative
contributions of each to the observed trauma.
Results: FEA models predicted increasing levels of force, distortion,
and strain with increasing blast energy. This is consistent with
the experimental finding that likelihood and severity of injury
increases with blast energy. Although not visible in the experiments,
computational results suggest compression of the peripapillary retina
is a potential injury mechanism even at low blast energies (Fig 1).
Conclusions: FEA models confirmed the potential for primary blast
ocular injury. The models suggest that different injuries are associated
with the different phases of the blast profile. Specifically, posterior
segment damage is associated with the positive phase and anterior
segment damage (angle recession) is associated with the negative
phase (Fig. 2). Computational results correlated with the physical
experiments providing insight into injury mechanisms not observable
in the experiments. Thus, FEA modeling is an essential supplement to
physical experiments.
Figure 1. Change in peripapillary retinal thickness as a function of
peak overpressure
Figure 2. Undeformed porcine eye geometry (left). Deformed
geometry and stress distribution in sclera when subjected to negative
phase of a hypothetical blast wave (right).
Commercial Relationships: Richard Watson, None; Walter
Gray, None; Randolph D. Glickman, None; Brian J. Lund, None;
William E. Sponsel, None; Matthew A. Reilly, None
Support: USAMRMC VRP Grant W81XWH-12-2-0055
Classification and clinical characteristics of epiphora with
punctal stenosis
Nam Yeong Kim, Mun Chong Hur, Yoon Hyung Kwon, Won Yeol
Ryu, Hee-Bae Ahn. Dong-A University Hospital, Busan, Republic of
Korea.
Purpose: To evaluate the classification of punctal stenosis according
to the shape of external punctum and related symptom and clinical
characteristics adding to investigated histopathologic finding of
stenotic punctum.
Methods: Patients who has tearing and diagnosed as punctal stenosis
between May 2013 and January 2014 were evaluated. Punctal
stenosis was diagnosed with slit lamp exam and classified according
to lower external punctum shape, membranous obstruction type,
operculum type, slit type, fibrotic circular type. Control group without
epiphora and punctal stenosis also evaluated. Clinical characteristics
like tear meniscus height, 2% fluorescine dye disappearance test(2%
FDDT) and lacrimal pathway irrigation were also done and statistical
correlation with dermographic data also analyzed. As treatment,
Punctal snip operation and silicone tube intubation was done and
histopathologic finding of punctum was evaluated.
Results: The mean age of punctum stenosis group(43 patients, 79
eyes) was 65.69 ± 7.84 years. and control group(30 Patients, 60
eyes) was 60.3 ± 6.31 years. Punctal stenosis was classified to four
types and it was divided to fibrotic circular type 26 eyes(34.17%),
operculum type 25 eyes(32.65%), membranous obstruction type 17
eyes(20.25%), slit type 11eyes(13.92%) and this distribution was
not statistically significant(p=0.054). And there was no statistically
significant correlation of clinical exam and types of punctal stenosis,
but tear meniscus was higher and 2% fluorescine dye was disappeared
slower at the type of membranous obstruction than other types. The
histopathologic finding of punctum showed loss of connective tissue
at the margin of conjunctiva, punctum and horizontal canaliculus.
and muscle of riolan was prominantly found within 0.1mm from
canalicular endothelium.
Conclusions: The fibrotic circular stenosis was represented as the
most common type of punctal stenosis in four different shapes
classification. Although these classification is not significant related
to tearing symptom, but, membranous obstruction group showed
elevated tear meniscus height and the histopathologic findings were
compatible to the shape.
Commercial Relationships: Nam Yeong Kim, None; Mun Chong
Hur, None; Yoon Hyung Kwon, None; Won Yeol Ryu, None; HeeBae Ahn, None
The Functional and Histopathological Change in the Levator
Palpebrae Superioris and Müller’s Muscle after Subconjunctival
Injection of Triamcinolone Acetonide in Rabbits
MINWOOK CHANG1, Jae Rock Do1, Youngseok Lee2, Sehyun Baek3.
1
Ophthalmology, Dongguk university, Goyang, Republic of Korea;
2
Pathology, Korea University College of Medicine, Seoul, Republic
of Korea; 3Ophthalmology, Korea University College of Medicine,
Seoul, Republic of Korea.
Purpose: The aim of this study was to evaluate the functional and
histopathological changes in the levator palpebrae superioris and
Müller’s muscles after subconjunctival injection of triamcinolone
acetonide (TA) in rabbits.
Methods: Twenty-four white New Zealand rabbits were divided
into two groups. In group A, a subconjunctival injection of 0.5 cc
TA (40 mg/cc) was administered to the right eye, while a normal
saline injection of the same volume was administered to the left eye.
In group B, the same procedures were done with a 1.0 cc injection
of TA or normal saline into each eyelid. Follow-up was done to
evaluate the histopathological changes in the levator and Müller’s
muscles, changes in the mean transectional area of Müller’s muscle,
and changes in upper lid height (MRD1) at one, two, four and six
weeks after injection. Western blot analyses were used to determine
the levels of myosin light chain phosphorylation (MLC-p) and alpha
smooth muscle actin (a-SMA), which are related to the contractility
of Müller’s muscle.
Results: No specific changes in MRD1 were noted in either group
A or B. No significant histopathological changes were found in
the levator muscles. However, significant atrophy and thinning of
Müller’s muscle were found, and MLC-p and a-SMA levels were
decreased. This was consistent with the histological changes of
Müller’s muscle observed in rabbits that received a TA injection.
These changes were reversible and influenced by the volume of the
injection.
Conclusions: Subconjunctival injection of TA into the upper eyelids
appears to be temporally influential on both the functional and
histopathological changes of Müller’s muscle. This may be explained
by the effect of improvement in lid retraction regardless of the
minimal specific change observed in the levator muscle.
Commercial Relationships: MINWOOK CHANG, None; Jae
Rock Do, None; Youngseok Lee, None; Sehyun Baek, None
Effect of mannitol on orbital volume
Adam Weber1, Bryan R. Costin1, Tal J. Rubinstein1, Khaled Asi2,
Julian D. Perry1. 1Cole Eye Institute, Cleveland Clinic, Cleveland,
OH; 2Neurological Institute, Cleveland Clinic, Cleveland, OH.
Purpose: This IRB-aproved retrospective chart review is designed to
ascertain if the administration of intravenous mannitol affects orbital
volume as determined by computed tomography (CT).
Methods: A list of patient’s who were administered intravenous
mannitol with CT scans of the head prior to and after mannitol was
obtained. Gender, age, and time between CT scans and mannitol
administration were recorded. Orbital volume was calculated
by calculating the area of the orbit for the five most superior CT
slices that clearly contain the globe, and multiplying the average
of consecutive slice areas by the thickness of each slice. This
measurement method has been validated in prior literature. Pre and
post-mannitol volumes were compared for each eye in each patient,
and the significance of the change was determined by t-test.
Results: Preliminary results were obtained from 9 patients (18
eyes). Five patients were male, and average age at time of mannitol
administration was 57.94 year old. Average time between initial CT
and mannitol administration was 15.47 hours, and average time from
mannitol to follow-up CT was 26.17 hours. Area calculations showed
an average gain in orbital volume of 19.08mm3 (+1.4%, p=0.36).
Conclusions: This limited data series shows no statistically
significant effect of mannitol on orbital volume. Scientific literature
shows that mannitol decreases intracranial soft tissue volume. These
preliminary results appear to show that orbital volume is affected
differently by mannitol than intracranial tissues. Further study is
required to determine if mannitol affects orbital volume.
Commercial Relationships: Adam Weber, None; Bryan R. Costin,
None; Tal J. Rubinstein, None; Khaled Asi, None; Julian D. Perry,
None
Impact of physiological oxygen level on the growth and the
transcriptome of melanocytes and fibroblasts from the choroid
Solange Landreville1, 2, Laurence Trudel-Vandal2, Constance BarryMarcheterre2, Renée Paradis2, Stephanie Proulx1, 2. 1Ophthalmology,
Université Laval, Québec, QC, Canada; 2CUO-Recherche et Centre
LOEX, Centre de recherche du CHU de Québec, Québec, QC,
Canada.
Purpose: Cell culture is traditionally performed in CO2 incubators
maintained at atmospheric oxygen level (21%), while oxygen is
transported by the blood and delivered to the cells at levels lower
than 14%. Some oxygen derivatives such as the reactive oxygen
species (ROS) can induce replicative senescence of cells when
produced in excess, presumably because of irreversible damages to
nucleic acids, lipids and proteins. The choroid is an interesting model
for understanding the molecular effects of oxygen because the cells
are exposed to oxygen levels varying between 4.5-11%. The goal
of this study was to recreate the physiological oxygen conditions
of the native choroid to study the growth and the transcriptome of
melanocytes and fibroblasts.
Methods: Melanocytes and fibroblasts were isolated from human
choroids (n=3) by successive digestions in trypsin and collagenase.
The cells were then exposed to physiological (<5%) or atmospheric
(21%) oxygen levels. Cell viability and proliferation under both
oxygen levels were measured by a colorimetric assay using the
MTS tetrazolium salt and by phospho-histone H3 immunostaining,
respectively. Then, gene expression profiling by microarray was
conducted using RNA prepared from melanocytes and fibroblasts
grown under both oxygen levels. Presence of oxidative DNA damage
was studied with the CellROX Green reagent that binds oxidized
DNA.
Results: A significant increase in the percentage of viability was
measured in melanocytes cultured at low oxygen level, compared
to cells grown at 21% oxygen (increases of 65%, 31% and 50%,
respectively). An opposite effect was observed with the fibroblasts
(decreases of 4%, 35% and 23%, respectively). Scatter plot analysis
of the microarray data indicated very few modifications in gene
expression between choroidal cells exposed to both oxygen levels
(R2=0.9715 and R2=0.9777, respectively). Finally, the level of
oxidative stress was reduced at physiological oxygen level.
Conclusions: This study demonstrated that choroidal cells could
grow in physiological oxygen conditions that more closely replicate
the native tissue environment. Choroidal cells exposed to different
oxygen levels shared larger similarities in their transcriptome than
we had predicted. Extensive work is warranted to understand the
discrepancy between adaptive responses of choroidal melanocytes
and fibroblasts to oxygen level.
Commercial Relationships: Solange Landreville, None; Laurence
Trudel-Vandal, None; Constance Barry-Marcheterre, None;
Renée Paradis, None; Stephanie Proulx, None
Support: Master Research Training Award from Université Laval
(LTV), Research Training Awards from FRQS Vision Research
Network and Fondation des maladies de l’oeil (CBM). Funding from
Fondation des Hôpitaux Enfant-Jésus/Saint-Sacrement, Fondation
“Ophtalmologie, Recherche & Développement”, Fondation des
maladies de l’oeil, Fondation de l’Université Laval, FRQS Vision
Research Network.
Interaction between resident macrophages and perivascular
mural cells of the choroid: Relevance to pathological choroidal
changes in retinal disease
Anil Kumar1, Lian Zhao1, Robert N. Fariss2, Wai T. Wong1.
1
UNGIRD, National Eye institute, Bethesda, MD; 2Biological
Imaging Core, National Eye institute, Bethesda, MD.
Purpose: Pathological choroidal vascular changes have been
associated with retinal diseases such as age-related macular
degeneration (AMD) and diabetic retinopathy (DR), including
changes in perivascular mural cells (PMCs) (i.e. pericytes and smooth
muscle cells). We investigated how resident macrophages in the
choroid may contribute to changes in PMC structure and function.
Methods: αSMA-GFP transgenic mice containing GFP+ PMCs
were used to visualize vascular interactions with resident choroidal
macrophages which were labeled with antibodies to MHC-II.. The
effects of polarized human THP1 macrophages on human retinal
pericyte cells (HRPC) were assessed in vitro using a TUNEL
assay (apoptosis), a MTT assay (cell survival), and a BrdU assay
(proliferation).
Results: Perivascular ramified MHC-II macrophages, resident
throughout the adult mouse choroid, was observed to contact PMCs
via branched and motile processes. At the level of choroidal arteries
and arterioles, the termini of macrophage processes made multiple
focal contacts on PMC somata and encircling processes. At the level
of choriocapillaris, stellate-shaped macrophages closely fasciculated
their processes with those of flattened pericytes located on the
sclerad surface of the choriocapillaris. These intimate and extensive
intercellular contacts indicated that macrophage-PMC interactions
occur constitutively in the choroid and may regulate vascular
structure and function. In in vitro experiments, conditioned media
from M1- and M2-polarized THP1 macrophages, were capable of
differentially affecting HRPC pericyte proliferation and survival. M2conditioned media induced in pericytes (1) increased proliferation (2)
increased survival, and (3) a transition to an elongated morphology,
while M1-conditioned media conversely (1) decreased proliferation,
(2) increased apoptosis, and (3) promoted a transition to a rounded
morphology.
Conclusions: Macrophages resident in the healthy adult choroid
demonstrate extensive physical interactions with PMCs, suggestive
of ongoing signaling. Alterations in macrophage polarization in
retinal and choroidal disease may influence pericyte density, survival
and function and drive pathological vascular change in the choroid.
Macrophage-PMC interactions may play a role in retinal disease
pathogenesis and constitute potential targets for intervention.
Commercial Relationships: Anil Kumar, None; Lian Zhao, None;
Robert N. Fariss, None; Wai T. Wong, None
Support: NEI intramural
Extracellular HMGB1 promotes choroidal angiogenesis
Younghee Kim1, Nagaraj Kerur1, Shengjian Li1, Ana BastosCarvalho1, Bradley D. Gelfand1, William W. Hauswirth2, Jayakrishna
Ambati1. 1Ophthalmology & Visual Sciences, University of Kentucky,
Lexington, KY; 2University of Florida, Gainesville, FL.
Purpose: High mobility group box 1 (HMGB1) protein acts as a
cellular alarm against pathologic inflammation and its extracellular
release by acetylation has been reported to be required for its
angiogenic cytokine function. HMGB1 has been recently suggested
as a novel therapeutic target for angiogenesis; however the
association between its acetylation and angiogenic function in vivo
is not clear. Here we aim to examine HMGB1 effect on choroidal
angiogenesis (CNV) and extracellular HMGB1 expression related to
its acetylation.
Methods: To investigate the effect of HMGB1 on angiogenesis, we
used a mouse model of laser coagulation-induced CNV and measured
CNV volume by FITC-conjugated GS-IB4 staining after treatment
of a neutralizing antibody or recombinant protein of HMGB1 in WT
mice. To confirm whether HMGB1 protein expression is related to
CNV induction, we examined not only total HMGB1 protein but also
extracellular HMGB1 protein and its acetylation in retina and retinal
pigment epithelium/choroid (RPE/C) from CNV eyes comparing
to WT by Western blotting. In addition, we measured CNV volume
by RPE-specific knockdown of HMGB1 by subretinal injection of
AAV1-VMD2-Cre to HMGB1f/f mice comparing to control AAV1VMD2-GFP group.
Results: We confirmed that antibody neutralization of HMGB1
reduced CNV, whereas administration of recombinant HMGB1
protein induced CNV, implying that both endogenous and exogenous
HMGB1 promote CNV. In Western blotting, total HMGB1 protein
abundance did not change in both retina and RPE/C by laser injury,
however extracellular HMGB1 release and its acetylation occurred in
retina and RPE/C by laser-induced CNV.
Conclusions: Taken together, these data suggest that extracellular
HMGB1 by its acetylation is related to CNV development, and
control of HMGB1 expression and localization can be a novel
therapeutic target for in vivo angiogenesis.
Commercial Relationships: Younghee Kim, None; Nagaraj Kerur,
None; Shengjian Li, None; Ana Bastos-Carvalho, None; Bradley
D. Gelfand, None; William W. Hauswirth, None; Jayakrishna
Ambati, University of Kentucky (P)
Support: NEI/NIH, Doris Duke Charitable Foundation, Burroughs
Wellcome Fund, Ellison Medical Foundation, Dr. E. Vernon and
Eloise C. Smith Endowed Chair, Foundation Fighting Blindness,
Carl Reeves Foundation, Harrington Discovery Institute; Research to
Prevent Blindness
Caveolae transport of albumin by choriocapillaris from serum to
Bruch’s membrane and RPE
Masataka Nakanishi, Imran A. Bhutto, Rhonda Grebe, Malia M.
Edwards, Scott D. McLeod, Gerard A. Lutty. Opthalmology, Johns
Hopkins University, Baltimore, MD.
Purpose: The choriocapillaris (CC) is the fenestrated capillary
system that adjacent to the retinal pigment epithelium (RPE),
separated from RPE by Bruch’s membrane (BM). Recent studies
show that dysfunction and eventual death of CC is related to
development of choroidal neovascularization (CNV) in aged-macular
degeneration (AMD). If CC transport is altered and whether it plays
a role in the development of AMD is not known. The aim of this
study was to examine the role of CC caveolae in transport of serum
macromolecules from the CC lumen to RPE.
Methods: Alexa647-conjugated to bovine serum albumin (A647BSA) and PBS were administrated in normal mice and caveolaedeficient mice (cav1-/-) intravenously. Mice were perfused with
PBS and sacrificed at 0.5, 1 and 4 hours post injection. Eyecups
were cryopreserved. 8 um thick sections were analyzed with a Zeiss
710 confocal microscope and autofluorescence was assessed in the
sections of PBS injected eyes. Five nanometer BSA-conjugated
gold nanoparticles (BSA-GNP) were administrated intra-arterially
via the carotid artery and mice were then perfused with PBS and
sacrificed at 1 hour post injection. Eyecups were fixed with 2.5 %
paraformaldehyde/2 % glutaraldehyde, post fixed with 0.5 % OsO4,
dehydrated and embedded in LX112 resin. 70 nm ultrathin sections
were stained with uranyl acetate and lead citrate and visualized on a
Hitachi H7600 TEM.
Results: In eyes of normal mice at 0.5 hour after intravenous
injection, A647-BSA was localized to the RPE and appeared to have
diffused into the region of photoreceptor outer segments. On the
other hand, most of A647-BSA was found in CC in eyes of caveolaedeficient mice, suggesting that albumin was only bound to its
receptor but not taken up nor transported by caveolae. The majority
of BSA-GNP found in normal mice was observed on the luminal not
abluminal side of the CC endothelium, however, some was located
in caveolae, some perivascular, and some in RPE. Conversely,
BSA-GNP was not observed outside the lumens of CC in caveolaedeficient mice.
Conclusions: BSA transport by CC was regulated by caveolae via
albumin receptor. The other transport systems of CC will be assessed
using different tracers and materials to determine the normal in-vivo
transport potential of CC in mice. Simultaneously, changes in CC
caveolae volume are being determined by TEM in human aged and
AMD subjects.
Commercial Relationships: Masataka Nakanishi, None; Imran
A. Bhutto, None; Rhonda Grebe, None; Malia M. Edwards, None;
Scott D. McLeod, None; Gerard A. Lutty, None
Support: NIH grants EY001765 and EY016151 and RPB
N-cadherin-mediated cell adhesion regulates epithelium polarity
and morphogenesis in the developing ciliary body of the mouse
eye
Yi Zhou1, 2, Christopher P. Tanzie1, 2, Ting Xie1, 2. 1Stowers Institute for
Medical Research, Kansas City, MO; 2Department of Anatomy and
Cell Biology, University of Kansas Medical Center, Kansas City, KS.
Purpose: Glaucoma is one of the leading causes of irreversible
blindness worldwide and is often associated with elevated intraocular
pressure (IOP). The ciliary body (CB), a bilayered epithelial
structure with underlying stroma, is involved in regulating IOP
and hence often serves as the pharmacologic target for glaucoma.
Despite the significant role it plays in ocular health and homeostasis,
its development and morphogenesis remains poorly understood.
Previously, we determined that Notch2 regulates CB morphogenesis
at least in part by regulating cell proliferation and BMP signaling.
Here we show that cell adhesion mediated by N-cadherin regulates
cell polarity and morphogenesis independently of Notch2 and BMP
signaling.
Methods: We used Trp1-cre mediated loxp recombination to
selectively knock-out N-cadherin in both CB epithelial layers.
Mutants and littermate controls were harvested at multiple
developmental stages for analysis. Standard procedures including
immunohistochemistry, in-situ hybridization, BrdU labeling, TUNEL
staining and Western blotting were performed to characterize the
mutant phenotype. To further support our hypothesis, we used the
human RPE cell line ARPE19 to knock-down N-cadherin by shRNA
and measured the changes of polarity caused by the reduction of
N-cadherin.
Results: N-cadherin mutants display about 50% penetrance with
regards to defective CB morphogenesis. Immunostaining results
show that N-cadherin depletion affects neither Notch2 nor BMP
signaling. BrdU labeling results indicate that N-cadherin is required
to maintain high levels of cellular proliferation. Interestingly,
localization of the apical polarity marker Par3 is compromised in
N-cadherin mutants, and its proper localization correlates well with
the degree of morphogenesis, suggesting that N-cadherin might
regulate morphogenesis, at least in part, through regulation of cell
polarity.
Conclusions: Our previous study demonstrated that Notch2 regulates
CB morphogenesis. These results identify the important role of
N-cadherin-mediated cell adhesion in regulating CB development.
N-cadherin regulates CB morphogenesis by stabilizing Par3-based
apical polarity. These findings expand our understanding of CB
development and provide insight into the cross-talk of important
signaling pathways, which may be applied in other systems as well.
Commercial Relationships: Yi Zhou, None; Christopher P.
Tanzie, None; Ting Xie, None
Mechanics of Optic Fissure Invagination
Benjamen A. Filas1, Jie Huang1, Larry A. Taber2, David C. Beebe1.
1
Ophthalmology and Visual Sciences, Washington University School
of Medicine, St. Louis, MO; 2Biomedical Engineering, Washington
University, St. Louis, MO.
Purpose: During early eye development the ventral optic cup
invaginates to form the optic fissure. Defects in invagination have
usually been studied because the deletion or mutation of a gene
induces a mutant phenotype (e.g. severe coloboma, or failure
of hyaloid artery and/or optic nerve formation). However, the
morphogenetic mechanisms that drive optic fissure formation (and
malformation) are unknown.
Methods: The optic fissure of developing chicken (HH13-21) and
mouse embryos (E9.5-12.5) was non-invasively imaged using an
optical coherence tomography system coupled to a Nikon FN1
microscope (Thorlabs). Optic cups were segmented using the
Computerized Anatomy Reconstruction Toolkit and surface curvature
was computed and mapped to the 3-D reconstructions with a custom
Matlab routine. A finite-element plane-strain model (Comsol
Multiphysics, v4.2) was used to simulate volumetric growth in the
ventral optic cup assuming a Blatz-Ko pseudoelastic strain energy
density function. Regional proliferation rates were specified from
morphological measurements made using ImageJ.
Results: OCT images showed that optic fissure invagination was
distinct; occurring after optic cup formation was complete (Fig. 1,
top). This invagination was asymmetric in transverse section and
characterized by epithelial elongation (up to 50%) (Fig. 1, middle).
Next, the fissure narrowed and deepened prior to closure (Fig. 1,
bottom). Noting that Morcillo et al. (Development, 133: 3179-3190,
2006) found reduced cell proliferation in the ventral optic cup of
Bmp7 KOs (fissure invagination blocked), and that we have found
reduced cell proliferation in optic vesicle Bmp4 KOs (fissure and
optic cup formation blocked, unpublished), we hypothesized regional
growth as a mechanism for fissure formation. To test this, we used
a finite-element model to simulate increased transverse growth on
the nasal vs. temporal side of the fissure, as guided by morphology
measurements. Resulting model shapes and curvatures were similar
to experiments throughout the invagination process (Fig. 1, bottom).
Conclusions: Morphological and computational analysis suggests
differential growth to be a driver of fissure invagination. This finding
is corroborated by abnormal regional cell proliferation patterns
observed when fissure invagination is blocked in BMP KO embryos.
Figure 1. Optic fissure invagination.
Commercial Relationships: Benjamen A. Filas, None; Jie Huang,
None; Larry A. Taber, None; David C. Beebe, None
Support: Knights Templar Career-Starter Research Award (BF), NIH
R01 EY004853 (DB)
Postnatal Overexpression of TGFα in Eyelid Stroma Disturbs
Eyelid Morphogenesis
Fei Dong1, 2, Chia-Yang Liu1, Wei Li2, Zuguo Liu2, Yongxiong Chen2,
Winston W. Kao1. 1university of Cincinnati, Cincinnati, OH; 2Xiamen
University, Xiamen, China.
Purpose: Growth factors play important roles in tissue
morphogenesis during development and maintenance of homeostasis
in adults. Epidermal growth factor receptor (EGFR) signaling
has pivotal role in eye lid morphogenesis, perturbation of genes
involved in the signaling pathways results in eye open at birth in
mouse. Transforming Growth Factor alpha (TGFα) is one of the
important ligands of EGFR, which has been found to have important
roles on the eyelid morphogenesis. This study aims to examine the
manifestation of excess TGFα in eyelid morphogenesis after birth.
Methods: : Bi-transgenic Kera-rtTA/tet-O-TGFα (KR/TGFα) mice
were bred by crossing Kera-rtTA (keratocan promoter) and Tet-OTGFα mice. The newborn KR/TGFα pups were induced to over
express TGFα by eyelid stromal cells via feeding doxycycline chow
to the nursing mother from P0 (postnatal day 0) through P15. Eyes
were collected at various time points and subjected to histological
and immunofluorescence staining.
Results: Excess TGFα resulted in abnormalities in eyelid
morphogenesis. The bi-transgenic mice displayed eyelid open
around P10 and swollen eyelids. Single transgenic mice eye open
around P14. Histology examination shows conjunctival epithelium
thickening, increase of goblet cells, and malformation of Meibomian
gland, formation of a cyst derived from lid stromal cells which
leads to levator aponeurosis dysfunction, and mimicking human
ptosis symptoms. Immunohistochemistry examination manifested
high proliferation property (PCNA positive), low expression
of differentiation markers e.g., collagenous matrix by Massontrichrome staining of the levator aponeurosis. The cells of the the
cyst are keratin negative and vimentin positive suggesting they are
mesenchyme origin.
Conclusions: Taken together, these observations suggest that excess
TGFα disrupts eyelid morphogenesis, especially the proper formation
and function of the levator aponeurosis.
Commercial Relationships: Fei Dong, None; Chia-Yang Liu,
None; Wei Li, None; Zuguo Liu, None; Yongxiong Chen, None;
Winston W. Kao, None
Support: NIH/NEI EY013755, Research to Prevent Blindness, Ohio
Lions eye Research Foundation
Junctional p120-catenin Recruitment of Shroom3 Facilitates
Apical Constriction During Lens Pit Morphogenesis
Timothy F. Plageman1, Albert Reynolds3, Richard A. Lang2. 1College
of Optometry, The Ohio State University, Columbus, OH; 2Pediatric
Ophthalmology, Cincinnati Children’s Hospital Research Foundation,
Cincinnati, OH; 3Cancer Biology, Vanderbilt University, Nashville,
TN.
Purpose: Apical constriction is an important epithelial cell behavior
characterized by the conversion of a cylindrical to wedge-like shape
that is necessary for normal embryonic morphogenesis of the eye.
During lens pit invagination cells undergo apical constriction under
the direction of the cytoskeletal protein Shroom3. Invertebrate
epithelial cells undergoing apical constriction depend on the
contraction of apical cortex spanning actomyosin filaments that
generate force on the apical junctions and pull them toward the
middle of the cell, effectively reducing the apical circumference.
A current challenge is to determine whether these mechanisms are
conserved in the vertebrate lens during invagination and to identify
the molecules responsible for linking apical junctions with the
Shroom3-dependent apical constriction machinery. This study tests
the hypothesis that p120-catenin is required for apical constriction
through the recruitment of Shroom3 to adherens junctions.
Methods: Utilizing the developing mouse eye as a model system,
the embryonic lens placode and lens pits of wild-type and mutant
embryos were analyzed by immunofluorescent labeling of wholemount and histologically sectioned embryos. A genetic interaction
screen utilizing Shroom3 and conditional adherens junction alleles
was also performed to determine which junctional proteins function
with Shroom3. Cultured epithelial cells expressing Shroom3 to model
apical constriction and test junctional localization were also utilized.
Results: Contractile actomyosin filaments spanning the apical cortex
are circumferentially positioned in the invaginating lens pit. Among
several junctional components, it was determined that p120-catenin
strongly genetically interacts with Shroom3 becoming a candidate
molecule for functionally linking the apical constriction machinery
with epithelial junctions. Further analysis revealed that like Shroom3,
p120-catenin is required for apical constriction of lens pit cells.
Finally, we determined that p120-catenin functions by recruiting
Shroom3 to adherens junctions.
Conclusions: These data demonstrate the existence of contractile,
apical cortex spanning actomyosin filaments that require linkage to
the Shroom3-dependent apical constriction machinery via p120catenin during lens pit invagination.
Commercial Relationships: Timothy F. Plageman, None; Albert
Reynolds, None; Richard A. Lang, None
Glutathione is a Critical Regulator of Ocular Morphogenesis
Ying Chen1, David Orlicky2, Monica Sandoval1, David Thompson3,
Vasilis Vasiliou1. 1Department of Pharmaecutical Sciences, University
of Colorado Anschutz Medical Campus, Aurora, CO; 2Department
of Pathology, University of Colorado Anschutz Medical Campus,
Aurora, CO; 3Department of Clinical Pharmacology, University of
Colorado Anschutz Medical Campus, Aurora, CO.
Purpose: During embryogenesis, the ocular surface tissues are
constructed from surface ectoderm (SE). Modulation of signal
transduction by the cellular milieu during embryogenesis remains
poorly understood; however, evidence indicates that key signaling
pathways are redox-sensitive. Glutathione (GSH), the most abundant
non-protein thiol, functions as the major redox buffer that maintains
cellular redox homeostasis. An essential role of GSH in early mouse
development is demonstrated by deaths of E7.5-8.5 embryos deficient
in Gclc, the gene that encodes the rate-limiting enzyme in GSH
biosynthesis. The presence of GCLC mRNA in the developing mouse
eye is suggestive of a possible role of GSH in the process of ocular
morphogenesis.
Methods: To elucidate such a role, we have developed a mutant
(Gclcle/le) mouse line rendered incapable of GSH synthesis in SEderived ocular structures. This was achieved by crossing Gclc-Floxed
mice with Le-Cre mice that express CRE recombinase driven by
the promoter of mouse Pax6 gene, the “master control” gene for the
development of eyes and some epidermal organs.
Results: Heterozygous (Gclcw/le) and homozygous (Gclcle/le) mutants
were born alive with an expected Mendelian frequency. These
mutants appear to grow normally and display eye opening around
postnatal day 14 (P14). Around weaning (P21), Gclcw/le and Gclcle/
le
mice show good overall health except that Gclcle/le mice exhibit
small eye phenotype. Starting around P35, Gclcle/le mice show
growth retardation, develop elevated fasting serum glucose levels
(diabetes). Around P63, they become moribund and die of diabetes
complications. At this age, small eye becomes apparent in Gclcw/
le
mice, which appear otherwise healthy. Histological examination
of Gclcle/le eyes at P23 and P56 revealed all ocular structures to be
present. However, pathologies were observed in multiple tissues,
specifically cornea, lens, iris, ciliary body and retina. These included
hypercellularity and cytoplasmic vacuolization associated with
disorganized cellular proliferation/differentiation/death. Further
characterization of the ocular phenotype at embryonic and early
postnatal stages in the Gclcle/le mutants is currently underway.
Conclusions: Collectively, our data demonstrate, for the first time, a
critical role for GSH in ocular development. Our Gclcle/le mutant line
represents a novel model by which the in vivo role of GSH in eye
development may be explored.
Commercial Relationships: Ying Chen, None; David Orlicky,
None; Monica Sandoval, None; David Thompson, None; Vasilis
Vasiliou, None
Support: NIH grants EY011490 and EY021688
Prohibitin is essential in zebrafish optic fissure closure
Mariana Rius, Sunit Dutta, Brian Brooks. Ophthalmic Genetics and
Visual Function Branch, National Eye Institute, Bethesda, MD.
Purpose: Uveal coloboma is a potentially blinding ocular
malformation that significantly contributes to childhood blindness.
It is caused by the failure of optic fissure closure during eye
morphogenesis. Optic fissure closure is a highly conserved and
complex biological process, yet the governing molecular mechanisms
and coloboma formation remain elusive. We previously used
developmental profiling to identify a zinc-finger-containing gene,
nlz1, as an important regulator of optic fissure closure. NLZ1 was
independently shown to interact with prohibitin (PHB/phb), another
gene suggested by our developmental screen. phb is thought to
regulate cell cycle progression and mitochondrial biogenesis. This
study evaluates phb as a candidate gene for coloboma using a
zebrafish model.
Methods: Whole mount in situ hybridization and RT PCR
determined prohibitin expression patterns across relevant
developmental stages. Antisense morpholino and RNA rescue
measured the loss-of-function effects and confirmed the specificity of
the reaction. The resulting phenotype was verified at the cellular level
using histological sectioning.
Results: Whole mount in situ hybridization confirmed phb1 and phb2
expression in the eye at 24 hours post fertilization (hpf), 26 hpf, 48
hpf, 72 hpf, and 120 hpf. RT PCR indicated maternal and zygotic
expression of phb1 and phb2, and morpholino knockdown revealed
a uveal coloboma phenotype. The morphant phenotype was rescued
through co-injection with phb1 RNA, verifying the specificity
of phb1 in the closure of the optic fissure. Histological analysis
confirmed failure of optic fissure closure throughout embryonic
development in the morphant embryos.
Conclusions: Loss of prohibitin function results in coloboma in
the zebrafish. The identification of a novel gene’s involvement
in the fusion of the optic fissure provides insight into the various
participants that may contribute to this complex, conserved, and
poorly understood mechanism.
Commercial Relationships: Mariana Rius, None; Sunit Dutta,
None; Brian Brooks, None
Association of Aqueous Humor Cytokines with the Development
of Retinal Ischemia and Recurrent Macular Edema in Retinal
Vein Occlusion
Sung Jae Yang1, Hyoung Jo2, Kyoung-A Kim2, Sang Hoon Jung2.
1
Ophthalmology, Gangneung Asan Medical Center, Gangneung,
Republic of Korea; 2Functional Food Center, Korea Institute of
Science and Technology (KIST) Gangneung Institute, Gangneung,
Republic of Korea.
Purpose: To evaluate the association of aqueous humor angiogenic
and inflammatory cytokine levels with the development of retinal
ischemia and recurrent macular edema in retinal vein occlusion
(RVO) patients.
Methods: Retrospective cross-sectional study, patients with RVO
(n=41) and age-matched control subjects (n=25) were included. The
concentrations of angiogenic and inflammatory cytokines,including
VEGF, PDGF-AA, IL-1a, IL-6, IL-8, MCP-1, TNF-α, and IP-10,in
the aqueous humor were obtained before intravitreal injection of
bevacizumab and measured using suspension array technology. After
the retinal hemorrhage disappeared, fluorescein angiography(FA)
images were obtained. Based on FA data, RVO patients were divided
into a non-ischemic group and an ischemic group. We investigated
the presence of recurrent macular edema based on optical coherent
tomography (OCT) during the follow-up period. We compared the
levels of cytokines of RVO patients and control subjects, and between
the non-ischemic and ischemic groups and the presence of recurrent
macular edema or not.
Results: The aqueous humor levels of VEGF, PDGF-AA, IL-1a,
IL-6, IL-8, MCP-1, TNF-α, and IP-10 were higher in the RVO group
than in the control group. The aqueous humor levels of IL-8, PDFGFAA, TNF-α, and VEGF were significantly higher in the ischemic
RVO group than in the non-ischemic RVO group. We did not observe
any association between the cytokine levels and recurrent macular
edema.
Conclusions: Angiogenic and inflammatory cytokines were
overexpressed in RVO patients. Additionally, increased aqueous
humor levels of IL-8, PDFGF-AA, TNF-α, and VEGF at the onset of
RVO were associated with the development of future retinal ischemia
in RVO patients.
Aqueous Humor Levels(pg/ml) of Angiogenic and Inflammatory
Cytokines in the Control and RVO Groups.
Aqueous Humor Levels (pg/ml) of Angiogenic and Inflammatory
Cytokines in BRVO and CRVO (Presence or Lack of Non-Perfusion).
Commercial Relationships: Sung Jae Yang, None; Hyoung Jo,
None; Kyoung-A Kim, None; Sang Hoon Jung, None
Clinical Trial: none
Involvement of classical and lectin pathway in an immune
mediated model of glaucoma
Sabrina Reinehr, H. Burkhard Dick, Stephanie C. Joachim. Ruhr
University Eye Hospital, Experimental Eye Research Institute,
Bochum, Germany.
Purpose: Latest studies assume a participation of the complement
system (CS) in the development of glaucoma. However, less is
known about the pathways which activated the CS. In this study we
wanted to investigate which complement pathways may play a role in
the activation of the CS in an autoimmune model of glaucoma.
Methods: Rats were immunized with optic nerve homogenate (ONA)
or S100 protein. The control group (Co) received sodium chloride.
After 7 and 14 days cross-sections of the retina were stained with
C1q (Quidel), mannose binding lectin (MBL; Biozol), and mannoseassociated-serine-proteases 2 (MASP2; Biozol) (n=5-6/group).
Complement components were counted using ImageJ Software.
Statistical analysis was performed using t-test.
Results: 7 days after immunization, no difference could be observed
in C1q staining in both immunized groups compared to Co (ONA:
p=0.09; S100: p=0.7). Regarding MBL, less cells could be seen in
the ONA group compared to Co (p=0.0005), no changes could be
noted in the S100 group (p=0.8). Although mean values showed no
difference, we noted distinct more C1q and/or MBL positive cells in
single immunized eyes compared to Co. Regarding MASP2 staining,
significant more positive area could be seen in the ONA group
compared to Co (Co: 4.24%±5.03; ONA: 9.45%±7.22; p=0.000001).
No difference was noted in the S100 group (p=0.4). 14 days after
immunization, no changes in C1q or MBL staining could be observed
in the ONA animals (C1q: p=0.8; MBL: 0.5). Less C1q and MBL
was present in the S100 group (C1q: p=0.002; MBL: p=0.046). At
this point in time MASP+ area significantly decreased in ONA group
(p=0.000001). No changes could be observed in the S100 group
compared to Co (p=0.4).
Conclusions: C1q and MBL, the marker for classical pathway or
lectin pathway, were not significantly increased in both immunized
groups at both points in time. But we could detect more MASP2
activation in ONA animals after 7 days. Nevertheless, we noted in
single immunized eyes significant more C1q and/or MBL depositions
compared to Co. Additionally, in previous studies, we could show a
significant increase of C3 and the terminal pathway of the CS. These
data suggest, that the CS might be activated via the classical and
lectin pathway at an earlier point in time in this model.
Commercial Relationships: Sabrina Reinehr, None; H. Burkhard
Dick, None; Stephanie C. Joachim, None
Support: German Research Foundation JO-886/1-1
Identification of Trauma-Related Biomarkers Following Blast
Injuries to the Eye
Randolph D. Glickman1, 6, Dustin Stidger1, Brian J. Lund2, Stephan
Bach3, Andrea Kelley3, Walter Gray4, William E. Sponsel5, 6, Matthew
A. Reilly6. 1Ophthalmology, Univ of Texas Hlth Sci Ctr SA, San
Antonio, TX; 2Institute of Surgical Research, US Army, Ft. Sam
Houston, TX; 3Chemistry, Univ TX San Antonio, San Antonio, TX;
4
Geological Sciences, Univ TX San Antonio, San Antonio, TX;
5
WESMD Professional Associates, San Antonio, TX; 6Biomedical
Engineering, Univ TX San Antonio, San Antonio, TX.
Purpose: Injuries to neural tissue result in the expression of specific
biomarkers, due to the activation of repair processes or apoptotic
signaling pathways. In the case of blast injuries to the eye, detection
of these biomarkers may provide useful metrics for diagnosis,
prognosis, and therapy. The present investigation is designed to use
matrix assisted laser desorption ionization time of flight (MALDI/
TOF) to identify protein biomarkers in the eyes of animal models
exposed to calibrated blasts from a shock tube.
Methods: To prepare for live animal experiments, ex vivo porcine
eyes were commercially obtained and exposed to blasts produced by
a 17” diameter shock tube, with peak pressure ranging from 48.3 to
151.5 kPa and positive pulse duration from 2.1 to 2.8 ms, depending
on the number of aluminum disks placed between the driver chamber
and the expansion chamber. Following blast or sham exposure, ocular
samples were flash frozen and cut into 20-mm thick sections. The
sections were transferred to indium titanium oxide-coated slides and
coated with sinapinic acid as the matrix agent. Protein signatures
were detected in these sections over a range of 7,000 to 50,000 m/z
using a Bruker Daltonics Ultraflextreme MALDI/TOF.
Results: The optic nerves of the eyes were probed with the laser set
to 28% of maximum power. A representative mass spectrum obtained
from a control eye (not subjected to a blast) is shown in Fig 1, while a
spectrum obtained from an eye subjected to a blast of 120.9 kPA peak
pressure and 2.5 ms duration is shown in Figure 2.
Conclusions: The spectra from the control eye (Figure 1) and eye
subjected to blast trauma (Figure 2) are similar, but not identical.
There is a peak at m/z = 37,500 in the control eye that is not present
in the blast-subjected eye, and there is a sharp peak at m/z = 12,200
present only in the blast eye. Because ex vivo tissue would not be
expected to express new proteins, these differences are unlikely to
result from blast effects, but rather may represent variations between
ocular samples or perhaps the specific metabolic state or stress level
of the animals when slaughtered. Nevertheless, these results indicate
this experimental approach has the sensitivity to detect traumainduced biomarkers. Ongoing work is directed at the identification of
the specific proteins observed in the MALDI spectra.
Figure 1. MALDI spectrum from control eye.
mediated signaling may play important roles that extend beyond the
well-established vascular effects of Ang 1-7.
Commercial Relationships: Tuhina Prasad, None; Amrisha
Verma, None; Qiuhong Li, None
Support: Supported in part by grants from American Diabetes
Association, American Heart Association, Research to Prevent
Blindness, NIH grants EY021752 and EY021721.
Figure 2. MALDI spectrum from blast eye.
Commercial Relationships: Randolph D. Glickman, None; Dustin
Stidger, None; Brian J. Lund, None; Stephan Bach, None; Andrea
Kelley, None; Walter Gray, None; William E. Sponsel, None;
Matthew A. Reilly, None
Support: DOD Vision Research Program, Award Number
W81XWH-12-2-0055
Expression and Cellular Localization of Mas Receptor in the
Adult and Developing Mouse Retina
Tuhina Prasad, Amrisha Verma, Qiuhong Li. Ophthalmology,
University of Florida, Gainesville, FL.
Purpose: Recent studies have provided evidence that a local rennin
angiotensin system (RAS) exists in the retina and plays an important
role in retinal neuro-vascular function. We have recently shown
that increased expression of ACE2 and Ang1-7, two components
of the protective axis of the RAS, in the retina via AAV mediated
gene delivery, conferred protection against diabetes- induced
retinopathy. We hypothesize that the protective molecular and cellular
mechanisms of Ang1-7 are mediated by its receptor, Mas, and its
expression level and cellular localization dictate the response to
Ang 1-7 and activation of subsequent protective signaling pathways.
We tested this hypothesis by examining the expression and cellular
localization of the Mas receptor in adult and developing mouse retina.
Methods: Eyes from adult and postnatal day 1(P1), P5, P7, P15
and P21 mice were enucleated and fixed in 4% paraformaldehyde
overnight at 4°C.The cellular localization of Mas receptor protein
was determined by immunofluorescence from OCT-embedded frozen
sections or paraffin embedded sections using Mas receptor specific
antibody, and the mRNA was detected by in situ hybridization from
paraffin embedded sections using RNAscope kit from Advanced Cell
Diagnostics Inc. Western blotting and real–time RT-PCR analysis
were performed to determine the relative levels of Mas protein and
mRNA in retina and cultured retinal cells.
Results: In the adult eye, Mas receptor protein is abundantly present
in the retinal ganglion cells (RGC) and the photoreceptor cells, lower
level of expression is seen in endothelial cells, muller glial cells and
other neurons in the Inner plexiform layer of the retina. In developing
retina, both Mas receptor mRNA and protein expression can be
detected in the inner retina at P1 and the expression levels increase
with age to reach the adult level by P21.
Conclusions: Mas receptor is expressed in both adult and developing
mouse retina, more abundant in retinal neurons than endothelial
and muller glial cells. These observations suggest that Mas receptor
Pre-gliotic Müller cell responses in the isolated adult rat retina
Linnea T. Taylor, Karin M. Arner, Fredrik K. Ghosh. Ophthalmology,
Lund University, Lund, Sweden.
Purpose: Upregulation of GFAP intermediate filaments in Müller
cells is a well known consequence of retinal insult. However, the
timeline of glial-related events preceeding this reaction and their
relation to cell death progression have not been extensively explored.
Methods: Full-thickness retinal sheets were isolated from adult rat
eyes. 6x6 mm retinal explants were cultured for 1h, 3h, 6h, 12h, 24h,
48h and 5 days in vitro (DIV) using a previously established protocol
with the photoreceptors positioned against the culture membrane.
Adult rat eyes fixed immediately after enucleation were used as a
baseline in vivo control. The grafts were analyzed morphologically
using hematoxylin and eosin staining (H&E), immunohistochemistry
with antibodies directed against several Müller cell proteins, and
apoptosis (TUNEL labeling).
Results: Already 1h after explantation, Müller cell expression of
carbonic anhydrase II and glutamine synthetase (GS) was found to be
strongly upregulated compared to baseline. Early events also included
cellular retinaldehyde-binding protein (CRALBP) upregulation
after 12h. These three Müller cell related proteins as well as basic
fibroblast growth factor (bFGF) were progressively downregulated
after 12h - 5 DIV compared to baseline whereas GFAP expression
was unchanged from 1h-24h, and upregulated late, at 2-5 DIV.
TUNEL labeling was evident in the ganglion cell layer (GCL) at 1h.
At 6 and 12h, numerous cells were labeled in the GCL and the inner
nuclear layer (INL). At 24h, scattered cells were labeled in the INL.
After 48h, isolated labeled cells were found in the INL and outer
nuclear layer, and at 5 DIV, a multitude of labeled cells were present
in all cell layers.
Conclusions: Several Müller cell proteins important for retinal
homeostasis alter their expression almost immediately after retinal
insult. These changes preceed GFAP upregulation which is a
comparably late event. The pre-gliotic events and their relation to
cell death dynamics provides important insight into early cell death
mechanisms after retinal injury.
Commercial Relationships: Linnea T. Taylor, None; Karin M.
Arner, None; Fredrik K. Ghosh, None
MMP-3 EXPRESSION IN THE DAMAGED MOUSE RETINA:
A ROLE IN GLIAL REACTIVITY?
Manuel Salinas-Navarro1, Lies De Groef1, Eline Dekeyster1, Ingeborg
Stalmans2, Inge Van Hove1, Lieve K. Moons1. 1Laboratory for Neural
Circuit Development and Regeneration, KU Leuven, Leuven,
Belgium; 2Department of Ophthalmology, KU leuven, Leuven,
Belgium.
Purpose: Matrix metalloproteinase-3 (MMP-3) has been associated
with neuro-inflammatory and neurodegenerative diseases. A recent
transcriptome profiling study also revealed highly upregulated
levels of MMP-3 mRNA in retinas after optic nerve axotomy. As the
involvement of MMP-3 to optic neuropathies has been understudied,
we investigated its role in RGC survival and glial reactivity in mice
subjected to optic nerve crush (ONC), a model to study glaucoma
pathophysiology
Methods: The left optic nerve was intraorbitally crushed in
MMP-3 deficient (MMP-3-/-) and wild type (WT) mice and
retinas were dissected at several survival intervals after crush (6,
12, 24 & 48h, 4 & 7d). Expression of MMP-3 was examined via
immunohistochemistry (IHC) and western blot (WB). RGC survival
was assessed at 4 and 7 days after ONC by quantifying the number of
surviving RGCs after Brn3a immunostaining on whole mount retinas
using distribution maps and automatical analyses. IHC for Iba-1,
GFAP and GS was performed on retinal whole mounts and/or radial
sections, which were all examined and photographed using confocal
microscopy. The expression of tissue inhibitor of metalloproteinase-1
(TIMP-1) is currently being analyzed via WB and IHC
Results: IHC revealed MMP-3 expression in the processes of Müller
glia throughout the entire retina of the healthy mouse eye. MMP-3
protein expression dramatically increases at early time points after
ONC, peaks at 4d after injury and is highly associated with glial
cells, as confirmed by both WB and IHC. Strikingly, the number of
surviving RGCs was found to be similar in MMP-3-/- and WT mice,
both at 4 days (70 ± 9% vs 72 ± 6%; n=15) and at 7 days (22 ± 6% vs
25 ± 4%; n=10) post-surgery. IHC stainings for the glial cell markers
Iba-1, GFAP and GS revealed a similar expression in both genotypes.
To further investigate the underlying mechanisms we are currently
analysing the spatio-temporal expression pattern of the endogenous
MMP-3 inhibitor TIMP-1, in the retina of MMP-3-/- and WT mice
subjected to ONC
Conclusions: MMP-3 expression, found in Muller glia cells in the
healthy retina, is highly upregulated in the injured retina. Given its
glial expression, MMP-3 could contribute to glial reactivity, implying
an involvement in retinal homeostasis. However, MMP-3 deficiency
did not protect retinas from ONC-induced RGC death. Future
experiments aim at unraveling the role of this proteinase in glaucoma
pathogenesis
Commercial Relationships: Manuel Salinas-Navarro, None; Lies
De Groef, None; Eline Dekeyster, None; Ingeborg Stalmans,
None; Inge Van Hove, None; Lieve K. Moons, None
Support: Belgian FWO fellowship
(CXCL12/CXCR4 and CCL2/CCR2) play a role in the modulation
of spinal pain. To date the precise localization of these chemokines
CXCL12 and CCL2 in the trigeminal sensory innervation of the
cornea has not been described
Methods: Tissue preparation: Adult male C57BL/6 mice were
deeply anesthetized and perfused with saline followed by 4%
paraformaldehyde. Tissues (eye, trigeminal ganglion and brainstem)
were kept overnight in 4% paraformaldehyde. Coronal brainstem
sections (40 mm) were cut on a vibratome. Eye and trigeminal
ganglion were cryoprotected, frozen and cryostat (16 mm) sections
were taken.
Light and immunofluorescence labeling : tissues sections were
incubated with CXCL12 (Torrey Pines) and CCL2 (Torrey Pines)
polyclonal antibodies. CXCL12 and CCL2 immunoreactivity
(IR) were detected either with 3,3ʹ-Diaminobenzidine (DAB) or
fluorescent probes.
Results: In corneal cryosections, CXCL12-IR and CCL2-IR are
detected in epithelial cells and keratocytes. Interestingly, we also
observed CXCL12 and CCL2 labelling in corneal nerve fibers. At
the trigeminal ganglion level, in the ophtalmic V1 region we noted
a basal/ constitutive production of CXCL12 and CCL2 in primary
afferent neurons and in glial satellite cells. Microscopic analysis
of brainstem sections immunostained with CXCL12 and CCL2
antibodies showed that both chemokines are detected in neurons
and nerve fibers at the ventral trigeminal subnucleus interpo-laris–
caudalis (Vi/Vc) transition and in the trigeminal subnucleus caudalis–
cervical cord (Vc/C1) junction region.
Conclusions: This anatomical study reported for the first time the
distribution of CXCL12 and CCL2 along the corneal trigeminal
neuronal pathways. Further investigations are now needed to evaluate
whether these chemokines may play a role in the neuromodulation of
trigeminal corneal pain.
436 Current Topics in Anatomy
Wednesday, May 07, 2014 11:00 AM–12:45 PM
Distribution of the CXCL12 and CCL2 chemokines in the corneal
trigeminal pathways : from the cornea to the brainstem
Annabelle Reaux-le Goazigo1, Pierre-Serge Launay1, Christophe
Baudouin1, 2, Stephane Melik-Parsadaniantz1. 1UPMC Univ Paris
06, UMR_S 968, CNRS, UMR_7210, Institut de la Vision, Paris,
F-75012, France; 2Centre Hospitalier National d’Ophtalmologie des
Quinze-Vingts, INSERM-DHOS CIC 503, Paris, F-75012, France.
Purpose: The sensory innervation of the cornea originated from the
ophtalmic division of the trigeminal ganglion whose neurons send
fibers centrally to terminate in the trigeminal brainstem complex.
Our recents studies showed that the chemokines CXCL12/SDF-1 and
CCL2/MCP-1 and their receptors CXCR4 and CCR2 are produced by
sensory neurons and glial cells in dorsal root ganglia and the spinal
cord (Reaux-Le Goazigo et al., 2012 ; 2013) where both couples
CXCL12 immunoreactivity in trigeminal ganglion
Conclusions: In this study, the diagnosis of eyelid SAs was rare.
However, the importance of this benign eyelid tumor stems from its
association with internal malignancies in Muir-Torre syndrome. For
this reason, it is essential that ophthalmologists initiate a systemic
work-up of all patients with eyelid SAs. To diagnose a SA, a biopsy
should be sent for histopathological assessment, as the clinical
impression is unreliable. Finally, immunohistochemical staining of
mismatch repair proteins MLH1 and MSH2 is a valid and accessible
strategy for investigating MTS.
CXCL12-IR in spinal trigeminal nucleus
Commercial Relationships: Annabelle Reaux-le Goazigo, None;
Pierre-Serge Launay, None; Christophe Baudouin, Alcon (C),
Allergan (C), Santen (C), Thea (C); Stephane Melik-Parsadaniantz,
None
Support: This study was supported by agence Nationale pour la
Recherche (Grant J12R135 Project CHEMOK PAIN 2) and LABEX
LifeSence Grant
Sebaceous adenomas of the eyelid and Muir-Torre syndrome
Lisa Jagan, Vasco Bravo-Filho, Patrick Logan, Mohammed Qutub,
Eman Al-Sharif, Miguel N. Burnier. Department of Ophthalmology,
McGill University, Montreal, QC, Canada.
Purpose: Sebaceous adenomas (SAs) are rare, benign sebaceous
gland tumors that typically manifest as yellow, circumscribed nodules
on the eyelid, face and scalp. Histopathologically, these lesions are
composed of multiple sebaceous lobules that vary in size, shape, and
differentiation. SAs may be associated with internal malignancies,
particularly gastric and colon cancer. This association, known as
Muir-Torre syndrome (MTS) is caused by the autosomal dominant
inheritance of defective genes encoding DNA mismatch repair
proteins. The purpose of this study was to assess the prevalence of
SAs and MTS, to determine the reliability of the clinical diagnosis
of SAs, and to evaluate the immunohistochemical staining patterns
of the DNA mismatch repair proteins MLH1 and MSH2 for a recent
MTS case.
Methods: The histopathology reports from all eyelid specimens
collected between 1993-2013 at the Henry C. Witelson Ocular
Pathology Laboratory were evaluated to determine the number of
SAs with or without associated MTS. The clinical diagnosis of
each eyelid lesion was compared to the confirmed histopathological
diagnosis in order to determine concordance. Immunohistochemical
staining for MLH1 and MSH2 was performed for each SA.
Results: Of the 5884 eyelid specimens collected, nine were
SAs (6 females, 3 males; 42-72 years old). The diagnosis of
SA was clinically suspected in only one of the nine cases.
MTS was discovered in one patient with an associated colon
adenocarcinoma. The recognition of the eyelid SA on histopathology
was the main reason for systemic investigation in this patient.
Immunohistochemical staining for this case revealed positive MLH1
expression and negative MSH2 expression, confirming a diagnosis of
MTS.
Commercial Relationships: Lisa Jagan, None; Vasco Bravo-Filho,
None; Patrick Logan, None; Mohammed Qutub, None; Eman AlSharif, None; Miguel N. Burnier, None
Topography of lymphatic markers in human anterior uvea
Falk Schroedl2, 1, Alexandra Kaser-Eichberger2, Andrea Trost2,
Clemens Strohmaier2, Barbara Bogner2, Christian Runge2, Martin
Laimer4, Simona L. Schlereth3, Ludwig M. Heindl3, Herbert A.
Reitsamer2. 1Anatomy, Paracelsus University Salzburg, Salzburg,
Austria; 2Ophthalmology, Paracelsus University Salzburg, Sallzburg,
Austria; 3Ophthalmology, University Cologne, Cologne, Germany;
4
Dermatology, Paracelsus University Salzburg, Salzburg, Austria.
Purpose: Reports of lymphatics in the anterior human uvea are
contradictory. This might be caused due to a certain topography
which has not been considered yet. Therefore we here systematically
analyze iris and adjacent ciliary body with immunohistochemistry by
combining various lymphatic markers.
Methods: Meeting the Declaration of Helsinki, human iris and
ciliary body were obtained from cornea donors and prepared for
cryosectioning. Cross sections of tissue blocks at 12, 3, 6, 9 o`clock
position and at all corresponding intersections (i.e., 1.30, 4.30, 7.30,
10.30) were processed for immunohistochemistry of the following
lymphatic markers: LYVE1, podoplanin, PROX1, FOXC2, VEGFR3,
CCL21. Additionally applying DAPI, double, triple and quadruple
combination of aforementioned markers were documented using
confocal microscopy.
Results: In the iris, LYVE 1 positive cells of various morphology
were distributed throughout the non-pigmented part. These cells
were lacking podoplanin. Numerous podoplanin positive cells were
mainly located at the anterior border of the iris. These cells were not
colocalized with LYVE1 or PROX1, FOXC2, CCL21, VEGFR3.
While podoplanin positive cells were only rarely detected posteriorly
of the iris root, many LYVE1 positive cells were also present
within the cilary muscle and ciliary body villi. In the ciliary muscle,
occasionally podoplanin+ vessel-like structures were detectable,
but these were never colocalized with LYVE-1. Similar vessel-like
structures immunoreactive for VEGFR3 never displayed PROX1
or CCL21. A certain topography of structures at the various uveapositions investigated was not obvious.
Conclusions: Structures colocalizing for at least two lymphatic
markers were not detectable at positions investigated, and further a
certain topography was not obvious. Putative lymphatic channels of
the anterior uvea therefore might display a different marker panel
than generally presumed.
Commercial Relationships: Falk Schroedl, None; Alexandra
Kaser-Eichberger, None; Andrea Trost, None; Clemens
Strohmaier, None; Barbara Bogner, None; Christian Runge,
None; Martin Laimer, None; Simona L. Schlereth, None; Ludwig
M. Heindl, None; Herbert A. Reitsamer, None
Support: FFG830770, PMU-FFF R13-/01/042-KAS, FuchsFoundation, Lotte-Schwarz Endowment
Absence of lymphatic vessels in the developing human sclera
Barbara Neuser1, Simona L. Schlereth1, Martina C. Herwig2, Annette
M. Müller3, Falk Schroedl4, Konrad R. Koch1, Claus Cursiefen1,
Ludwig M. Heindl1. 1Department of Ophthalmology, University
of Cologne, Cologne, Germany; 2Department of Ophthalmology,
University of Bonn, Bonn, Germany; 3Department of Pediatric
Pathology, University of Bonn, Bonn, Germany; 4Departments
of Ophthalmology and Anatomy, Paracelsus Medical University,
Salzburg, Austria.
Purpose: The adult human sclera contains a network of small sized
blood vessels and LYVE1+ macrophages mainly in the episclera, but
lacks LYVE1+/podoplanin+ lymphatic vessels. Since it is not known
whether the fetal sclera is primarily alymphatic or contains lymphatic
vessels during the intrauterine scleral development, we investigate for
blood and lymphatic vessels in human fetal scleral tissue at different
gestational ages.
Methods: Meeting the declaration of Helsinki, fetal human sclera
from 35 abortions / stillborns (12-38 weeks of gestation (WoG);
12-18 WoG, n=10; 19-23 WoG, n=13; and 24-38 WoG, n=11) was
analyzed immunhistochemically for blood (CD31+) and lymphatic
vessels (LYVE+, podoplanin+) at anterior, equatorial, and posterior
location. The number of CD31+blood vessels or LYVE1+cells was
correlated with gestational age.
Results: The human sclera contained CD31+ blood vessels as early
as WoG 13. Until 18 WoG, the scleral stroma is enriched with blood
vessels in a similar amount as the episclera (p=0.4). After 19 WoG
the amount of stromal CD31+ blood vessels decreased significantly
compared to episclera (p<0.02 at 19-23WoG and p<0.001 at 2438WoG). In the lamina fusca blood vessels were absent at any time
point investigated. And further, LYVE1+ or podoplanin+ lymphatic
vessels were not detectable in the sclera at any gestational ages
analyzed. However, single LYVE1+cells were identified primarily
in the episclera their amount decreasing significantly with increasing
gestational ages (12-18 WoG compared to 24-38WoG: p<0,01). In
contrast to the conjunctiva where LYVE1+podoplanin+ lymphatics
were detectable as early as week 17, the amount of LYVE1+cells
in the sclera was highest in early pregnancy (12-18WoG), with a
significant decrease during pregnancy (p<0.001).
Conclusions: The fetal human sclera contains CD31+blood vessels
as early as week 13, but is primarily alymphatic. However, single
LYVE1+ cells could be identified in early pregnancy, decreasing
in number with ongoing pregnancy. Compared to the conjunctiva,
with high amounts of LYVE1+cells in early pregnancy and growth
of lymphatic vessel as early as week 17, our findings within
the sclera suggest an early and strong expression of selective
antilymphangiogenic factors during development present in the
sclera.
Commercial Relationships: Barbara Neuser, None; Simona L.
Schlereth, None; Martina C. Herwig, None; Annette M. Müller,
None; Falk Schroedl, None; Konrad R. Koch, None; Claus
Cursiefen, None; Ludwig M. Heindl, None
Support: DFG grant # HE 6743/2-1 to L.M. H., DFG grant
#CU47/6-1, Bayer Graduate School Pharmacology, Ruth and Helmut
Lingen Stiftung Cologne to C. C., Cologne GEROK program to S. L.
S.
Optic Nerve Sheath Mechanics and Permeability in VIIP
Syndrome
Julia Raykin1, Lauren Best2, Rudy Gleason1, Lealem Mulugeta3, Jerry
Myers2, Emily Nelson2, Brian C. Samuels4, C R. Ethier1. 1Department
of Biomedical Engineering, Georgia Institute of Technology/
Emory University, Atlanta, GA; 2NASA Glenn Research Center,
Cleveland, OH; 3Universities Space Research Association, Houston,
TX; 4Department of Ophthalmology, U. Alabama at Birmingham,
Birmingham, AL.
Purpose: To investigate the biomechanical properties of the optic
nerve sheath (i.e. fluid permeation across the meninges and tissue
stiffness) at various CSF pressures. Such information is relevant to
understanding ophthalmic changes associated with long-duration
space flight (i.e. Visual Impairment and Intracranial Pressure
syndrome; VIIP), thought to be largely due to cephalad fluid shifts
causing altered ocular, cardiovascular, lymphatic and cerebrospinal
fluid (CSF) pressures. The hypothesis is that increased CSF pressure
drives connective tissue remodeling of the posterior eye and optic
nerve sheath.
Methods: The meninges of fresh porcine eyes (n=10) was reflected
to expose the optic nerve, which was truncated 3-4 mm posterior
to the sclera. The meninges were repositioned leaving a “hollow”
cylinder of connective tissue attached to the posterior sclera. The
distal end was cannulated, sealed, and attached to a pressure control
system that simulated CSF pressure changes. The anterior chamber
of the eye was also cannulated for independent control of intraocular
pressure (IOP). While IOP remained stable, a CCD camera recorded
the meningeal diameter as the CSF pressure cycled between 7-50
mmHg. In a second set of experiments, the rate of fluid permeation
across the meninges was recorded by observing the drainage of an
elevated fluid reservoir connected to the meninges.
Results: Cyclic pressure-diameter curves showed a preconditioning
effect, with repeatable behavior in cycles 4-6 (Figure). The meninges
showed marked nonlinear stiffening, particularly at CSF pressures
>15 mmHg. The tangent moduli extracted from these data were
318, 745, and 1273 kPa at CSF pressures of 7, 15 and 30 mmHg,
respectively. Permeability experiments determined a flow rate
of ~2ml/hr through the intact meninges at a driving pressure of
30mmHg, corresponding to a permeability of 7.34x10-5 ml/min/cm2/
mmHg.
Conclusions: The meninges demonstrate biomechanical properties
typical of other soft tissues, with nonlinear stiffening and appreciable
hysteresis on pressure cycling. This tissue is surprisingly permeable,
suggesting that there could be important CSF drainage through the
meninges into the periorbital fat. These experimental measurements,
extended to cadaveric eyes, will be critical in informing
computational models aimed at identifying the pathophysiology of
VIIP syndrome.
Increased or decreased Cyp1b1 inhibited retinal and NC survival.
Additional studies assessed substrates and targets of Cyp1b1 in eye
development. While Cyp1b1 regulation of eye development was
not mediated by estradiol, Cyp1b1 overexpression partially rescued
defects due to retinoic acid (RA) deficiency. However, Cyp1b1
did not regulate transcript expression of RA synthesis (raldh2) or
degradation (cyp26c1) enzymes or pitx2, a known downstream target
of RA in the periocular mesenchyme. In addition, alterations of
Cyp1b1 did not significantly change transcript expression of genes
known to be associated with colobomas such as vsx2, otx2, dvr1,
gdf6, and pax6.
Conclusions: We demonstrated that cyp1b1 expression spatially and
temporally correlated with the ocular fissures and regulated fissure
closure. Further, Cyp1b1 had a non-cell autonomous effect on NC
proliferation and migration. Although this effect is partially mediated
by RA, additional downstream targets remain to be elucidated. These
studies give insight into how mutations in CYP1B1 disrupt ocular NC
development in the pathogenesis of congenital glaucoma.
Pressure-diameter curves for a single eye, tested over six 7-50 mmHg
pressure cycles.
Commercial Relationships: Julia Raykin, None; Lauren Best,
None; Rudy Gleason, None; Lealem Mulugeta, None; Jerry
Myers, None; Emily Nelson, None; Brian C. Samuels, None; C R.
Ethier, None
Support: NASA NNX13AP91G
Cyp1b1 Regulates Ocular Fissure Closure and Subsequent
Neural Crest Migration
Brenda L. Bohnsack, Bahaar Chawla. Ophthalmology and Visual
Sciences, University of Michigan, Ann Arbor, MI.
Purpose: CYP1B1 gene mutations are a common cause of congenital
glaucoma, but the mechanism by which CYP1B1 regulates eye
development is unknown. In these studies, we used zebrafish to study
the role of cyp1b1 in the eye.
Methods: Morpholino oligonucleotides (MO) and mRNA injections
were used with exogenous regulators and analogs of estradiol and
retinoic acid (RA). Embryos were analyzed by time-lapse imaging, in
situ hybridization, immunostaining, TUNEL assay, and histology.
Results: Cyp1b1 was expressed in the retina with localization in
the ocular fissures by 24 hours post fertilization (hpf). Cyp1b1
expression peaked at 36hpf and then disappeared in the dorsal and
ventral retina by 60 and 72hpf, respectively. Cyp1b1 knockdown
did not inhibit early (12-24hpf) NC migration into the craniofacial
region. However, Cyp1b1 was required for later (24-60hpf) NC
migration through the ocular fissures and iridocorneal angle
formation. Cyp1b1 overexpression prevented closure of the inferior
ocular fissure resulting in prominent colobomas (Fig). While Cyp1b1
overexpression did not inhibit NC migration, failure of fissure
closure disrupted NC organization around the lens. Knockdown
of Cyp1b1 significantly increased retinal and NC cell proliferation
while overexpression did not affect the number of cells in S-phase.
Methods: 20 myopic subjects (n=40 eyes) without other identified
pathologies participated in this study: 20 eyes of ≤3 diopters [D] (low
myopic), 10 eyes between -3D and -6D (mildly myopic), and 10 eyes
of ≥6 D (highly myopic). The mean age of subjects was 30.2 years
(±7.6 years; range, 24 to 46 years). A 100-kHz SD-OCT system,
capable of 6-mm ranging distance and 12-mm-wide field of view, was
used to acquire 3D datasets from the subjects. The 3D OCT image
of choroid was segmented into superior, inferior, nasal and temporal
quadrants. The measured CT represented the normal distance between
outer retinal pigment epithelium (RPE) layer and inner scleral border.
Measurements were made within concentric regions centered at the
fovea, extended to 5 mm away from the fovea at 1 mm intervals in
the nasal and temporal directions. Statistical analysis was performed
to evaluate CT at each region and observe the relationship between
CT and the degree of myopia.
Results: For low myopic eyes, CT is the thickest under the fovea
(273.85±49.01μm) as is in the case of healthy eyes. Peripheral to
the fovea, the mean CT decreases rapidly along the nasal direction,
reaching a minimum of 180.65±58.25μm at 5 mm away from the
fovea; this value is 234.25±42.27μm in the temporal direction. In
contrast, the thickest CTs lie in the temporal region for mildly and
highly myopic eyes (where CT=194.94±27.28μm and 163±34.89μm,
respectively), whilst the thinnest CTs appear in the nasal region
(where CT= 100.84±16.75μm and 86.64±42.6μm, respectively). The
CT reduction rate is calculated as 31.28μm/diopter from low to mild
myopia, whilst it is 13.49μm/diopter from mild to high myopia.
Conclusions: The CT decreases gradually with the increased myopia.
The current results support the theory that choroidal abnormality may
play an important role in the pathogenesis of myopic degeneration.
Commercial Relationships: Brenda L. Bohnsack, None; Bahaar
Chawla, None
Support: NH Grant K08EY022912-01, Research to Prevent
Blindness Career Development Award, Edward Mallinckrodt Jr
Foundation Grant
Measuring Choroidal Thickness in Myopic eyes using 1060-nm
Spectral Domain Optical Coherence Tomography
qinqin zhang1, Maureen Neitz2, Jay Neitz2, Ruikang K. Wang1.
1
Department of Bioengineering, university of washington, seattle,
WA; 2Department of Ophthalmology, university of washington,
seattle, WA.
Purpose: To measure macular choroidal thickness (CT) using
1-micron SD-OCT system and to investigate the geographic CT
differences among low, mildly and highly myopic subjects.
Fig.1 Typical geographic maps of CT of (a) low, (b) mild and (c) high
myopic eyes.
Commercial Relationships: qinqin zhang, None; Maureen Neitz,
None; Jay Neitz, None; Ruikang K. Wang, Carl Zeiss Meditec (P),
Optovue Inc. (F)
470 Uveal melanoma: Pathophysiology, Managment and
Prognosis
Wednesday, May 07, 2014 3:45 PM–5:30 PM
Effects of various MAPK and PI3K/Akt inhibitors on the
constitutive secretion of VEGF by uveal melanoma cells
Dan-Ning Hu, Steven A. McCormick, Tommaso Vagaggini, Richard
B. Rosen. Pathology & Ophthalmology, New York Eye & Ear
Infirmary, New York, NY.
Purpose: VEGF plays a critical role in angiogenesis and in the
growth and metastasis of uveal melanoma (UMa). Studying the
regulation of secretion of VEGF by UMa cells with MAPK and
PI3K/Akt signal pathways is important for understanding the
pathogenesis of and target therapy for UMa.
Methods: The constitutive levels of VEGF secretion were tested
in six human immortal UMa cell lines (MP17, M21, M23, SP6.5,
SP8.0 and TP3.1). Cells were seeded into 24-well plates and cultured
with serum-free culture medium. After 24 hr, the conditioned culture
medium was collected, and the amount of VEGF was measured
using a sandwich enzyme-linked immunosorbent assay kit (R & D
Systems). In a separate signal pathway study, cells were cultured
siminarly with or without various signal pathway inhibitors,
including LY294002 (PI3K/Akt inhibitor), UO1026 (ERK inhibitor),
SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor).
The conditioned media were collected 24 hr later, and the amount of
VEGF was measured as described above. All tests were performed in
triplicate.
Results: VEGF could be detected in the conditioned medium at a
relatively high concentration from all cultured UMa cells (1989 ± 71
pg/106 cells/24 hr). Treatment of cells with PI3K/Akt inhibitor and
ERK inhibitor significantly decreased VEGF levels in the conditioned
media to 41% and 46% of that in controls (P < 0.05). JNK1/2
inhibitor and p38 MAPK inhibitor did not cause a significant change
in the VEGF production by UMa cells (P > 0.05).
Conclusions: Uveal melanoma cells have a significantly constitutive
secretion of VEGF. Inhibition of PI3K/Akt and ERK pathways, but
not JNK1/2 and p38 MAPK pathways, decreases the constitutive
secretion of VEGF by UMa. This indicates that PI3K/Akt and
ERK pathways play an important role in the constitutive secretion
of VEGF by UMa cells and might be considered as a target in the
prevention and treatment of UMa.
Commercial Relationships: Dan-Ning Hu, None; Steven A.
McCormick, None; Tommaso Vagaggini, None; Richard B. Rosen,
Clarity (C), OD-OS (C), Optovue (C)
Support: Pathology Research Fund, New York Eye and Ear
Infirmary and the Bendheim-Lowenstein Family Foundation, New
York
The expression of FoxC1 in Uveal Melanoma
Silvin Bakalian, Dana Faingold, Pablo Zoroquiain, Matthew Balazsi,
Natàlia Vilà, Emilia Antecka, Miguel N. Burnier. McGill University,
St. Laurent, QC, Canada.
Purpose: Forkhead box transcription factor C1 (FoxC1) plays an
important role in the eye differentiation. FoxC1 overexpression was
linked to poor survival in hepatocellular carcinoma and breast cancer.
Other reports showed decreased metastasis of breast cancer cells
following FoxC1 overexpression. The aim of this study is to evaluate
the expression of FoxC1 inuveal melanoma (UM).
Methods: Fifty one cases of paraffin-embedded, human UM
were immunostained with a polyclonal FoxC1 antibody.
Immunohistochemical expression was assessed based on staining
extent and intensity. Extent was evaluated as the percentage of
cells showing positive staining (0=none, 1= ≤50% of the tumour
cells; 2=51-75% of the tumor cells; and 3=≥76% of the tumor
cells). Staining intensity was assessed as follows (0=none; 1=weak;
2=moderate; and 3=strong). The cases were grouped into low FoxC1
expression when combined extent and intensity scores were between
1 and 4, and high FoxC1 expression when total score was 5 and 6.
Clinical-pathological data were obtained, including age, gender, cell
type, and largest tumor dimension (LTD), which were correlated with
FoxC1 expression.
Results: The expression of FoxC1was positive in all cases of
UM samples (51 specimens). The immunostaining was always
cytoplasmic. In addition, weak intranuclear staining was seen in 16
cases. Twenty specimens showed low FoxC1 expression, while 31
specimens showed high FoxC1 expression. The mean age of UM
patients was 62 years, and the mean follow up period was 52 months.
The mean LTD was 11mm. There were 8 (16%) cases of spindle UM,
26 (51%) cases of mixed UM and 17 (33%) cases of epithelioid cell
type UM. All intratumoral vessels as well as retinal and choroidal
vessels displayed negative immunostaining for FoxC1. Survival
analysis did not show statistically differences between the two groups
of patients (low expression of FoxC1 versus high expression of
FoxC1). However, there was a trend with a longer survival rate for
the group of patients with low expression of FoxC1.
Conclusions: To the best of our knowledge this is the first study
on the expression of FoxC1 in primary uveal melanoma. A positive
correlation of intranuclear staining with metastasis has been reported
in other malignancies. Due to the fact that FoxC1 is expressed in the
cytoplasm of uveal melanoma cells, it is possible that FoxC1 plays a
different role in this particular malignancy.
Commercial Relationships: Silvin Bakalian, None; Dana
Faingold, None; Pablo Zoroquiain, None; Matthew Balazsi, None;
Natàlia Vilà, None; Emilia Antecka, None; Miguel N. Burnier,
None
FOXO1 expression in uveal melanoma and ocular tissues of
normal eyes
Vasco Bravo-Filho1, 2, Silvin Bakalian1, Paula Blanco1, Li-Anne Lim1,
Emilia Antecka1, José João Mansure1, Miguel N. Burnier1, 2. 1Ocular
Pathology, McGill University, Montreal, QC, Canada; 2Department
of Ophthalmology and Visual Sciences, Federal University of São
Paulo, São Paulo, Brazil.
Purpose: Forkhead transcription factor class O1 (FOXO1) is
necessary for the normal development of the vascular system.
Loss of FOXO1 may be associated with tumorigenesis and
cancer progression. Also, FOXO1 is involved in cancer cell
chemoresistance. Our purpose was to evaluate and compare the
expression of FOXO1 in eyes with uveal melanoma (UM) to normal
eyes.
Methods: Twenty-six eyes with UM and pertinent clinical
information were retrieved from the Henry C. Witelson
Ocular Pathology Laboratory (McGill University, Canada).
Immunohistochemical expression of FOXO1 in eyes harboring UM
was compared to 25 normal eyes (>50 years old; obtained from The
Eye Bank of Canada). FOXO1 staining was scored by an ocular
pathologist according to intensity and extent: low (1), moderate
(2), or strong (3) for the former; and less than 50% (1), between
50 and 80% (2), and more than 80% (3), for the latter. Data were
then converted to a numeric final score by multiplying the intensity
and extent staining scores, resulting in a score ranging from 0 to 9
(0=negative;1–3=weak;4–6=moderate; 7–9=strong). The Student’s
t-test was used to determine statistical significance. A KaplanMeier survival analysis using the log-rank test was used to evaluate
differences in survival curves according to staining score.
Results: FOXO1 was expressed in 57.3% of UM cells. Choroidal and
retinal vessels in normal eyes had higher FOXO1 scores compared
to tumor harboring eyes (P=0.0001). In UM eyes, tumor cells had
higher FOXO1 scores compared to normal choroidal melanocytes
(P=0.0001). There was no statistical difference in expression in
the other eye structures between the two groups and there was no
association between expression of FOXO1 and metastases or FOXO1
and cell type.
Conclusions: Uveal melanoma cells showed higher expression
of FOXO1 than normal melanocytes. Due to the evidence that
FOXO1 decreases apoptosis via the PI3K/Akt pathway, the higher
expression of FOXO1 in melanoma cells may indicate increased cell
proliferation and viability of the malignant cells. The low expression
of FOXO1 in normal choroidal and retinal vessels in uveal melanoma
cases should be further investigated.
Commercial Relationships: Vasco Bravo-Filho, None; Silvin
Bakalian, None; Paula Blanco, None; Li-Anne Lim, None; Emilia
Antecka, None; José João Mansure, None; Miguel N. Burnier,
None
Support: CAPES and Sean Murphy Ocular Pathology Pan-American
Fellow
SIRT2 Expression is Higher in Uveal Melanoma Than Ocular
Melanocytes
Henry A. Wood, Pablo Zoroquiain, Patrick Logan, Shawn C
Maloney, Nouf AlSaati, Miguel N. Burnier. Henry C. Witelson Ocular
Pathology Laboratory, McGill University, Montreal, QC, Canada.
Purpose: Current research has illustrated the potential of Sirtuins to
contribute to neoplastic progression in different cancers. SIRT2 is a
sirtuin that has been shown to be involved in cell cycle progression
and changes in protein acetylation. Uveal Melanoma (UM) is the
most common primary intraocular tumour in adults, with 45% of
afflicted patients developing metastasis. The aim of this study was to
characterize the expression of SIRT2 in UM cases and compare with
expression of SIRT2 in the uveal tract of normal human eyes (NHE).
Methods: Twenty-one formalin-fixed, paraffin-embedded human UM
cases were immunostained for SIRT2, along with 15 NHE obtained
from the Eye Bank of Canada. The uveal tract was evaluated in all
eyes and tumor-specific staining was evaluated in the UM cases.
Immunostaining was graded based on intensity and extent of staining.
Intensity was classified as 0 (negative), 1 (weak), 2 (moderate), or
3 (strong). Extent was classified as 0 (negative), 1 (<50% positive
cells), 2 (50-80% positive cells), or 3 (>80% positive cells). A final
immunoreactive score (IRS) was calculated as follows: intensity x
extent. Mean IRS was calculated for each group and differences were
evaluated using analysis of variance (ANOVA).
Results: The mean SIRT2 IRS for each cell type in UM cases (+/standard deviation) was as follows: 4.4 +/- 2.9 for UM cells, 0.9 +/0.7 for choroidal melanocytes, and 1.0 +/- 1.7 for iris melanocytes.
For NHE, IRS for choroidal melanocytes was 0.5 +/- 0.6. Iris
melanocytes were not evaluated due to absence of the anterior
segment in those eyes. UM cells had a significantly higher IRS than
all groups of evaluated normal melanocytes in both UM and NHE
cases (p<0.05). No significant difference in IRS was found when
comparing normal melanocytes in UM and NHE cases.
Conclusions: The results of this study demonstrate that SIRT2
expression is significantly stronger in uveal melanoma cells than
normal ocular melanocytes. This finding may indicate an important
role of SIRT2 as a prognostic marker in uveal melanoma progression.
Commercial Relationships: Henry A. Wood, None; Pablo
Zoroquiain, None; Patrick Logan, None; Shawn C Maloney,
None; Nouf AlSaati, None; Miguel N. Burnier, None
VEGF-A expression is positively correlated with metastasis in
uveal melanoma
Patrick Logan, Shawn Maloney, Vasco Bravo-Filho, Natàlia Vilà,
Matthew Balazsi, Miguel N. Burnier. McGill University, Montreal,
QC, Canada.
Purpose: Vascular endothelial growth factor (VEGF)-A expression in
uveal melanoma (UM) has previously been described; however, there
is a lack of consensus about whether or not expression correlates with
metastasis. In the present study, we developed a custom algorithm
for objectively quantifying VEGF-A immunostaining in UM and
determined if VEGF-A expression correlates with metastasis.
Methods: The inclusion criteria for patient samples were: choroidal
UM diagnosis, minimum 5-year follow-up, data on the presence of
metastases, and sufficient tumor size to obtain 3 non-overlapping
images occupying 90% of the image at 200× magnification. Tumors
were stained with a monoclonal VEGF-A antibody and all images
were obtained using the identical camera and settings. A custom
algorithm was created using ImageJ software to identify and quantify
the number of positively stained UM cells. The hue, saturation, and
brightness (HSB) were adjusted in order to isolate positive cells.
HSB values were determined by trial and error; however, once
set, they remained consistent for all images. The percentage of the
image that was immunostained and the number of positively stained
cells were determined using the algorithm. An ocular pathologist
evaluated the slides to confirm that the majority of cells that stained
positive and were isolated by the algorithm were UM cells. A binary
logistic regression was performed with the presence of metastasis as
the dependent variable and average cell count and average positive
staining fraction as the independent variables.
Results: Of the 29 cases that met the inclusion criteria, eight UM
patients had metastases (28%). The quantification algorithm was
capable of identifying the positively stained cells and rendered
cell counts and area stained even in samples with strong melanin
presence. Using positive immunostaining values generated by the
algorithm, we determined that VEGF-A presence in the primary
tumor was significantly associated with metastasis; the predictive
accuracy of the model was 79.3% using positive VEGF-A staining
average cell count and average positive area (P<0.05).
Conclusions: The presence of VEGF-A in primary uveal melanoma
can be used to retrospectively predict metastasis. The custom
algorithm used in this study facilitates meta-analyses and eliminates
potential bias when grading immunohistochemical staining. This
information may help clinicians to select patients for anti-VEGF
therapy.
Commercial Relationships: Patrick Logan, None; Shawn
Maloney, None; Vasco Bravo-Filho, None; Natàlia Vilà, None;
Matthew Balazsi, None; Miguel N. Burnier, None
Increased HIF-1α Expression Correlates with Cell Proliferation
and Vascular Markers CD31 and VEGF-A in Uveal Melanoma
Frederic Mouriaux1, 4, Francois Sans Chagrin2, Caroline Diorio3,
Solange Landreville4, François Comoz1, Edwige Petit5, Myriam
Bernaudin5, Alain P. Rousseau4, Dan Bergeron4, Mohib Morcos2.
1
Ophthalmology, CHU, Caen, France; 2Pathology, Hopital St
Sacrement, Québec, QC, Canada; 3URESP, Centre de recherche
FRSQ du CHU de Québec, Québec, QC, Canada; 4Ophthalmology,
Université Laval, Québec, QC, Canada; 5CNRS, UMR 6301 ISTCT,
CERVOxy. GIP CYCERON, Caen, France.
Purpose: Overexpression of hypoxia inducible factor-1 alpha (HIF1α) has been found in several cancers and is thought to correlate with
aggressive disease. The purpose of our study was to investigate the
influence of HIF-1α on clinical outcome in uveal melanoma (UM)
along with proliferative (MIB-1) and vascular (CD31, VEGF-A)
markers
Methods: A retrospective analysis was carried out on UM tumors
from 88 patients. HIF-1α, MIB-1, CD31 and VEGF-A expression,
as well as necrosis were assessed by H&E immunohistochemistry
on paraffin-embedded UM tumor sections using a Tissue Microarray
(TMA). The bivariate analysis involving HIF-1α expression and
clinicopathological covariates was performed using the Chi-square
test. The association of clinicopathological covariates and HIF-1α
expression with patient survival was evaluated using the KaplanMeier approach and Cox proportional-hazards regression analysis.
Results: Among our study population, 56 patients (63.6%) had
high levels of HIF-1α expression. High expression of HIF-1α was
associated with high expression of MIB-1 (P=0.04), CD31 (P=0.03)
and VEGF-A (P<0.0001), as well as necrosis (P=0.04). However,
high HIF-1α expression was not correlated with cell type, largest
macroscopic tumor dimension or thickness, anterior localization,
pigmentation, mitotic figures or microcirculation patterns. Patients
with high HIF-1α expression did not showed a reduced survival when
compared with patients with low HIF-1α expression (P=0.92).
Conclusions: An increase in HIF-1α expression is significantly
associated with proliferative (MIB-1) and vascular (CD31 and
VEGF-A) markers, as well as necrosis in UM. However, there is no
correlation between high HIF-1α expression and patient survival.
Commercial Relationships: Frederic Mouriaux, None; Francois
Sans Chagrin, None; Caroline Diorio, None; Solange Landreville,
None; François Comoz, None; Edwige Petit, None; Myriam
Bernaudin, None; Alain P. Rousseau, None; Dan Bergeron, None;
Mohib Morcos, None
Support: The Québec Uveal Melanoma Infrastructure is financially
supported by the Réseau de recherche en santé de la vision from the
Fonds de recherche du Québec – Santé (FRQS)
MicroRNA-135a suppresses Uveal Melanoma Cell Proliferation
and Migration
Xiaoyan Chen1, Jiao Wang1, Lihua Wang1, Dan-Ning Hu2, Dongsheng
Yan1. 1Sch of Ophthal & Optometry, Wenzhou Medical College,
Wenzhou, China; 2Tissue Culture Center, The New York Eye and Ear
Infirmary, New York, NY.
Purpose: MicroRNAs (miRNAs) can act as either oncogenes or
tumor suppressors in tumorigenesis. Evidence indicates that some
miRNAs are essential for uveal melanoma cell proliferation and
migration. Nevertheless, the role of miR-135a in uveal melanoma
remains unknown. Here, we investigated the function of miR-135a in
uveal melanoma cells.
Methods: Realtime RT-PCR was carried out to detect the expression
of miR-135a in uveal melanoma specimens as well as normal
controls. Transfection of miR-135a into uveal melanoma cells was
performed by Lipofectamine RNAiMAX reagent. The proliferation
of uveal melanoma cells was examined by MTS assay. Cell cycle
was analyzed by flow cytometry. Cell migration was examined by
transwell migration assay.
Results: miR-135a was downregulated in uveal melanoma specimens
as compared with normal controls. Transfection of miR-135a into
uveal melanoma cells remarkably inhibited cell proliferation and
blocked cell cycle at G1 phase. Furthermore, miR-135a suppressed
cell migration.
Conclusions: Our results demonstrated that miR-135a may act as a
tumor suppressor in uveal melanoma cell proliferation and migration.
Commercial Relationships: Xiaoyan Chen, None; Jiao Wang,
None; Lihua Wang, None; Dan-Ning Hu, None; Dongsheng Yan,
None
Support: National Natural Science Foundation of China(81077682
& 81272286)
The effect of 17-AAG on focal adhesion kinase expression in
uveal melanoma cell lines
Dana Faingold, Silvin Bakalian, Vasco Bravo-Filho, Henry A.
Wood, Maria Eugenia Orellana, Emilia Antecka, Miguel N. Burnier.
Ophthalmology, McGill University, Montreal, QC, Canada.
Purpose: We have previously reported that focal adhesion kinase
(FAK) was overexpressed in primary uveal melanoma (UM)
and that phosphorylated FAK (pFAK) was detected in the most
aggressive UM cell lines. The aim of this study is to compare the
immunohistochemical profile of FAK and pFAK in UM to normal
ocular tissues and to determine the effect of an Hsp90 inhibitor, 17AAG, on the expression of FAK and pFAK in UM cells.
Methods: Immunohistochemical expression of FAK was evaluated
in 39 UM specimens, pFAK expression was assessed in 51 UM
specimens, and both FAK and pFAK expression were assessed in
the uveal tract of 20 normal eyes. Immunohistochemical staining
was assessed based on staining extent and intensity. For extent,
specimens were scored “1” when less than 50% of the tumor cells
stained, “2” when 50% to 80% of tumor cells stained, and “3”
when more than 80% of tumor cells stained. Intensity was scored
as follows: weak staining, “1”; moderate staining, “2”; and intense
staining, “3”. The cases were grouped into low FAK expression when
combined intensity and extent scores were between 1 and 4; and high
FAK expression when total score was 5 and 6. Five UM cell lines
(92.1, OCM-1, MKT-BR, SP6.5, and UW-1) were incubated with 10
mmol/L 17-AAG for 24, 48, and 72 hours. The expression of FAK
and pFAK following treatment was determined by Western blot.
Results: FAK was expressed in 87.2% of UM specimens, and
69.2% (n=27) were classified as having high FAK expression. pFAK
was expressed in 90% of UM specimens and 60.8% (n=31) of the
cases had high expression. We observed low FAK expression in
non-tumor structures, such as choroidal melanocytes and the ciliary
body, adjacent to UM tumors. Counterpart structures in normal eyes,
including choroidal and ciliary body melanocytes, displayed negative
or low cytoplasmic FAK and pFAK immunostaining. FAK and pFAK
protein levels in all five cell lines decreased following treatment with
10 mmol/L of 17-AAG at 24, 48 and 72 hours.
Conclusions: Higher expression of FAK and pFAK was observed
in uveal melanomas compared to normal ocular melanocytes.
Hsp90 inhibition downregulated FAK and pFAK expression in UM
cell lines. Therefore, we hypothesize that there is a role of high
expression of FAK in uveal melanoma pathogenesis. Future studies
are needed to explore the use of Hsp90 inhibitors for modulating
FAK in uveal melanoma.
Commercial Relationships: Dana Faingold, None; Silvin
Bakalian, None; Vasco Bravo-Filho, None; Henry A. Wood, None;
Maria Eugenia Orellana, None; Emilia Antecka, None; Miguel N.
Burnier, None
Comparing the lymphangiogenic impact of conjunctival and
uveal melanoma cells
Nasrin Refaian1, Konrad R. Koch1, Simona L. Schlereth1, Deniz
Hos1, Jacobus J. Bosch2, Claus Cursiefen1, Ludwig M. Heindl1.
1
Department of Ophthalmology, University of Cologne, Cologne,
Germany; 2Department of Internal Medicine 5, Hematology and
Oncology, University of Erlangen-Nuremberg, Erlangen, Germany.
Purpose: A clinically relevant difference between malignant
melanomas within the eye (uveal melanoma, UM) and those on
the ocular surface (conjunctival melanoma, CM) consists in their
distinct metastatic behaviour. CM has a propensity to first spread into
regional lymph nodes. Contrarily, UM almost exclusively spreads
into the liver via the hematogenic path, whereas lymphatic metastases
have only been described in advanced cases with extrascleral tumor
extension. The outgrowth of new lymphatics (lymphangiogenesis)
is a major prerequisite for lymphatic spread (LS), which is driven
by tumor-derived VEGF-C/-D secretion. The goal of this study
was to evaluate whether diverging prolymphangiogenic potentials
of CM and UM cells could contribute to the higher rate of LS in
CM, as assessed via VEGF-C/-D expression, and the proliferation
of lymphatic endothelial cells (LECs) cultivated in CM/UM cell
conditioned medium.
Methods: Human primary UM cells (OCM1, Mel270) and CM cells
(CM2005.1) cultured in RPMI medium (10% FCS) were harvested
for mRNA isolation followed by cDNA synthesis. Quantitative realtime PCR was performed to measure VEGF-C/-D expression. For
MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide)
based proliferation assays, LECs were seeded on a 96-well-plate
(4000 cells/well), left to attach overnight, and then cultivated for 48
hours in 25%, 50%, or 75% UM/CM cell conditioned medium (1%
FCS). MTT was added resulting in intracellular formazan deposition.
The reaction was stopped after 3 hours. Absorbances were measured
at 570 nm.
Results: VEGF-C and -D were expressed by all three cell lines.
VEGF-C expression was significantly higher in Mel270 (p<0.01)
compared with OCM1 and CM2005.1. VEGF-D expression was
similar in all analyzed cell lines (p>0.05). The proliferation rate
of LECs was positively correlated with increasing proportions of
Mel270-, OCM1-, CM2005.1-conditioned medium (p<0.05 for each
melanoma cell line). CM2005.1-conditioned medium stimulated LEC
proliferation to a significantly lower extent than UM cell lines OCM1
and Mel270 (p<0.05).
Conclusions: CM cells did not show a higher prolymphaniogenic
potential than UM cells. Accordingly, the higher rate of LS in CM is
more likely attributable to the specific microenvironment at the ocular
surface, which includes preexisting lymph vessels as potential access
point for malignant cells, as opposed to the lymphatic free intraocular
vicinity of UM.
Commercial Relationships: Nasrin Refaian, None; Konrad
R. Koch, None; Simona L. Schlereth, None; Deniz Hos, None;
Jacobus J. Bosch, None; Claus Cursiefen, None; Ludwig M.
Heindl, None
Hemangiogenic profile of uveal versus conjunctival melanoma
cell lines
Ludwig M. Heindl1, Nasrin Refaian1, Simona L. Schlereth1, Deniz
Hos1, Jacobus J. Bosch2, Claus Cursiefen1, Konrad R. Koch1.
1
Department of Ophthalmology, University of Cologne, Cologne,
Germany; 2Department of Internal Medicine 5, Hematology and
Oncology, University of Erlangen-Nuremberg, Erlangen, Germany.
Purpose: Uveal and conjunctival melanoma (UM, CM) are distinct
malignant entities, which show diverging patterns of metastatic
spread. Hematogenic metastases, which are dependent on tumor cell
driven formation of blood vessels (hemangiogenesis) within and
surrounding the tumor, often occur in UM. In contrast, spreading of
CM is less frequent and primarily affects regional lymph nodes. The
purpose of the present study was to analyze whether these differences
of hematogenic spread, which are of high prognostic relevance, could
be attributable to differential hemangiogenic potentials of UM and
CM cells. This was assessed by measuring (1) VEGF-A expression
in UM and CM cells and (2) proliferation rates of blood endothelial
cells (BECs) incubated with UM/CM cell conditioned medium.
Methods: mRNA expression of VEGF-A by human primary UM
cells OCM-1 and Mel-270 as well as CM cells CM2005.1 was
measured by quantitative real-time PCR. For proliferation analysis,
BECs were seeded on a 96-well-plate (4000 cells/well) and were left
to attach for 24 hours. Hereafter cells were incubated with 50 % of
BEC medium containing 1 % fetal calve serum and 50 % UM or CM
cell conditioned medium. BrdU was added 1 hour later. Colorimetric
analysis was performed after 48 hours of incubation at a measuring
wavelength of 450 nm.
Results: VEGF-A mRNA was expressed in all three cell lines.
Mel-270 cells expressed VEGF-A mRNA to a lower extent than
OCM-1 and CM2005.1 (p<0.05). The proliferation of BECs was
significantly increased by adding conditioned medium of CM2005.1,
OCM-1, or Mel-270 cells (p<0.05 for all groups). CM2005.1- and
OCM-1-conditioned medium had a similar impact, while a smaller
proliferative increase of BECs was found under the influence of Mel270 (p<0.05).
Conclusions: Both UM and CM cells expressed VEGF-A, and
increased BEC proliferation. Higher VEGF-A levels (independent
of tumor cell origin) were associated with a stronger increase of
proliferation. A reduced hemangiogenic potential of CM cells was
not detectable. Other additional mechanisms within the tumor
microenvironment might thus account for the lower hematogenic
metastatic rate in CM.
Commercial Relationships: Ludwig M. Heindl, None; Nasrin
Refaian, None; Simona L. Schlereth, None; Deniz Hos, None;
Jacobus J. Bosch, None; Claus Cursiefen, None; Konrad R. Koch,
None
Support: German Research Foundation HE 6743/2-1
Origin of Tumor-Associated Vessels and Vasculogenic Mimicry in
an Intraocular Murine Melanoma Model
Martina C. Herwig1, Marta M. Kilian1, Frank G. Holz1, Daniela
Wenzel2, Karin U. Loeffler1. 1University Eye Clinic, University of
Bonn, Bonn, Germany; 2Institute of Physiology I, University of
Bonn, Bonn, Germany.
Purpose: To study the origin of tumor-associated vessels and
vasculogenic mimicry, which is associated with prognosis in human
uveal melanoma, in an intraocular murine melanoma model.
Methods: Six Bac-Flt1-GFP mice (with vascular endothelium
specifically labeled by eGFP) with a C57Bl/6 background received
an intraocular injection of HCmel12 cells (which represent a skin
melanoma cell line) in order to induce melanoma growth. The eyes
were removed 10 days after tumor cell injection and the vascular
pattern of the murine vessels were investigated with a Zeiss Axio
Zoom microscope. Following our standardized imaging protocol, the
eyes were immediately fixed in 4% paraformaldehyde and embedded
in paraffin or submitted for cryoconservation. Immunohistochemical
stains for von Willebrand factor (vWf), vascular endothelial cadherin
(VEC) which is supposed to stain vasculogenic mimicry and vascular
endothelial cell adhesions, and Green Fluorescent Protein (GFP) as
well as immunofluorescent stains for vWf and VEC were performed
to characterize and distinguish between host-derived (GFPexpressing) vessels and tumor-derived vessels.
Results: In this model, tumor growth could be induced in all
experimental eyes. Macroscopic imaging showed a network of GFPlabelled vessels on the tumor surface. Histologic tumor characteristics
comprised tumor cell pleomorphism, infiltration by inflammatory
cells, cooption of blood vessels, and vasculogenic mimicry. Analysis
of the immunohistochemical/immunofluorescence stains revealed
an expression of GFP in all blood vessels including tumor vessels.
Vasculogenic mimicry was neither labelled by GFP nor vWf.
However, extracellular structures were partially stained with VEC.
Conclusions: This preliminary study used a novel mouse model
for intraocular melanoma which allows for discrimination between
host- and tumor-derived vascular structures. The findings indicate
that true intratumoral vessels had apparently a host-derived origin
while vasculogenic mimicry structures were not derived from host
vessels and are probably tumor-derived. Further studies in this model
are needed to address potential factors for tumor vasculature and
microcirculation and to further analyze the origin of vasculogenic
mimicry.
Commercial Relationships: Martina C. Herwig, None; Marta M.
Kilian, None; Frank G. Holz, None; Daniela Wenzel, None; Karin
U. Loeffler, None
Support: BONFOR grant (in-house grant from the University
Hospital Bonn)
Cytological Characteristics of Uveal Melanoma
Carlos A. Medina Mendez1, Charles V. Biscotti2, Nakul Singh3,
Arun D. Singh1. 1Ophthalmology, Cole Eye Inst, Cleveland Clinic,
Cleveland, OH; 2Department of Anatomic Pathology, Cleveland
Clinic, Cleveland, OH; 3Harvard School of Public Health, Harvard,
Cambridge, MA.
Purpose: To report on the cytological characteristics of choroidal
melanoma.
Methods: All patients with clinical diagnosis of choroidal melanoma
from May 2009 to July 2012 who underwent prognostication FNAB
were included. In this prospective, single center study of consecutive
patients, clinical and diagnostic cytological characteristics of were
analyzed.
Results: A total of 141 FNAB samples of 141 patients. Fifty-three
percent of the patients were male and the average age for all patients
was 60 years. Transcorneal (7), transcleral (69) and transvitreal (63)
approaches were used (Table 1).
Of 141 samples, 129 adequate samples where obtained (91 %
true positive). These included 121 samples that were positive for
melanoma and 8 samples that contained atypical cells consistent
with melanoma. Twelve samples were inadequate (7 unsatisfactory
for interpretation, 2 negative for melanoma, and 3 non-diagnostic
atypical cells). Average tumor height was 6.3mm for adequate
samples and 3.3 mm for inadequate samples (p=0.0001). Median
tumor height was 5 and 2.8mm for adequate and inadequate saples
respectively. Sample adequacy was varied significantly (p=0.029)
by FNAB approach (100% transcorneal, 97% transcleral, and 84%
transvitreal). Spindle cells were observed in 98% of the samples
(63% mixed and 25% spindle only) where as only epithelioid cells
were observed in 2% samples). Melanin was present in 80% of
samples (Table 2). Tumor nuclear grade (atypia) increased with tumor
height and by tumor locations (least atypia with iris tumors).
Conclusions: Cytological features such as spindle cells and melanin
which where present in 98% and 80% of samples respectively are
important diagnostic features. Iris melanoma has bland fetaures as
compared to ciliary and choroidal melanoma. Thinner tumors (less
than 3 mm in height) are likely to yield inadequate diagnostic FNAB
sample.
Commercial Relationships: Carlos A. Medina Mendez, None;
Charles V. Biscotti, None; Nakul Singh, None; Arun D. Singh,
None
Support: Research to Prevent Blindness and The Falk Trust
Uveal Melanoma Cytology : Computer Assisted Diagnosis
Arun D. Singh1, 2, Nathan Tenley3, Charles V. Biscotti4. 1Cole Eye Inst,
Cleveland Clinic, Cleveland, OH; 2Ophthalmic Oncology, Cole Eye
Institute, Cleveland, OH; 3Image IQ, Cleveland Clinic, Cleveland,
OH; 4Anatomic Pathology, Cleveland Clinic, Cleveland, OH.
Purpose: To develop a fully automated, customized image
processing algorithm to quantify the total number of cells in a given
cytological slide and to detect spindle cells as an aid in the diagnosis
of uveal melanoma.
Methods: Comparative masked study of ocular FNAB cytopathology
slides prepared using standard laboratory techniques in a
cytopathology laboratory. 10 cases of primary uveal melanoma and 5
cases of non-melanoma uveal tumors. The manual counts of all cells
and spindle cells by an independent masked reader were recodered
as standrad. The manual counts were compared to the automated
counts (CAD) for each image tile to analyze the detection rates for
any cell and spindle cells (Figure 1). The CAD algorithm was tuned
to minimize the difference between the manual and computer assisted
diagnosis (CAD) outputs for each image tile.
Results: The manual counts for any cell ranged from 5.7 to 401.8
/ μm2 (mean = 112.8) for non-melanoma cases and 7.1 to 112.1 /
μm2 (mean = 49.5) for melanoma cases. The computer assisted and
the manual counts had an average cell detection difference of 9.9 ±
19.2 for non-melanoma cases and 4.0 ± 7.1 for the melanoma cases
(average 9.8%). There was no significant correlation between the
cellularity of the slide and the percentage difference in counts. The
spindle cell detection difference between computer assisted and the
manual counts was 5.2 ± 4.6 and -7.6 ± 6.8 for the non-melanoma
cases and melanoma cases, respectively. The computer assisted
counts calculated the average spindle cell composition rate for
non-melanoma and melanoma cases to be 4.53 ± 2.43% and 23.04 ±
9.65%, respectively.
Conclusions: Automated digital methods can identify, quantify, and
characterize spindle cells in an ocular FNAB cytopathology slide.
Such automated system may be used to identify melanoma cases that
could be verified by an expert cytopathologist at a remote site. Use of
this computer assisted diagnostic tool can potentially be expanded for
cytopathologic assessment of other tumors.
Figure 1. Manual count training of cell and spindle cells. (A)
Large field-of-view image comprised of 41,412 um2<\>> field-ofview (15 grids) at 0.185 um/pixel resolution. (B) Magnified
view of single grid with manual cell counts (cyan dots) and spindle
counts (red dots). (C) Analysis output from CAD algorithm associated to manual counts tile in B with cell counts (green dots) and
spindle counts (red outline).
Commercial Relationships: Arun D. Singh, None; Nathan Tenley,
None; Charles V. Biscotti, None
Support: RPB
Morphologic Changes of Cultivated Uveal Melanoma Cells with
Increasing of Passages
Sahar Balagholi1, Narges Fazili1, Abouzar Bagheri1, Somayeh Asadi1,
Mozhgan Rezaei Kanavi1, Hamid Ahmadieh1, Zahra-Soheila Soheili2,
Shahram Samiei3. 1Ophtalmic Research Center, Shahid Beheshti
University of Medical Sciences, Tehran, Islamic Republic of Iran;
2
National Institue of Genetic Engineering and Biotechnology, Tehran,
Islamic Republic of Iran; 3Iranian Blood Transfusion Organization
Research Center, Tehran, Islamic Republic of Iran.
Purpose: This study was conducted to investigate the morphologic
changes of cultivated uveal melanoma (UM) cells with increasing
number of passages.
Methods: Immediately after enucleation of an eye with mixed celltyped UM, tumor tissue was obtained, digested with 1.25% trypsin,
and cultivated in DMEM F12 with 20% FBS. The cultivated cells
were passaged to reach the fifth passage. Immunocytochemistry
for melan-A monoclonal antibody, as a melanoma marker, was
performed to identify melanoma cells. All the experiments were
done in duplicate. With increasing number of passages, morphologic
changes of cultivated melanoma cells were investigated.
Results: In the first passage, there was a mixture of epithelioid
melanoma cells with owl’s eye nucleoli and spindle-shaped neurallike melanoma cells with dendritic processes. With increasing
of passages, the predominant population of cultivated cells were
epithelioid melanoma cells. However, the numbers of spindle-shaped
neural-like melanoma cells were significantly reduced. Colony
formation of tumor cells was observed in the third passage (Figure
-1).
Conclusions: Homogeneity of cultivated melanoma cells can be
obtained through increasing number of passages. It seems that
differentiated neural-like melanoma cells are more vulnerable to the
increasing number of passages as compared to the less differentiated
epithelioid melanoma cells.
Figure 1. A- C: Cultivated UM cells in passages 1 (A), 2 (B), and
3 (C). Note the presence of epithelioid melanoma cells with owl’s
eye nucleoli (circle 1), neural-like melanoma cells with dendritic
processes (circle 2), and colony formation of cultivated UM cells
(arrow). FITC of melan-A expression in cultivated UM cells (D),
DAPI of nucleus (E) and merged FITC and DAPI of UM cells (F).
Commercial Relationships: Sahar Balagholi, None; Narges Fazili,
None; Abouzar Bagheri, None; Somayeh Asadi, None; Mozhgan
Rezaei Kanavi, None; Hamid Ahmadieh, None; Zahra-Soheila
Soheili, None; Shahram Samiei, None
The effects of macrophages on tumor growth characteristics in a
mouse-model of intraocular melanoma
Marta M. Kilian1, Karin U. Loeffler1, Hans E. Grossniklaus2,
Frank G. Holz1, Christiane Pfarrer3, Christian Kurts4, Martina C.
Herwig1. 1Ophthalmopathology, University Eye Hospital Bonn,
Bonn, Germany; 2Department of Ophthalmology, Emory University,
Atlanta, GA; 3Department of Anatomy, University of Veterinary
Medicine Hannover, Hannover, Germany; 4Departments of Molecular
Medicine and Experimental Immunology, University of Bonn, Bonn,
Germany.
Purpose: To investigate the influence of tumor associated
macrophages and age dependency on clinicopathological factors of
intraocular melanoma using a murine ocular tumor model
Methods: Groups of 8-12 either untreated or systemically
macrophage depleted CX3 CR1 GFP knock-in mice were examined at
two different ages (young 8-10 weeks, old >10 months), respectively.
After intravitreal injection of 1 x 105 HCmel12 melanoma cells,
enucleation was performed at day 9. In histology (HE, PAS without
hematoxylin), maximum intraocular tumor area was determined as
well as co-option of retinal vessels, intratumoral endothelial lined
vasculature and vasculogenic mimicry including its prognostic
significant microcirculatory patterns. Microvasculature (VE
Cadherin-Ab) and macrophage infiltration (GFP-Ab) in untreated
mice were assessed by immunohistochemistry.
Results: Over all groups, intratumoral endothelial-lined vasculature,
vasculogenic mimicry and presence of microcirculatory patterns
were not correlated with tumor size. Untreated old mice showed
significantly more microcirculatory patterns compared with their
younger counterparts. Macrophage depleted old mice exhibited
significantly less patterns than untreated old mice. Compared to
untreated mice of both age groups all macrophage depleted mice
exhibited significant less patterns. Macrophage infiltration appeared
to be more numerous in old than in the young mice.
Conclusions: The findings in this mouse-model indicate that
tumor associated macrophages seem to have impact particularly on
the formation of microcirculatory patterns. In untreated old mice
microcirculatory patterns were distinctive in contrast to macrophage
depleted mice. Tumor associated macrophages may have influence
on the presence of these patterns, which in humans are known to be
correlated with metastasis and therefor with worse prognosis.
Commercial Relationships: Marta M. Kilian, None; Karin U.
Loeffler, None; Hans E. Grossniklaus, None; Frank G. Holz,
None; Christiane Pfarrer, None; Christian Kurts, None; Martina
C. Herwig, None
Support: BONFOR Forschungsförderprogramm
Morphologic and immunohistochemical features in the ageing
Hgf-Cdk4 mouse eye
Karin U. Loeffler1, Marta M. Kilian1, Frank G. Holz2, Thomas
Tüting3, Martina C. Herwig1. 1Ophthalmology, Division of
Ophthalmic Pathology, University of Bonn, Bonn, Germany;
2
Ophthalmology, University of Bonn, Bonn, Germany; 3Experimental
Dermatology, University of Bonn, Bonn, Germany.
Purpose: Searching for an animal model of spontaneous or inducible
uveal melanoma, we evaluated the ocular findings in an established
mouse model of spontaneous skin melanoma with melanocytic
proliferation and knock-out of a tumour suppressor gene (Hgf-Cdk4).
This should also further clarify the role and impact of hepatocyte
growth factor/scatter factor/deregulated receptor tyrosine kinase and
cyclin-dependent kinase 4 on ocular melanocytes in comparison with
dermal melanocytes.
Methods: In total, 15 Hgf-Cdk4 mouse eyes were investigated,
the emphasis being on the aged eye (11 months, n=12), and were
compared with three 10 months old eyes from “regular” C57Bl/6
mice with a similar genetic background. Step sections were stained
with H&E and PAS, and sections inbetween were labeled with
antibodies (Abs) to Ki67 (proliferation marker), MART1 (‘melanoma
antigen’) and F4/80 (macrophages). Prior to immunohistochemistry
(IH), sections were bleached for better visualization of the
chromogen (AEC).
Results: Morphologically, the most marked difference in melanocytemodified mouse eyes was the appeareance of heavily pigmented
cells in the anterior corneal stroma and along the chamber angle.
No significant increase in epithelial pigmentation of either corneal
or conjunctival epithelium was noted. Unequivocal proliferation of
uveal melanocytes or RPE cells was also not observed however, the
choroid appeared even somewhat less pigmented than in C57Bl/6
mice with some conspicuous cellular features. IH so far showed no
significant difference in labeling between Hgf-Cdk4 and C57Bl/6
mouse eyes, and surprisingly, using our 3 Abs, the origin of the
pigmented corneal cells (or the pigment) remains unclear. Also, Ki67
was negative while the overlying corneal/conjunctival epithelium
showed some proliferative activity in all specimens.
Conclusions: In Hgf-Cdk4 mouse eyes, no major difference in
pigmentation or cellular components was observed in the ocular
epithelium or in the posterior segment, and subtle findings so far were
equivocal. Thus, although the Hgf-Cdk4 mouse develops spontaneous
cutaneous melanomas, it does not qualify as a model for spontaneous
conjunctival or uveal melanoma. However, remarkable changes
regarding pigmentation were noted in the cornea and chamber angle
in these animals, and further manipulation of the anterior segment
might allow creating models of anterior segment tumors and possibly
also glaucoma.
Commercial Relationships: Karin U. Loeffler, None; Marta
M. Kilian, None; Frank G. Holz, None; Thomas Tüting, None;
Martina C. Herwig, None
An Agonist of Toll-Like Receptor 5 Suppresses Hepatic
Metastases in a Mouse Model with Ocular Melanoma
Hua Yang1, Hans E. Grossniklaus1, Camille A. Benjamin2, Lyudmila
Burdelya2, Qing Zhang1, Andrei V. Gudkov2. 1Ophthalmology, Emory
University Eye Center, Atlanta, GA; 2Cell Stress Biology, Roswell
Park Cancer Institute, Buffalo, NY.
Purpose: CBLB502, an agonist of toll-like receptor 5, is a
polypeptide drug derived from Salmonella flagellin that binds to Tolllike receptor 5 (TLR5) and activates nuclear factor–kB signaling,
and has been shown to have an effect on several animal models of
liver metastasis, regardless of TLR5 expression. In this study, we
investigate whether CBLB502 can inhibit hepatic metastases in a
mouse model with ocular melanoma.
Methods: To know whether CBLB502 is able to activate TLR5 in
melanoma cells, an in vitro P65 translocation response was checked.
Mouse melanoma cell lines B16LS9 and Queens were cultured
and treated with 100ng/ml of CBLB502 or 10ng/ml of TNF alpha
or PBS for 30 minutes, fixed and stained with anti-P65 antibody,
and the intracellular location of P65 were determined by confocal
microscopy. B16LS9 cells were inoculated into the choroid of right
eye in C57BL/6 mice. 40 mice were divided into 4 groups including a
PBS control group (n=10), CBLB502 starting at the day before tumor
inoculation (n=10), CBLB502 starting at the day of tumor inoculation
(n=10), CBLB502 starting at 3 day after tumor inoculation (n=10).
Each mouse received sc 1ug/100ul of CBLB502 or PBS every 3 days.
The inoculated eye was removed at 7 day and checked tumor growth
with histology methods. The liver was collected at 21 days and the
number of metastasis were microscopically enumerated. IHC for
S100 and CD49b was performed.
Results: After CBLB502 or positive control TNF alpha treatment,
P65 was observed to translocate from the cytoplasm to the nucleus
in B16LS9 and Queens cells. Animal experiments showed that
CBLB502 significantly decreased the number of hepatic metastasis in
three treatment groups compared with the PBS group. S100 staining
confirmed the metastases were from ocular melanoma. We observed
CD49b+ cells in liver or hepatic metastases in the treatment groups
but not in the control group.
Conclusions: The agonist of toll-like receptor 5, CBLB502 is able
to activate TLR5 in melanoma cells, and suppress hepatic metastasis
from ocular melanoma in a mouse model through attracting natural
killer cells to the liver. CBLB502 may be a potential treatment for
metastastic uveal melanoma to the liver.
Commercial Relationships: Hua Yang, None; Hans E.
Grossniklaus, None; Camille A. Benjamin, None; Lyudmila
Burdelya, None; Qing Zhang, None; Andrei V. Gudkov, None
Support: NIH R01CA176001 and RPB
Purpose: To test the hypothesis that the histological and
immunohistochemical findings in metastatic human uveal melanoma
to the liver in a mouse model are similar to metastatic uveal
melanoma to the liver in humans.
Methods: Human uveal melanoma cell line Mel290 were cultured,
and inoculated into the superchoroidal space in the right eyes
of NU:NU mice via trans-scleral technique. The tumor-burden
eyes were enucleated at the 7th day to determine primary tumor
growth, and the livers were collected at the 1, 2, 6, or 12 weeks to
determine the hepatic metastases. Histological examination and
immunohistochemical staining were then performed.
Results: Stage 1 metastases (defined as tumor clusters less than 50
μm in diameter) were identified in the sinusoidal spaces of 10 of 12
mice (83%). Stage 1 metastases were avascular and lacked mitotic
activity. Stage 2 metastases (defined as tumors measuring 51-200
μm in diameter) were found in 8 mice sacrificed at the 6 or 12
weeks. Stage 3 metastases (defined as tumors measuring greater than
200 μm in diameter) were found in 4 mice sacrificed at 12 weeks.
Immunohistochemical stains were positive for S100 or HMB45 in all
tumors. Overall, stage 1 metastases out-numbered stage 2 metastases,
which out-numbered stage 3 metastases (P less than 0.0001 and P
less than 0.005, respectively). The mean vascular density and mitotic
index increased from stage 1 to stage 3 metastases (P less than 0.01).
The architecture of stage 2 metastases mimicked the surrounding
hepatic parenchyma, with pseudosinusoidal spaces metastases stained
with reticulin and lined by smooth muscle actin-positive activated
stellate cells (myofibroblasts), whereas stage 3 metastases exhibited
either lobular or portal growth patterns with tumor vascularization.
Conclusions: The in vivo mouse model of human uveal melanoma
is reliable and reproducible, and forms hepatic metastases which can
be categorized as stage 1 micro-, stage 2 intermediate-, or stage 3
vascularized and mitotically active macro-metastases with a lobular
or portal pattern of growth that mimics metastatic uveal melanoma to
the liver in humans.
Progression of Ocular Melanoma Metastasis to the Liver in
Mouse Model
Hans E. Grossniklaus1, 2, Qing Zhang1, Hua Yang1, Shou You2, Shin
Kang1. 1Dept of Ophthal, School of Med, Emory University, Atlanta,
GA; 2Winship Cancer Institute, Atlanta, GA.
Support: NIH R01CA176001 (HEG); P30EYE06360 (HEG);
T32EY007092 (JML)
Progression of metastatic uveal melanoma in mouse liver. Left:
lobular pattern. Right: portal pattern. Top: Stage 1 micrometastases
(arrow). Middle: Stage 2 intermediate tumors Bottom: Stage 3
metastases with vascularization (arrow).
(asterisk=venule;hematoxylin and eosin, 100X)
Commercial Relationships: Hans E. Grossniklaus, None; Qing
Zhang, None; Hua Yang, None; Shou You, None; Shin Kang, None
Support: NIH R01CA176001 and RPB, Inc.
The Role of a High Fat Diet in Liver Metastasis of Ocular
Melanoma in C57BL/6 Mice
John Lattier, Hua Yang, Hans E. Grossniklaus. Department of
Ophthalmology, Emory University, Atlanta, GA.
Purpose: We have previously demonstrated increased progression
of liver metastasis of ocular melanoma in mice deficient for pigment
epithelium-derived factor (PEDF). Since PEDF null mice exhibit
obesity and fatty liver, we hypothesized that fatty liver alone could
provide an environment conducive for metastatic progression.
Methods: C57BL/6 mice were placed on a high fat (60% kcal
from fat) diet or a control (10%) diet for six weeks. Mice were
then injected with B16-LS9 melanoma cells into the posterior
compartment of the eye, and followed for one month while
maintaining their diets. Afterwards, livers were collected for
histological examination.
Results: Mice on a high fat diet were significantly obese after two
weeks. At the end point, their livers contained an increased amount
of fatty lipid droplet content. However, the number and size of
metastases in both high fat and low fat mice were equivalent.
Conclusions: Fatty liver alone does not cause an increased number or
size of liver metastases in a mouse model of ocular melanoma. Thus
the increased metastatic progression seen in PEDF null mice must be
due to another PEDF-related phenomenon such as angiogenesis or
stromagenesis.
Commercial Relationships: John Lattier, None; Hua Yang, None;
Hans E. Grossniklaus, None
Chick embryo model systems to study uveal melanoma metastasis
Haleh Shahidipour1, Sarah E. Coupland1, Diana Moss1, Bertil E.
Damato2, Helen Kalirai1. 1The University of Liverpool, Liverpool,
United Kingdom; 2University of San Fransisco, San Fransisco, CA.
Purpose: Almost fifty percent of patients with uveal melanoma (UM)
will develop fatal metastatic disease, which occurs predominantly
in the liver. Despite significant improvements in the treatment of
the ocular tumour, patient mortality has not improved and most
metastatic lesions remain untreatable. Little is known about the
mechanisms by which UM cells metastasise to and colonise the liver.
We have developed a chick embryo model of spontaneous and direct
metastasis allowing for the processes of UM tumour development,
intravasation, extravasation and dissemination to be studied.
Methods: At embryonic day 3, a small window was made in the shell
and GFP labelled Omm1 UM cells were either: 1) inoculated onto the
lowered chorioallantoic membrane (CAM) – spontaneous metastasis
model; or 2) injected directly into the circulation of the chick embryo
– direct metastasis model. At embryonic day 7, tumour nodules that
had formed on the CAM were excised, fixed in 10% neutral buffered
formalin and embedded for immunohistochemical analyses. Internal
organs were examined for the presence of UM cells in both model
systems under a fluorescence microscope, prior to fixation and
embedding. All experiments were repeated at least three times.
Results: Omm1 UM cells formed tumour nodules within the CAM,
at embryonic day 14, which were associated with an overlying
network of chick embryo blood vessels. Immunohistochemical
analyses of cross sections of the tumour nodules demonstrated the
presence of MelanA positive cells surrounded by chick embryo
CAM, internal blood vessels and a high Ki67 staining index. Omm1
cells were not observed in any internal organs in the spontaneous
metastasis model after seven days. Following direct injection of
Omm1 cells into the chick embryo circulation, tumour cell deposits
were observed microscopically in the eye, liver and kidneys after
seven days. Immunohistochemistry showed numerous micro- and
macro- metastatic liver deposits which stained positively for MelanA
and demonstrated a low Ki67 growth fraction.
Conclusions: Chick embryo metastasis model systems can be used to
provide important information on the specific behavioural patterns of
UM cells during individual stages of metastatic dissemination. Future
studies will utilise these systems to study the functional mechanisms
of individual metastasis-related genes.
Commercial Relationships: Haleh Shahidipour, None; Sarah E.
Coupland, None; Diana Moss, None; Bertil E. Damato, None;
Helen Kalirai, None
Support: Eye Tumour Research Fund A0814
sIL-2R an immuno-biomarker for prediction of metastases in
uveal melanoma
Vivian Barak1, Jacob Pe’er2, Inna Kalickman1, Shahar Frenkel2.
1
Immunology Laboratory for Tumor Diagnosis, Hadassah-Hebrew
University Medical Center, Jerusalem, Israel; 2Ophthalmology,
Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Purpose: High serum levels of sIL-2R have been reported in
acute inflammations and in several types of cancer, especially in
the metastatic stage. The aim of the present study was to evaluate
the potential of increased sIL-2R levels to predict metastatic uveal
melanoma (UM).
Methods: Serum levels of sIL-2R were analyzed by ELISA. The
study included a total of 152 subjects: 74 patients with metastasis
(Mets), 42 10-year disease-fee (10yDF) patients, and 36 healthy
controls (CTRL). Patients were followed up biannually with liver US
for the presence of Mets. Blood samples were obtained from the time
of primary diagnosis, and on every follow-up visit. Sera from before
and after the diagnosis of metastases were assessed by a matched
pairs analysis for the 74 Mets patients.
Results: The mean±SE (range) sIL-2R levels for the 3 groups were:
575±41 (107- 1257), 780±60 (313- 1830), and 1401±146 (380- 8969)
U/ml for CTRL, 10yDF, and Mets, respectively (p= 0.03).
Conclusions: Significantly higher serum sIL-2R levels were shown
in UM pts with metastases. Significant increases in sIL-2R levels
on serial evaluations indicate the development of UM metastases,
enabling earlier treatment for a better survival of those patients.
Commercial Relationships: Vivian Barak, None; Jacob Pe’er,
None; Inna Kalickman, None; Shahar Frenkel, None
Lack of BAP1 germline gene mutation in patients with early
onset uveal melanoma
Elaine M. Binkley1, Colleen M. Cebulla1, Robert Pilarski2, James
B. Massengill1, Meghan J. Marino3, Arun D. Singh3, Mohamed
H. Abdel-Rahman1, 2. 1Department of Ophthalmology and Visual
Science, The Ohio State University, Columbus, OH; 2Division of
Human Genetics, The Ohio State University, Columbus, OH; 3The
Cole Eye Institute, The Cleveland Clinic, Cleveland, OH.
Purpose: Germline mutations in the tumor suppressor gene BAP1
have been found to be important to the pathogenesis of uveal
melanoma and an associated familial cancer syndrome. This BAP1
syndrome results in a predisposition to uveal melanoma, cutaneous
melanoma, mesothelioma, and renal cell carcinoma. Early onset
of uveal melanoma is a feature of hereditary cancer predisposition
syndromes. This study aims to determine the prevalence of BAP1
germline mutations in a series of young patients with uveal
melanoma, diagnosed before age 30. We hope to help better define
risk factors for disease pathogenesis and risk for transmission to
offspring in this subset of uveal melanoma patients.
Methods: Approval for this project was obtained from the
Institutional Review Board of the Ohio State University. Peripheral
blood samples were obtained from a series of 11 patients with early
onset uveal melanoma (average age 22.6 years, range 3 months
to 29 years). Germline DNA was extracted from peripheral blood
leukocytes. BAP1 sequencing was then carried out using direct
sequencing of all exons and adjacent intronic sequences.
Results: All 11 patients were negative for pathogenic germline
mutation in BAP1. One patient had an intronic variant of uncertain
significance (c.123-48T>G). Similarly, family history of the most
specific BAP1 syndrome cancers (uveal melanoma, mesothelioma,
and renal cell carcinoma) was negative in all patients, while 2
families had family history of cutaneous melanoma. Other cancers
present in the families included lymphoma, lung, breast, colon,
pancreatic, cervical, uterine, ovarian, prostate, bone, and brain.
Conclusions: This work suggests that germline mutation in genes
other than BAP1 likely play a greater role in the underlying genetic
predisposition to early onset uveal melanoma. Future work will be
directed at identifying additional genes which may be altered in these
patients.
Commercial Relationships: Elaine M. Binkley, None; Colleen M.
Cebulla, None; Robert Pilarski, None; James B. Massengill, None;
Meghan J. Marino, None; Arun D. Singh, None; Mohamed H.
Abdel-Rahman, None
Support: Ohio Lions Eye Research Foundation; K08EY022672;
Patti Blow Research Fund; American Cancer Society IRG-67-003-47;
Melanoma Know More Foundation; Ocular Melanoma Foundation
Elevated NG2 expression and Rho-kinase activity in the uveal
melanoma cells with monosomy-3
Aysegul Tura, Julia Lueke, Mihaela Reinsberg, Matthias Luecke,
Salvatore Grisanti. Department of Ophthalmology, University Clinic
Schleswig-Holstein, Luebeck, Germany.
Purpose: Presence of monosomy-3 in the uveal melanoma (UM)
cells is associated with a higher risk of mortality due to metastasis
particularly into the liver. Currently, there are no effective treatment
options to restrain the UM metastasis. The major liver-borne trophic
factors like hepatocyte growth factor and insulin-like growth factor-1
(IGF-1) can activate the Rho/Rho-kinase pathway in diverse cell
types. In this study, we analysed the outcomes of the conditioned
medium from hepatocytes (HCM) on the metastatic potential of the
UM cells together with the Rho-kinase activity and monosomy-3
status.
Methods: Circulating uveal melanoma cells (CMC) were
immunomagnetically isolated from the freshly collected blood of
n=21 UM patients. Cultures of UM cells were established from the
primary tumor of a UM-patient operated in our clinic. Immuno-FISH
was performed to detect chromosome-3 in the cells specifically
expressing NG2, Ki67, or pMYPT1, the major downstream target of
Rho-kinase. Cultured UM cells were incubated with the Rho-kinase
inhibitor H-1152 for 1 to 3 days. Proliferation was analysed by Ki67
immunostaining and EdU incorporation. Cell motility was evaluated
by the scratch assay. Levels of vimentin, beta-catenin, cleaved
caspase-3, and IGF-1 were determined by immunocytochemistry and
blotting.
Results: The melanoma associated chondroitin sulphate proteoglycan
(NG2), an upstream activator of RhoA/RhoC, was expressed at
significantly higher levels in both the CMC and the cultured UM
cells having monosomy-3. HCM led to a significant increase in
the expression of vimentin and beta-catenin and the motility of the
UM cells possibly due to the elevated levels of IGF-1. HCM also
promoted the proliferation of UM cells having monosomy-3, together
with an increase in Rho-kinase activity. Inhibition of Rho-kinase with
H-1152 suppressed the proliferation of UM-cells with monosomy-3
(p<0.05), reduced the expression of beta-catenin and vimentin, and
promoted apoptosis.
Conclusions: UM cells with monosomy-3 exhibited a higher
expression level of NG2 and an associated increase in Rho-kinase
activity, which may be involved in the elevated metastatic potential
of these cells in response to hepatocyte-derived growth factors.
Pharmacological inhibition of Rho-kinase therefore emerges as a
noteworthy strategy to suppress UM metastasis into the liver.
Commercial Relationships: Aysegul Tura, None; Julia Lueke,
None; Mihaela Reinsberg, None; Matthias Luecke, None;
Salvatore Grisanti, None
Comparison of uveal melanoma prognostication test results of the
‘paired’ intraocular biopsy with the subsequently enucleated eye
Sarah E. Coupland1, Helen Kalirai1, Vivian Ho2, Bertil E.
Damato3, Heinrich Heimann2. 1Molecular and Clinical Cancer
Medicine, University of Liverpool, Liverpool, United Kingdom;
2
Ophthalmology, Royal Liverpool University Hospital, Liverpool,
United Kingdom; 3Ophthalmology, University of California, San
Francisco, CA.
Purpose: To compare the accuracy of genetic results for chromosome
3 aberrations from a fine needle aspiration biopsy (FNAB) with those
from a subsequent enucleation specimen in uveal melanoma (UM)
patients who underwent prognostic testing in Liverpool, UK.
Methods: UM patients who underwent routine genetic testing for
prognostic purposes of an initial biopsy specimen and a subsequent
enucleation specimen between 2010 and 2013 were included in this
study. Depending on DNA yield, specimens underwent molecular
testing by either microsatellite analysis (MSA) or multiplex ligation
dependent probe amplification (MLPA). Data for chromosome 3
status were then compared between the ‘paired’ tumour samples.
During this period of 3 years, 111 UM patients seen at the Liverpool
Ocular Oncology Centre had undergone genetic tumour testing by
MSA, and an additional 429 patients by MLPA.
Results: Paired biopsy and enucleation data were available for 17
patients (5 female and 12 male). The median largest basal diameter of
the tumours was 15.8mm (range 9.2-20.4mm). The median followup time was 1.5 years (range 0.15-3.3 years). Epithelioid cells were
present in nine tumours. Seven biopsy specimens were analysed
by MLPA, the remainder being analysed by MSA. All seventeen
enucleation specimens were analysed by MLPA for chromosomes
1p, 3, 6 and 8. Chromosome 3 data showed complete concordance
between the biopsy samples and enucleated eyes in all 17 cases. Eight
patients were classified as monosomy 3 (M3) and nine as disomy
3. These results were incorporated for patient further management
purposes in the Liverpool Uveal Melanoma Prognostication Online
model. During the follow-up period, one of the eight patients with a
tumour classified as M3, died at the time of data analysis.
Conclusions: It is feasible to obtain DNA of sufficient quality and
quantity from FNAB specimens of UM with no loss of prognostic
accuracy compared with DNA extracted from the enucleated eyes. In
this cohort, tumour cell- or clonal heterogeneity did not play a major
role in affecting the results.
Commercial Relationships: Sarah E. Coupland, None; Helen
Kalirai, None; Vivian Ho, None; Bertil E. Damato, None; Heinrich
Heimann, None
Results: A total number of 151 patients with either Ciliary or
choroidal melanoma were treated at Copenhagen University
Hospital between 1 jan 2009 and 31 dec 2012. in 12 cases no
genetic information were found. 27.5% of the patients had a normal
chromosome status. Normal chromosomal status was found in 9 of 26
stage 1, 28 of 91 stage 2, 4 of 32 stage 3 and 0 of 2 stage 4 patients.
A overall survival rate of 63 % after 4 years were observed. Only
1 melanoma related death was observed in the group with normal
chromosomal status. There was a direct correlation between AJCC
staging and death and number of abnormal chromosomes and death.
A combination of AJCC staging and chromosomal status gave the
best prediction of melanoma related death.
Conclusions: AJCC staging and chromosomal status are good
predictors of survival. For prediction of melanoma related death a
combination of AJCC staging and chromosomal status gave the best
results
The correlation between survival and AJCC staging and genetic
status in uveal melanoma patients
Jens F. Kiilgaard1, 4, Mette Bagger1, 2, Morten T. Andersen2, Steffen
Heegaard3, 4, Mette K. Andersen2. 1Dept of Ophthalmology,
Rigshospitalet, Copenhagen, Denmark; 2Dept of Clinical Genetics,
Rigshospitalet, Copenhagen, Denmark; 3inst of Eye Patology,
Copenhagen University, Copenhagen, Denmark; 4Dept of
Ophthalmology, Glostrup Hospital, Glostrup, Denmark.
Purpose: To evaluate the predictive value of genetic status and AJCC
Staging in patients with uveal melanoma.
Methods: Patient charts for patients treated during 2009 and
2012 were reviewed. Information on survival, AJCC stage and
Chromosomal status were collected.
The value of whole body (18) fluorodeoxyglucose (FDG) positron
emission tomography (PET)/ computed tomography (CT) and
abdominal ultrasound in staging of patients with uveal melanoma
Efthymia Pavlidou1, 2, Amit Arora1, 2, Elizabeth Somerville1, 2,
Mandeep S. Sagoo1, 2, Peter Szlosarek3, Victoria Cohen1, 2. 1Ocular
Oncology, St Bartholomew’s, London, United Kingdom; 2Ocular
Oncology, Moorfields Eye Hospital, London, United Kingdom;
3
Oncology, St Bartholomew’s, London, United Kingdom.
Purpose: To determine the value of whole body (18)
fluorodeoxyglucose (FDG) positron emission tomography (PET)/
computed tomography (CT) and abdominal ultrasound in staging of
patients with uveal melanoma.
Methods: From January 2012 patients with uveal malignant
melanoma over 4 mm in thickness, were staged with whole body
Commercial Relationships: Jens F. Kiilgaard, None; Mette
Bagger, None; Morten T. Andersen, None; Steffen Heegaard,
None; Mette K. Andersen, None
(18) fluorodeoxyglucose (FDG) PET/CT and abdominal ultrasound.
The incidence and location of metastatic disease at diagnosis was
recorded. The imaging findings were compared.
Results: 108 patients had a whole body FDG PET/CT and abdominal
ultrasound at primary diagnosis. 3 patients (2.8%) were found to have
metastatic disease. All 3 had liver metastases, confirmed with biopsy
in only one case. 1 of 3 had additional extrahepatic widespread
metastases in the lungs, lymph nodes and bone seen on whole body
FDG PET/CT.
In the 3 patients with metastatic disease, the liver findings using both
imaging techniques were consistent in one patient. In the second
case abdominal ultrasound misdiagnosed metastatic disease as
liver cysts; however, FDG PET/CT revealed several foci of intense
metabolic activity in both lobes of the liver. In the third case, PET/
CT missed the presence of liver metastases as no metabolic activity
was detected, however a hypodense liver lesion was identified
on abdominal ultrasound. An abdominal CT scan with contrast
confirmed the presence of an enlarging liver metastasis.
PET/CT identified second primary malignancies in 9 patients (8.3%)
and incidental extrahepatic pathology in another 9 patients (8.1%).
Coincidental liver findings, such as fatty liver and haemangiomata,
were seen in 20 patients (18.5%) using abdominal ultrasound but
only 4 (3.7%) using whole body PET/CT.
Conclusions: Whole body PET/CT and abdominal ultrasound
complement each other in the staging of uveal melanoma. PET/CT is
useful to detect extrahepatic disease including widespread metastases
and other primary malignancies. The metabolic activity of uveal
melanoma metastases is variable. Abdominal ultrasound provides
more detailed description of the liver and detected an abnormality in
all 3 cases with liver metastases.
Commercial Relationships: Efthymia Pavlidou, None; Amit
Arora, None; Elizabeth Somerville, None; Mandeep S. Sagoo,
None; Peter Szlosarek, None; Victoria Cohen, None
Capture of circulating melanoma cells using a microfluidic device
Qing Zhang1, 2, Shuo You3, 4, Wilbur A. Lam5, Jordan Ciciliano6, Shin
Kang1, Hua Yang1, Hans E. Grossniklaus1, 3. 1Opthalmology, Emory
University School of Medicine, Atlanta, GA; 2Ophthalmology,
Central South University the Second Xiangya Hospital, Changsha,
China; 3Winship Cancer Institute, Emory University, Atlanta, GA;
4
Endocrinology, Central South University, the Second Xiangya
Hospital,, Changsha, China; 5Pediatrics, Emory University School of
Medicine, Atlanta, GA; 6Laney Graduate School, Emory University,
Atlanta, GA.
Purpose: Circulating tumor cells (CTCs) are present in metastatic
cancers and may potentially be used for disease profiling and
prognostication. Herein we developed a microfluidic device to isolate
circulating melanoma cells (CMCs).
Methods: Human uveal melanoma Mel 270 and Mel 290, and
mouse melanoma B16F10, B16LS9 cell lines were cultured. Murine
intraocular xenografts were performed via trans-scleral inoculation
or mice were injected with melanoma cells via the tail vein. A
microfluidic system, casted in polydimethylsiloxane (PDMS), was
used to capture viable tumor cells from PBS, spiked blood and
peripheral whole blood samples mediated by the interaction of
target CMCs with antibodies-coated microchannels under precisely
controlled flow conditions.
Results: The microfluidic-based system exhibited high capture rates
for B16F10, B16LS9, Mel 270 and Mel 290 cell lines in PBS- or
blood-spiked screening experiments. Increased cell capture rates were
found with relatively slow flow rates and high cell concentrations.
Using an experimental metastasis model established by tail vein
injection in severe combined immunodeficient mice, we successfully
isolated CMCs from peripheral blood samples. The system further
allowed detection of CMCs in a mouse model of xenografts with
metastatic melanoma.
Conclusions: The study shows antibody-based capture of circulating
melanoma cells using a microfluidic device in tumor-bearing mice.
This microfluidic-based system exhibits promise as a clinical tool for
capturing circulating uveal melanoma cells.
Commercial Relationships: Qing Zhang, None; Shuo You, None;
Wilbur A. Lam, None; Jordan Ciciliano, None; Shin Kang, None;
Hua Yang, None; Hans E. Grossniklaus, None
Support: NIH R01CA170006, RPB, Inc., National Natural Science
Funds for Young Scientist, and a Central South University Lieying
pilot grant.
Small metastasizing choroidal melanomas: Report of 6 cases
Ariane Malcles, Anh-Minh Nguyen, Laurent Kodjikian, Philippe
Denis, Jean-Daniel Grange. Ophtalmology, Croix Rousse Hospital,
LYON, France.
Purpose: Small choroidal melanomas have a better prognosis than
large tumors. However these small tumors can spread, often late in
their course.The aim of the study was to analyse survival and tumor
characteristics of 6 cases of late metastatic diseases after conservative
treatment.
Methods: A retrospective study was conducted among 523 patients
treated between 1991 and 2010 by proton beam therapy (508) or
brachytherapy with 106 Ru/Rh (15) for uveal melanomas. We have
selected patients with small choroidal melanoma (thickness ≤ 3
mm and diameter ≤ 9 mm) (59 patients), who have developed liver
metastases (6 out of 59). All tumors were classified T1a at the time of
diagnosis (according to the 7th edition of the TNM stagging system).
Results: At the time of diagnosis, median age was 57 years (range,
37-82 years). Four patients had visual symptoms (blurred vision,
metamorphopsia, floaters). The mean tumor thickness was 2.9 mm
(range 2.5mm – 3mm) and the mean diameter was 7 mm (5mm8mm). Orange pigment was observed in 4 cases, subretinal fluid in 2
cases, and one tumor touched the optic disc.
Five patients had proton beam therapy. One patient had beta
brachytherapy (106 Ru/106 Rh). Average follow-up was 8.3 years
(range 4.2 to 11.8 years). Local ocular tumor control was excellent,
none tumor recurred after local treatment.
The mean survival time after diagnosis of melanoma was 9.8 years
(range, 4.9-14.6 years).
The average time from treatment of primary tumor to detection of
liver metastasis was 7 years (range 3.9 to 12 years).
The mean survival time from the diagnosis of metastasis was 35,2
months (range 9 to 101 months).
Small melanoma-related death was 0% at 3 years, 1.7% at 5 years,
5.1% at 10 years, and 10.2% at 15 years in our series.
Conclusions: Despite a small tumoral size and an early and
effective local treatment 6 out of 59 small choroidal melanomas
have developed metastasis several years after local treatment. Small
tumors represent a significant risk of metastasis.
Commercial Relationships: Ariane Malcles, None; Anh-Minh
Nguyen, None; Laurent Kodjikian, None; Philippe Denis, None;
Jean-Daniel Grange, None
Efficacy and Safety of Transpupillary Thermotherapy for Small
Choroidal Melanomas
Yi Jiang, Rebecca Deutsch, Jack A. Cohen. Ophthalmology, Rush
University Medical Center, Chicago, IL.
Purpose: To investigate the efficacy and the complications of
transpupillary thermotherapy on small sized choroidal melanomas
and nevomas used as primary treatment.
Methods: We retrospectively reviewed all patients seen from January
2000 to September 2013 that received transpupillary thermotherapy
for small choridal melanomas or nevomas. We limited the inclusion
size of the melanomas to 4 millimeters in thickness. We also excluded
any patient that had received other treatments prior to receiving
transpupillary thermotherapy.
Results: There were 34 eyes from 34 patients. 17 (50.0%) patients
were male and 17 (50.0%) were female. The mean age was 58.3
years (23-89). The mean follow up time was 60.8 months (4-149).
Average tumor thickness was 2.89mm (1.1-4.0).
Of all patients, 18 (52.9%) patients ultimately achieved local tumor
control with transpupillary thermotherapy alone while 16 (47.1%)
patients had to undergo other treatments to control their melanoma.
6 patients (37.5%) ultimately had enucleations, 7 patients (43.8%)
underwent brachytherapy, 2 patients (12.5%) had photodynamic
therapy, and 2 patients (12.5%) had proton beam radiation therapy.
Of the 18 patients which were controlled with transpupillary
thermotherapy, 10 patients (55.6%) had good control with one
treatment session. 8 patients ultimately had more than one treatment
session – 6 patients had two sessions (33.3%), 1 patient (5.6%) had
3 treatments sessions, and 1 patient (5.6%) had 7 treatment sessions.
Of the 34 total patients, 17 (50.0%) had control without recurrence
while 15 patients (44.1%) had at least one recurrence and 2 patients
(5.9%) had primary treatment failure.
In total, 59 sessions of transpupillary thermotherapy performed
and there were 10 reported complications – 5 incidences (8.5%)
of vitreous hemorrhage, 2 incidences (3.4%) of retinal striae
formation, 1 (1.7%) branch retinal vein occlusion, 1 (1.7%) subretinal
hemorrhage, and 1(1.7%) rhegmatogenous retinal detachment.
Conclusions: Transpupillary thermotherapy has a good safety
profile with a low complication rate and can be employed when
brachytherapy, proton beam therapy, or enucleation is not desired.
The treatment is limited, however, by the significant recurrence rate
and need for repeat treatments.
Commercial Relationships: Yi Jiang, None; Rebecca Deutsch,
None; Jack A. Cohen, None
Outcomes of Medium Choroidal Melanomas Treated with EyePhysics Ruthenium Plaque Brachytherapy
Andrew Browne1, 2, Jesse L. Berry1, 2, Savita Dadapanai3, Marta
Stevanovic4, Thomas C. Lee5, 1, Melvin Astrahan3, A. Linn Murphree5,
1
, Jonathan W. Kim5, 1. 1Ophthalmology, Doheny Eye Institute, Los
Angeles, CA; 2Ophthalmology, Univ of Southern California, Los
Angeles, CA; 3RadiationOncology, Keck School of Medicine,
University of Southern California, Los Angeles, CA; 4Harvard
College, Harvard University, Cambridge, MA; 5Ophthalmology,
Children’s Hospital Los Angeles, Los Angeles, CA.
Purpose: The Collaborative Ocular Melanoma Study (COMS)
established Iodine 125 plaque brachytherapy as the standard eye
preservation treatment for medium sized choroidal melanomas in
the United States. Plaque Simulator (PS) software guided treatment
utilizing Eye Physics (EP) plaques provides optimal treatment by
enabling customizable radiation profiles for a variety of tumor shapes
and sizes. Berry et. al. recently reported EP customized Iodine-125
therapy with similar results to the COMS Iodine-125 non-custom
plaques. Herein we report results from a series of 15 patients treated
with EP custom Ruthenium Plaque Brachytherapy.
Methods: Fifteen patients with medium sized choroidal melanomas
(2.84-5.5 mm in apical height and a basal diameter of 7.8-12.6mm)
treated with ruthenium-based brachytherapy from 2003-2005 were
evaluated in a retrospective chart review. Baseline and follow-up
data were collected and evaluated for: tumor height, best corrected
visual acuity, radiation retinopathy, radiation optic neuropathy, postradiation cataract formation, diplopia and ptosis. Tumor response
kinetics for EP ruthenium plaques, EP iodine plaques were evaluated
and compared.
Results: Incidences of adverse radiation effects were documented:
optic neuropathy (6.7%), retinopathy (20%), and cataracts (33%)
(Table). Tumor height after therapy demonstrated regression profiles
equivalent to tumors treated with Iodine-125 plaques. Five year
tumor heights for radiation treated tumors were approximately 0.61
+/- 0.29 (iodine, n=16) and 0.53 +/- 0.17 (ruthenium, n= 6) relative to
their heights at diagnosis (Figure).
Conclusions: This patient subset had background characteristics
very similar to those from the COMS and patients treated with
EP iodine-125 plaques. Treatment response was equivalent and
radiation complications occurred slightly less frequently than those
patients treated with iodine-125 radiation. Tumors treated with
EP brachytherapy shrink to about 50-60% of their initial height at
diagnosis. EP plaque brachytherapy demonstrates similar results with
COMS plaques regardless of radiation source.
Table: a. Baseline characteristics of patients b. Tumor response and
clinical outcomes.
Conclusions: In our cohort of patients with large CMM treated
with primary plaque brachytherapy, 15.1% required secondary
enucleation. This information allows us to better counsel our patients
about the pros and cons of primary brachytherapy as opposed to
primary enucleation, help to minimize the potential burden of
additional surgical procedures, and improve quality of life.
Commercial Relationships: Brian Tse, None; Yevgeniy Shildkrot,
None; Matthew W. Wilson, None
Support: Research to Prevent Blindness
Figure: Tumor response to plaque brachytherapy with Iodine (blue)
and Ruthenium (Green). The subgroup of patients who were treated
with Iodine and developed metastasis are plotted in Red.
Commercial Relationships: Andrew Browne, None; Jesse L.
Berry, None; Savita Dadapanai, None; Marta Stevanovic, None;
Thomas C. Lee, None; Melvin Astrahan, Eye Physics, LLC (I); A.
Linn Murphree, None; Jonathan W. Kim, None
Enucleation After Iodine-125 Plaque Brachytherapy in Patients
with Large Choroidal Melanomas
Brian Tse1, 2, Yevgeniy Shildkrot3, Matthew W. Wilson1, 2. 1Dept of
Ophthalmology, University of Tennessee Health Science Center,
Memphis, TN; 2Dept of Surgery, St. Jude Children’s Research
Hospital, Memphis, TN; 3Dept of Ophthalmology, University of
Virginia, Charlottesville, VA.
Purpose: Treatment of choroidal melanoma (CMM) with either
iodine-125 (I125) plaque brachytherapy or enucleation yields similar
survival outcomes. As such, we have used brachytherapy as primary
treatment for large CMMs for more than a decade. Herein, we
evaluate our cohort of patients with large CMM initially treated with
plaque brachytherapy to determine rate of and factors associated with
secondary enucleation.
Methods: A retrospective chart review of patients with large CMMs
diagnosed and treated at our institution from January 1, 1988 to
February 1 2013. Main outcome measures were need for secondary
enucleation, local tumor recurrence, all cause mortality, development
of metastases, initial apical tumor height and maximal basal diameter.
Results: 245 patients with large CMM were treated primarily
with plaque brachytherapy. Local control was achieved in 230
patients (93.8%). Of the 15 patients (6.2%) with local recurrence, 8
underwent secondary enucleation and 7 deferred further treatment. In
all, 37 patients (15.1%) underwent secondary enucleation. Reasons
for secondary enucleation included: neovascular glaucoma or blind,
painful eye (21); local tumor recurrence (8), and scleromalacia (3).
All cause mortality was 116 (47.3%). Metastatic disease was seen in
68 (27.9%). Median time to death was 2.8 years (mean 3.6 +/- 2.7
(0.08-16.7)). Enucleation was performed more frequently in women
(20.2% vs 10.3%; p=0.03). Rate of metastases (p>0.7) and death were
not increased in patients undergoing enucleation, although time to
death appeared to be prolonged in patients who were enucleated (5.6
years vs 3.2 years; p<0.0001). There was no significant difference
(p>0.1) in baseline apical tumor height, maximal basal diameter,
collar-button configuration, or age at diagnosis
Evaluation of a Vision Prognostication Model for Choroidal
Melanoma after Plaque Brachytherapy
Saloomeh Saati1, Jesse L. Berry1, 4, Savita V. Dandapani2, Marta
Stevanovic3, A. Linn Murphree4, Melvin Astrahan2, Jonathan W. Kim4,
1 1
. Ophthalmology, Keck School of Medicine, University of Southern
California, Los Angeles, CA; 2Radiation Oncology, Keck School
of Medicine, University of Southern California, Los Angeles, CA;
3
Harvard College, Harvard University, Cambridge, MA; 4The Vision
Center, Children’s Hospital Los Angeles, Los Angeles, CA.
Purpose: To retrospectively evaluate and apply a vision
prognostication model for patients with choroidal melanoma treated
with Iodine-125 university of southern california (USC) eye physics
plaque brachytherapy.
Methods: A visual nomogram was generated to predict a percentage
of likely visual loss to the level of 20/200 or worse after plaque
brachytherapy for choroidal melanoma by Khan et al. Gender, tumor
height, and dose to macula were found to be the most significant
predictors for vision loss after therapy in their nomogram.To evaluate
the effectiveness of this nomogram for our patient population, we
included 64 patients with primary choroidal melanomas treated
with Iodine -125 eye physics plaques at USC from January 1, 1990
through December 30, 2010. Final endpoint was comparing actual vs.
predicted visual acuity (VA) per the nomogram worse than or equal
20/200 at 1 year post brachytherapy. All patients with initial pretreatment VA less than 20/200 were excluded.
Results: Of 64 patients with pre-treatment VA better than 20/200,
44 % (28) had post- treatment VA equal or worse than 20/200 and
56% (36) had post- treatment VA better than 20/200. Concordance
index of 0.60 and 0.56 was calculated between actual and predicted
loss of vision worse than or equal to 20/200 at 1 year at the level of
50% and 75% respectively according to the nomogram described
by Khan et al. Concordance was not significantly different between
the two levels. Both a threshold of 50% and 75% predicted visual
loss correctly in our patients approximately 60% of the time.
Lack of vision loss to the level of 20/200 or worse was predicted
correctly 87% of time at the 50% threshold and 94% of time at the
75% threshold. Loss of vision to the level of 20/200 or worse was
predicted correctly 12% and 5% of time at the threshold of 50% and
75% respectively.
Conclusions: The described visual prognostication model by Khan
et. al. is a practical, easy to use, first step for clinicians and patients
to predict vision loss after brachytherapy for choroidal melanomas.
In our application of this nomogram, the prediction of vision loss to
the level of 20/200 or worse was correct 60% of the time at both a
predicted threshold of 50% and 75%. In our series, the nomogram
predicted <lack> of vision loss more accurately than vision loss.
Further research to refine the nomogram may be helpful to develop a
valuable model for patients and clinicians.
Commercial Relationships: Saloomeh Saati, None; Jesse L. Berry,
None; Savita V. Dandapani, None; Marta Stevanovic, None; A.
Linn Murphree, None; Melvin Astrahan, Eye Physics, LLC (I);
Jonathan W. Kim, None
EDI-OCT for early evaluation of recurrence after ruthenium
brachitherapy for choroidal melanoma
Monica M. Pagliara, Maria Grazie Sammarco, Francesca Leonardi,
Andrea Scupola, Caputo G. Carmela, Aldo Caporossi, Maria
Antonietta Blasi. Department of Ophthalmology, Catholic University
of the Sacred Heart, Rome, Italy.
Purpose: To compare the use of EDI-OCT and ultrasonography for
early detection of marginal recurrence in posterior uveal melanoma
treated with brachitherapy.
Methods: recently available EDI-OCT has allowed to detect early
recurrences of uveal melanoma after brachytherapy in 3 eyes of 3
patients (1 male and 2 female). Inclusion criteria were: posterior pole
localization and rapidly regression to a flat scar. Every 4 months,
all patients underwent complete follow-up visit including fundus
examination, fundus photography (Panoret), ultrasonography and
EDI using SD OCT with the Heidelberg Spectralis (Heidelberg
Engineering, Heidelberg. Germany).
Results: in these patients, EDI-OCT was able to confirm recurrences
already detected by ultrasonography, however the improved
resolution of EDI-OCT allowed a more precise measurements of
these lesions. In addition, EDI-OCT pointed out other small marginal
nodules deemed undetectable by ultrasonography.
Conclusions: EDI-OCT is particularly useful in identifying small
posterior tumors as its axial resolution is approximately 3 to 4 mm
compared with 10 mm for time-domain OCT and approximately 50
to 200 mm for ultrasonography, as reported in literature. Furthermore,
in our study EDI-OCT documented its ability to located small
recurrences before ultrasonography detection.
In selected cases EDI-OCT proved to be a valid non invasive tool for
monitoring uveal melanoma treated with brachytherapy.
Commercial Relationships: Monica M. Pagliara, None; Maria
Grazie Sammarco, None; Francesca Leonardi, None; Andrea
Scupola, None; Caputo G. Carmela, None; Aldo Caporossi, None;
Maria Antonietta Blasi, None
A Biological Tissue Adhesive and Dissolvent System for
Intraocular Tumor Plaque Radiotherapy
Ido Didi Fabian, Vicktoria Vishnevskia-Dai, Michael Belkin, Ofira
Zloto. The Goldschleger Eye Institute, The Sheba Medical Center and
The Sackler Faculty of Medicine, Tel Aviv University, Ramat Gan,
Israel.
Purpose: To examine the feasibility of a novel technique for plaque
radiotherapy placement and removal, using fibrin glue and urokinase,
respectively, to explore quantitative variables, and compare these to
conventional suturing.
Methods: The feasibility of the surgical technique was tested in 6
enucleated porcine eyes. Plaques without radioactive seeds were
placed on the sclera beneath the conjunctiva, fibrin glue was then
applied on the plaque, covering it and its immediate environment
(Figure 1A). The eyes were then submerged in plasma for 3 days.
Thereafter, attempts were made to dissolve the glue and release
the plaque using urokinase (10,000 units/10cc normal saline (NS))
or NS only, by a surgeon masked as to the nature of the dissolving
fluid (Figure 1B). In a second group of eyes, adhesion strength was
measured using a system constructed of a tension transducer, a pulley,
a transmission wire and an electrical winch (Figure 2). The eyes
were prepared as described above, after which they were submerged
in plasma for 5 days (replaced every 36 hours). Measurements were
performed subsequently on 5 glued eyes and 5 eyes in which the
plaques were sutured, and the results were recorded and analyzed.
Results: One to 2 ml of fibrin glue was used in order to fix each
plaque in place. In all cases the urokinase syringe was identified,
as the NS had no effect on the plaque–glue–eye complex, whereas
the urokinase dissolved the adhesion between the glue layer and
surrounding tissues. The plaques were then delivered out easily,
after which a glue “blanket” was removed in full. No tissue defects
were observed thereafter. The volume of urokinase used per eye was
0.38±0.08 ml. In the second part, the weights needed to detach the
plaques were 0.35±0.17 kg and 0.41±0.08 kg for the glue and sutures,
respectively (P=0.59).
Conclusions: A novel biological adhesive and dissolvent system
using fibrin glue and urokinase was found to be a feasible technique
for plaque surgery in an ex-vivo animal model. Fibrin glue was as
durable as sutures, but may be preferable to the use of sutures in
enabling precise plaque positioning and in the lack of complications,
such as globe perforation or plaque displacement. Further in-vivo
experiments are warranted.
A - application of fibrin glue. The blue color is due to food coloring
(for visualization purposes)
B - application of urokinase
developed SNEC, 8 after I-125 plaque and 1 after SRT (p=0.3).
Thicker tumors (>6.5mm) were more likely to develop SNEC (n=7)
than thinner tumors (p=0.05). In the studied group, the cumulative
probability of developing radiation induced SNEC was 6.2% at 6
months, 7.4% at 12 months, 10.1% at 18 months, and 14.3% at 24
months remaining stable after that (14.3% at 42 months). Meanwhile
the cumulative probability of developing radiation induced SNEC
among thicker tumors was 23.5% at 45 months. Five patients were
treated by implantation of a scleral patch graft, 3 (with limited
SNEC) were managed by observation (33.3%), and 1 was enucleated.
The mean follow-up time among patients who did not develop SNEC
was 22.1 months (±14.0). The overall cumulative probability of
survival in this group was 77.3%.
Conclusions: In our series, tsFNAB appeared to have a role in the
development of radiation induced SNEC in patients evaluated treated
by focal radiation. Patients with thicker tumors seem to be more
likely to develop SNEC after focal radiation treatment for posterior
uveal melanoma. Observation has been our choice for limited SNEC,
but scleral patch graft is an alternative for eye preservation in cases of
extensive SNEC.
adhesion strength measuring system
Commercial Relationships: Ido Didi Fabian, None; Vicktoria
Vishnevskia-Dai, None; Michael Belkin, None; Ofira Zloto, None
Scleral necrosis in patients with posterior uveal melanomas
evaluated by transcleral FNAB and treated by focal radiation:
incidence and management
Zelia M. Correa1, James J. Augsburger2. 1Ophthalmology, University
of Cincinnati, Cincinnati, OH; 2Ophthalmology, University of
Cincinnati, Cincinnati, OH.
Purpose: Evaluate the incidence and management of scleral necrosis
(SNEC) in posterior uveal melanomas treated by radiation (I-125
plaque or Stereotactic Radiation Therapy [SRT]) and evaluated by
transcleral fine needle aspiration biopsy (tsFNAB) prior to treatment.
Methods: We reviewed all of our posterior melanomas treated
by focal radiation from July 2006 to July 2013 and selected those
evaluated by tsFNAB prior to focal radiation treatment. Statistical
analysis included computation of conventional descriptive statistics;
cross-tabulation and Chi-square test of factors possibly related
to the development of SNEC and summarization of management
approaches and results. Incidence of SNEC was calculated using the
Kaplan-Meier method.
Results: A total of 435 patients with posterior uveal melanoma were
treated during the 7-year study interval. Eighty-eight of them were
evaluated by tsFNAB and subsequent focal radiation treatment.
Fifty-one percent of patients were females and median patient age
was 62.3 years (± 13.9). Median largest basal diameter was 13.3 mm
(±2.7) and tumor thickness was 6.8 mm (±2.4). Nine patients (10.2%)
Commercial Relationships: Zelia M. Correa, None; James J.
Augsburger, None
Support: This work was supported in part by an unrestricted grant
from Research to Prevent Blindness to James J. Augsburger, MD,
Chairman, Department of Ophthalmology, University of Cincinnati
College of Medicine.
Gene Expression Profiling and Regression Rate of Irradiated
Uveal Melanomas
Rajesh C. Rao1, 2, Shahed N. Badiyan3, J William Harbour4.
1
Ophthalmology & Visual Sciences, W.K. Kellogg Eye Center,
University of Michigan, Ann Arbor, MI; 2Pathology, University
of Michigan, Ann Arbor, MI; 3Radiation Oncology, Washington
University School of Medicine, St. Louis, MO; 4Ocular Oncology
Service, Ophthalmology, Bascom Palmer Eye Institute, Sylvester
Comprehensive Cancer Center, University of Miami Miller School of
Medicine, Miami, FL.
Purpose: To determine whether gene expression profiling (GEP)
is associated with rate of tumor regression following I-125 plaque
brachytherapy for posterior uveal melanoma.
Methods: Retrospective review of of 83 patients with posterior uveal
melanoma treated with I-125 plaque brachytherapy in which GEP
class and 3-month post-radiation ultrasonographic tumor thickness
were available. Recorded data included baseline patient and tumor
characteristics and tumor thickness at 3-months after treatment.
Statistical analysis was performed using T-test and Fischer’s exact
test.
Results: GEP class assignment was class 1 in 83 (60.1%) and class
2 in 55 (39.9%) of patients. Mean patient age was 60.9 years for
class 1 and 68.1 years for class 2 tumors (P=0.002). Mean initial
tumor diameter was 13.0 mm for class 1 and 14.1 mm for class 2
tumors (P=0.02). Mean initial tumor thickness was 5.3 mm for class
1 and 6.1 mm for class 2 tumors (P=0.09). Mean reduction in tumor
thickness at 3-month post-radiation 26.5% for class 1 and 13.0% for
class 2 tumors (P=0.01). Complete tumor regression with flat residual
tumor was observed for four class 1 tumors and no class 2 tumors.
Conclusions: Class 1 tumors exhibit more rapid early tumor
regression than class 2 tumors following I-125 plaque radiotherapy.
This observation may reflect molecular and/or immunologic
differences between the two tumor classes.
Commercial Relationships: Rajesh C. Rao, None; Shahed N.
Badiyan, None; J William Harbour, Castle Biosciences (C), Castle
Biosciences (I), Castle Biosciences (P)
Support: J.W.H. - National Cancer Institute (R01CA125970),
Melanoma Research Foundation, Melanoma Research Alliance and
the Tumori Foundation, R.C.R. and S.N.B. - None
Evaluation of Cytokines and Chemokines in the Vitreous
Fluid of Eyes With Radiation Maculopathy Following Plaque
Radiotherapy for Uveal Melanoma
Hakan Demirci1, Ryan J. Fante1, Thomas W. Gardner1, Jeffrey
Sundstrom2. 1Department of Ophthalmology and Visual Sciences,
W.K. Kellogg Eye Center, University of Michigan, Ann Arbor, MI;
2
Department of Ophthalmology and Visual Science, Penn State
University, Hershey, PA.
Purpose: To evaluate the role Inflammatory cytokines and
chemokines in eyes with radiation maculopathy following plaque
radiotherapy for uveal melanoma.
Methods: Vitreous sample were obtained from five patients who
developed radiation maculopathy following plaque radiotherapy for
uveal melanoma immediately prior intravitreal treatment. Vitreous
from five patients undergoing pars plana vitrectomy for macular hole
were used as controls. Cytokines were analyzed using a 42 plexcytokine bead array.
Results: The clinical manifestations of radiation maculopathy varied
among patients. Two patients had macular edema without nerve
fiber layer infarcts or retinal dot-blot hemorrhages, two patients
had macular edema, nerve fiber layer infarcts and retinal dot-blot
hemorrhages and one patient had nerve fiber layer infarcts and retinal
dot-blot hemorrhages without significant macular edema.
In all patients with radiation maculopathy, fractalkine, GRO, MDC,
PDGF-AA, IL-6, IL-8, IP-10, MCP-1 and VGEF were increased in
comparison to the controls. Patients with macular edema but without
nerve fiber layer infarcts or retinal dot-blot hemorrhages were noted
to have RANTES, MDC, sCD40L, IL-15 and Flt-3L increased in
comparison to controls. In the patient with nerve fiber layer infarcts
and retinal dot-blot hemorrhages without significant macular edema,
IFN-a2, GRO, PDGF-AA, IL-6, IL-8 and MCP-1 were increased
in comparison to controls and the other 4 patients. Interestingly,
VEGF was highest in the patient who had nerve fiber layer infarcts
and retinal dot-blot hemorrhages without significant macular edema,
while it was lowest in the patients who had macular edema without
nerve fiber layer infarcts or retinal dot-blot hemorrhages.
Conclusions: These study shows that it is safe and feasible to
study vitreous cytokines and chemokines from vitreous samples
obtained in the clinical setting. Cytokines and chemokines expression
pattern seems to be different in various manifestations of radiation
maculopathy. With sufficient sample size and power, future studies
may help to identify biomarkers predictive of treatment response and
identify novel treatment targets in radiation maculopathy.
Commercial Relationships: Hakan Demirci, None; Ryan J. Fante,
None; Thomas W. Gardner, None; Jeffrey Sundstrom, None
Support: Ey20582, RPB Physician-Scientist Award and Taubman
Medical Research Institute.
Secondary Glaucoma after Cyberknife Radiotherapy for Uveal
Melanoma - Preliminary Analysis
Simon F. Leicht1, Veronika Reiterer1, Alexander Muacevic2, Paul
Foerster1, Anselm Kampik1, Kirsten Eibl-Lindner1. 1Department of
Ophthalmology, Ludwig-Maximilians-University, Munich, Germany;
2
European CyberKnife Center, Munich, Germany.
Purpose: Uveal melanoma represents the most common primary
intraocular malignancy. For tumor treatment, different modes of
radiation are applied such as brachytherapy (ruthenium106 or
iodine 125 plaque) or teletherapy with proton therapy or Cyberknife
radiotherapy. Cyberknife radiotherapy is administered as a frameless
single session outpatient procedure and represents one of the
latest available treatment techniques. Data on complications after
Cyberknife radiotherapy are unavailable so far and thus we aimed
to determine the incidence of secondary glaucoma after treatment,
a significant complication regarding life quality and long-term
preservation of vision.
Methods: We performed a retrospective chart review of 143
consecutive patients with uveal melanoma from our ocular oncology
service who had successfully undergone Cyberknife radiotherapy
between 2007 und 2013. The follow up period, tumor thickness as
determined by standardized ultrasound examination before treatment,
incidence of secondary glaucoma and time until onset of secondary
glaucoma were determined.
Results: 143 patients were included in the study. The follow up
period ranged from 2.73 to 85.22 months with a mean of 22.34
months. Tumors had an average thickness of 6.49 mm prior to
therapy. Of all patients, 27% (38 of 143 patients) developed
secondary glaucoma during the follow up period. The total
enucleation rate due to secondary glaucoma was 8% (12 of 143
patients). Otherwise, IOP was controlled effectively by topical and/
or Laser treatment. Secondary glaucoma occured at a mean of 21.8
months after Cyberknife radiotherapy ranging from 2.79 to 64.44
months.
Conclusions: Cyberknife radiotherapy allowed for tumor control in
all patients included in this retrospective chart review. The incidence
of secondary glaucoma was 27% at a mean follow-up time of 21.8
months. Secondary glaucoma is a critical parameter in the long-term
assessment for vision preservation in these patients.
Commercial Relationships: Simon F. Leicht, None; Veronika
Reiterer, None; Alexander Muacevic, None; Paul Foerster, None;
Anselm Kampik, None; Kirsten Eibl-Lindner, None
Intravitreal bevicizumab anti-vascular endothelial growth factor
therapy for radiation associated neovascular glaucoma
Ekaterina A. Semenova, Sonali Nagendran, Paul T. Finger. Ocular
Oncology, The New York Eye Cancer Center, New York, NY.
Purpose: To describe the effects of intravitreal bevicizumab antivascular endothelial growth factor therapy on radiation associated
neovascular glaucoma.
Methods: This is a retrospective single centre interventional case
series examining 12 eyes of 12 patients with ocular malignancies
(10 uveal melanoma, 1 conjunctival melanoma, 1 ciliary body
adenocarcinoma) who developed neovascular glaucoma after
radiation therapy (11 plaque brachytherapy and 1 external beam
teletherapy). All patients were treated with periodic 1.25 mg
intravitreal bevicizumab injections. Outcome measures assessed
included regression of iris neovascularization, change in intraocular
pressure (IOP), visual acuity, pain control and enucleation.
Results: Regression of iris neovascularization occurred in 9/12
patients after the first injection. The intraocular pressure (IOP)
decreased in 8/12 patients (67%) with a mean reduction of 9.25
mmHg across the study group. Visual acuity improved in 1/12 (8%),
remained stable in 5/12 (42%) and deteriorated in 6/12 (50%).
Patients were monitored for 24 months on average after the first
injection. Six eyes subsequently underwent enucleation for pain
control (4 patients, 66%), chronic uveitis (1 patient, 17%) and
tumor recurrence (1 patient, 17%). The 6 patients that retained their
eyes reported good pain control. Visual acuity ranged from 20/160
to no light perception. Intraocular pressures remained within the
normal range in 4 patients, 3 of whom required continued periodic
bevicizumab injections. Two patients had significantly raised but
stable IOP and opted to stop treatment.
Conclusions: Intravitreal bevicizumab is a promising treatment for
patients with radiation associated neovascular glaucoma who wish
to avoid enucleation. Although visual acuities did not improve, there
was clinical evidence of regression of iris neovascularization and
treatment achieved pain control and acceptable IOP control in the
majority of patients.
Commercial Relationships: Ekaterina A. Semenova, None; Sonali
Nagendran, None; Paul T. Finger, None
Support: The Eye Cancer Foundation
Long-term Experience with Intravitreal Anti-VEGF Therapy for
Radiation Retinopathy
Kimberly J. Chin, Paul T. Finger, Ekaterina A. Semenova, Sonali
Nagendran. New York Eye Cancer Center, New York, NY.
Purpose: To present a subgroup long-term analysis of patients with
uveal melanoma treated with intravitreal anti-VEGF therapy using
escalation strategies for radiation retinopathy (RR).
Methods: At a single ophthalmic oncology center, 127 patients
underwent intravitreal anti-VEGF injections for RR over 7 years.
A subgroup analysis was performed on 29 patients who met the
following inclusion criteria: diagnosis of uveal melanoma and
subsequent RR; pre-anti-VEGF acuity of 20/200 or better; followup of at least 6 months and at least 4 intravitreal injections; initial
and follow-up OCT central foveal thicknesses (CFT) on a single
spectral domain device. In that treatment was initiated at the first sign
of RR and/or edema of the macula or optic nerve, patients treated
for longstanding RR were not included. Our treatment protocol
has been refined over 7 years as follows: patients initially received
1.25 mg bevacizumab at 4 week intervals, increasing to 5-8 weeks
if improved. If two successive exams demonstrated worsening of
RR (by comparative ophthalmoscopy, photography, fluorescein
angiography) or increased CFT, patients were first shortened to a
4-week treatment interval, followed by increased dose to 2.0 mg,
2.5 mg, and lastly 3.0 mg. Best corrected visual acuity (ETDRS),
fundus photography, and CFT was recorded at each visit; fluorescein
angiography at 6 month intervals.
Results: Our subgroup analysis of 29 patients revealed a mean age
of 65 years with mean follow-up 22 months (range 9-48). By AJCC
criteria, there were 19 T1, 7 T2, and 3 T3-sized uveal melanomas.
Patients were treated with palladium-103 (n=27) or iodine-125
(n=2) to a mean foveal and optic nerve dose of 56 Gy and 43
Gy, respectively. Mean initial and final acuity (logMar) was 0.1
(20/25); acuity did not significantly change at any interval (p>0.05).
There was a general trend towards increased dose: at 1 year 68%
(19/28) and at 2 years, 42% (8/19) were still on baseline dose. CFT
significantly improved at 3 and 12 months (both p=0.04), but not
at the longer follow-up intervals (>2 years). This may be due to the
small number of patients at these intervals.
Conclusions: Intravitreal anti-VEGF dose escalation strategies may
offer patients additional time to forestall vision loss. Long-term
stability of visual acuity was achieved despite the progressive, natural
course of radiation retinopathy which ultimately leads to subsequent
severe vision loss.
Commercial Relationships: Kimberly J. Chin, None; Paul
T. Finger, The New York Eye Cancer Center (P); Ekaterina A.
Semenova, None; Sonali Nagendran, None
Endoresection Surgery for Intra Ocular Choroidal Tumors. Short
Term Results
Andre A. Vidoris1, Andre Maia1, Rubens Belfort, Jr.1, Marcia Lowen1,
Bruno Fernandes2, 1, Rafaello Salas1, Marcio Costa1. 1Ocular
Oncology, Federal University of Sao Paulo, Sao Paulo, Brazil;
2
Mcgill Univesity, Montreal, QC, Canada.
Purpose: Internal eyewall resection or endoressection has been
proposed as an alternative therapy to irradiation by Peyman et al. and
Damato et al.
Endoressection is a surgical technique where the tumor is excised by
a vitreous cutter during pars plana vitrectomy, which provides direct
access to posterior tumors.
This study aims to evaluate the endoressection technique in the
treatment of choroidal melanoma.
Methods: Thirteen patients with choroidal melanoma, diagnosed
by indirect ophthalmoscopy, ocular ultrasonography, and fundus
photography were selected for this study. None of the patients
showed evidence of metastasis on systemic screening. The same
surgeon performed all surgical procedures. Patients were evaluated by
best-corrected visual acuity (EDTVRS chart), slit lamp microscopy
(including the sclerotomy sites), indirect ophthalmoscopy,
retinography, and ocular-ultrasonography at the inclusion. Each
patient was seen at baseline, d30, d60, d90, and in 3-month intervals
afterwards for at least 6 months. The systemic-workup (liver tests,
abdominal ultrasonography, chest X-ray) was done every 3 months
starting for at least 6 months.
The vitreous aspirate from every case was sent to histopathological
evaluation. The fluid was centrifuged and fixed in formaline, and
embedded in paraffin. A trained ocular pathologist evaluated the
Hematoxilin-eosin stained slides by light microscopy.
Results: Thirteen patients, including eight (08) woman’s (61,5%%)
and one male (38,5%) were included in the study. The age ranged
from 42 to 65 years old, the mean age was 50,25. The tumor size
by ultrasonography measures ranged from 3.3 to 9.7 millimeters in
high, the mean high was 6.03 millimeters. In all cases the pathologist
was able to make a histological diagnose. The eye retention rate was
100% in twelve months. In a twelve-month follow up, one patient
developed liver metastasis.
Conclusions: Endoresection could be a safe and eye-sparring
treatment for patients with medium to large choroidal melanoma.
Every globe was saved and no recurrences were detected. Moreover,
the material obtained during the surgical procedure was able to
yield an accurate pathological diagnosis in all patients. Our pilot
study confirms the usefulness of this therapeutic modality for local
control of the disease. Larger series and longer follow-up periods will
determine whether the natural history of the disease is affected.
detection of metastases, the type and outcome of therapies, the time
of last follow-up or death, and the cause of death were included. Data
were analysed with a Cox regression model to identify prognostic
factors that may influence final outcome.
Results: The median age at detection of primary uveal melanoma
was 61.5 years (range, 25.3-82.5 years); metastatic disease was
detected at median age of 63.6 years (range, 34.3-83.0 years)
and median survival from metastasis detection was 18.5 months
(range, 1.2-86.4 months). The most frequent first metastatic site
was liver (79%). Patients received loco-regional hepatic treatments
(12.1%), systemic therapy (39.7%), both (33.3%) or no treatment
(14.9%). Liver substitution entity (hazard ratio (HR) 1.3), multiple
organ involvement (HR 2.0), increasing levels of serum lactate
dehydrogenases(LDH) (HR 1.8) and WHO performance status = 1
(HR 1.6) or 2-3 (HR 4.7) were associated with worse prognosis. Long
disease-free interval (HR 0.9) and loco-regional treatment for liver
metastases (HR 0.5) conferred a survival advantage (22.6 months)
compared to systemic therapies alone (13.4 months) (p=0.038). A
nomogram to predict death probability within 6, 12 and 24 months
was developed (C-index=0.74).
Conclusions: Life prognosis of metastatic uveal melanoma depends
on the percentage of liver involvement, spread to multiple organs,
serum LDH, performance status, disease-free interval and locoregional treatment.
Commercial Relationships: Raffaele Parrozzani, None; Sara
Valpione, None; Vanna Chiarion-Sileni, None; Olympia Kotsafti,
None; Edoardo Midena, None
Commercial Relationships: Andre A. Vidoris, None; Andre Maia,
None; Rubens Belfort, Jr., None; Marcia Lowen, None; Bruno
Fernandes, None; Rafaello Salas, None; Marcio Costa, None
Metastatic Uveal Melanoma: a Prognostic Model and Nomogram
Raffaele Parrozzani1, Sara Valpione2, Vanna Chiarion-Sileni2,
Olympia Kotsafti1, Edoardo Midena1, 3. 1GB Bietti Foundation,
IRCCS, Roma, Italy; 2Melanoma Unit, Veneto Region Oncology
Research Institute (IOV-IRCCS), Padova, Italy; 3Department of
Ophthalmology, University of Padova, Padova, Italy.
Purpose: The aim of this study was to identify independent
prognostic factors to formulate a prognostic nomogram to treat
metastatic uveal melanoma patients
Methods: Prospectively recorded data of 141 patients with metastatic
uveal melanoma, who received different therapy (loco-regional
hepatic treatments, systemic therapy, both or no treatment) as firstline approach, were retrospectively analysed. Metastatic disease
was confirmed by biopsy or fine needle aspiration cytology. The
characteristics of primary uveal melanoma, the site and time of
Clinical Predictors of Survival after Hepatic Arterial
Chemoembolization for Stage IV Uveal Melanoma
Scott D. Walter1, Tahira Mahten2, J William Harbour1, 3. 1Department
of Ophthalmology, Bascom Palmer Eye Institute, University of
Miami, Miami, FL; 2Department of Ophthalmology and Visual
Sciences, Washington University School of Medicine in St. Louis,
Saint Loius, MO; 3Sylvester Comprehensive Cancer Center,
University of Miami, Miami, FL.
Purpose: To determine factors predicting response to hepatic arterial
chemoembolization (HACE) in patients with metastatic uveal
melanoma.
Methods: A retrospective analysis of 37 patients who underwent
HACE for metastatic uveal melanoma. Primary outcome measure
was months from first HACE to death. Statistical analysis was
performed using Cox proportional hazards analysis.
Results: Factors associated with poor outcome following HACE
included epithelioid cell type (HR 3.34, p=0.02) and ciliary body
involvement in the primary tumor (HR 3.04, p=0.01). Factors
associated with better outcome included nodular angiographic pattern
of liver metastasis (HR 0.22, p<0.001), fewer than 10 liver metastases
(HR 0.16, p=0.001), pre-treatment liver enzymes less than twice the
upper limit of normal (alkaline phosphatase HR 0.42, p=0.05; alanine
aminotransferase HR 0.17, p<0.001), and greater number of HACE
treatments (HR 0.36, p<0.001). The use of Gelfoam drug-eluting
particles was a significant predictor of longer survival, even after
controlling for number of HACE treatments (HR 0.58, p=0.03).
Conclusions: HACE for metastatic uveal melanoma is most effective
in patients with a nodular angiographic pattern, fewer than 10 liver
metastases, and lower pre-treatment liver enzymes. HACE is more
effective in treating metastatic uveal melanoma when combined with
the use of drug-eluting Gelfoam particles.
Commercial Relationships: Scott D. Walter, None; Tahira
Mahten, None; J William Harbour, Castle Biosciences (P)
Support: NIH Center Core Grant P30EY014801, Research to
Prevent Blindness Unrestricted Grant, Department of Defense Grant
DOD#-W81XWH-09-1-0675
Has survival time of patients following detection of metastatic
uveal melanoma really increased in recent years?
James J. Augsburger, Zelia M. Correa. Ophthalmology, University of
Cincinnati, Cincinnati, OH.
Purpose: During the past decade, numerous treatments that were
not available previously have been introduced for management of
metastatic uveal melanoma. Many clinicians are convinced (usually
on the basis of one or a few anecdotal cases in spite the absence of
published reports of any large scale randomized clinical trials) that
the various newer treatments prolong patient survival substantially.
The authors attempted to determine whether patients with metastatic
uveal melanoma encountered in recent years truly have prolonged
survival relative to patients encountered during the preceding two
decades.
Methods: Retrospective analysis of two sequential series of cases
of metastatic uveal melanoma encountered in the authors’ private
practice. The earlier series (Wills Eye Hospital series, 1980 – 1999)
consisted of 320 patients while the more recent series (University of
Cincinnati series, 1999-2013) consisted of 81 cases.
Results: The clinical characteristics of the patients (age, gender) and
their tumors (largest basal diameter, thickness, intraocular location)
at baseline were all quite similar. The mean interval between initial
treatment of the primary intraocular tumor and detection of metastasis
in the earlier series was substantially longer (49.6 months) than that
in the more recent series (35.2 months) . At the same time, the mean
interval between detection of metastasis and death was shorter in
the earlier series (7.7 months) than in the more recent series (9.5
months). However, comparative actuarial survival curves for length
of survival following detection of metastasis showed only a slight
difference between the curves with a median survival improvement in
the more recent series of only about 2 months.
Conclusions: While this study revealed a slight improvement in
survival time of patients with metastatic uveal melanoma in recent
years compared with that observed in earlier years, one cannot tell
whether this improvement is due to improved effectiveness of current
treatments, detection of metastasis at a less advanced substage due to
increased and improved surveillance testing, or other factors.
Commercial Relationships: James J. Augsburger, None; Zelia M.
Correa, None
Support: Research to Prevent Blindness, Inc. (Unrestricted
Departmental Grant to Department of Ophthalmology, University of
Cincinnati (J. Augsburger, MD, Chairman)
503 Conjunctival and ocular adnexal tumors
Thursday, May 08, 2014 8:30 AM–10:15 AM
Childhood Orbital, Ocular and Optic nerve tumors in Egypt
Ahmad S. AlFaar1, 2, Mohamed S. Bakry1, Sameera Ezzat1, 3. 1Research
Department, Children Cancer Hospital - Egypt 57357, Cairo, Egypt;
2
Ophthalmology, Cairo University School of Medicine, Cairo, Egypt;
3
Public Health, National Liver Institute, Shebin Elkom, Egypt.
Purpose: Our aim is to study the incidence of different ocular, orbital
and optic nerve tumors in childhood age group (between 0-18 years
old) in the largest Egyptian pediatric oncology referral center and
correlate the features of each disease and its survival outcomes.
Methods: Institutional cancer registry database was reviewed for
patients who presented with orbital tumors as a primary site of
involvement between July 2007 and November 2013. REDCap
(Research Electronic Data Capture) system was used for data
collection and organization. Integration between clinical information
system and RedCap was established for real-time registry updating.
Relevant ICD-O-3 topography codes were used to identify the sites.
Data were presented in terms of frequencies and percentages. Other
demographic properties were presented. Survival was demonstrated
using kaplan-meier curves.
Results: Among 7277 patients presented with different childhood
tumors 425 had the mentioned lesions. Males were affected more
than females. Retinoblastoma, Astrocytomas of optic nerve and
Rhabdomyosarcoma of orbit were the most frequent tumors. Mean
age of retinoblastoma cases was 1.4 years while it was higher in
other diseases except germ cell tumors. We’ve presented correlations
of our findings with the current Egyptian population-based cancer
registry and previous results from Cancer in five continents report.
Overall Survival of Retinoblastoma was 95.4% while it was better in
Germ cell tumors and less in all other tumor categories. Orbital bones
masses were hard to identify due to the broad classification in current
ICD-O topography coding.
Conclusions: Childhood orbital tumors distribution was similar
to international publications except increased incidence of orbital
lymphomas. Special attention should be paid to updating the ICD-O
classification system to present different skull bones.
Commercial Relationships: Ahmad S. AlFaar, None; Mohamed S.
Bakry, None; Sameera Ezzat, None
Successful management of ocular juvenile xanthogranuloma
using off-label bevacizumab: a report of 2 cases
Noy Ashkenazy, Christopher M. Henry, Ashkan M. Abbey, Craig A.
McKeown, Audina M. Berrocal, Timothy G. Murray. Bascom Palmer
Eye Institute, Miami, FL.
Purpose: To present off-label bevacizumab as a potential therapeutic
modality for ocular JXG refractory to management with local
corticosteroid therapy.
Methods: Retrospective case series
Results: Case 1: A four-year-old male presented with pain and
decreased vision in the right eye. Examination revealed a 1-mm
hyphema, which initially cleared with topical atropine 1% drops
BID. Upon recurrence 1 week later, gonioscopy revealed a yellow
mass on the surface of the iris (Figure 1a), and OCT demonstrated
hyperreflective mass on the iris surface, abutting the trabecular
meshwork (Figure 1b). Topical atropine 1% drops BID and
prednisolone acetate 1% QID were used. Nonetheless, 1 month after
discontinuing steroid therapy, the patient returned with recurrent,
spontaneous hyphema. Growth of the iris lesion with overlying
focal hemorrhage was seen (Figure 1c). Intravitreal bevacizumab
(1.25mg/0.05cc) was injected via the pars plana with a 30-gauge, 0.5inch needle. There was complete involution of the lesion (Figure 1d),
with improved visual acuity.
Case 2: A 6-month-old female with a history of multifocal cutaneous
lesions presented with recurrent subconjunctival hemorrhages OD.
There was a 3-mm elevated inferotemporal episcleral lesion (Figure
2a), with gonioscopy demonstrating involvement of the posterior
peripheral cornea, angle, and iris (Figure 2b). Following failed
therapy with sub-Tenon’s injection of triamcinolone acetonide
(40mg/1cc) and topical prednisolone acetate 1% ophthalmic drops,
off-label intracameral bevacizumab (1.25mg/0.05cc) was delivered
on a 30-gauge needle. 3 months later, gonioscopy showed flattening
of the epibulbar component (Figure 2c) and involution of the
intraocular lesion (Figure 2d), with minimal residual scarring and no
recurrent hemorrhages over the next 36 months. The patient showed
reduced astigmatism and no longer required topical glaucoma drops.
Conclusions: There is presently no standard treatment for the
ophthalmic manifestations of JXG. The current case series is the
first report to demonstrate the efficacy of off-label intraocular
bevacizumab for ocular JXG, even in refractory cases. Intraocular
bevacizumab offers significant advantages in its lower associated risk
of cataract and secondary glaucoma compared to local steroid therapy
or radiation. Additionally, it spares the adverse effects of systemic
chemotherapeutic agents.
Figure 1 (a-d).
Figure 2 (a-d).
Commercial Relationships: Noy Ashkenazy, None; Christopher
M. Henry, None; Ashkan M. Abbey, None; Craig A. McKeown,
None; Audina M. Berrocal, None; Timothy G. Murray, None
Giant Orbital Hydrocystoma in Children: Report of Three Cases
Mehrdad Malihi1, Roger Turbin1, Neena Mirani2, Paul D. Langer1.
1
The Institute of Ophthalmology and Visual Sciences, New Jersey
Medical School, Newark, NJ; 2Department of Pathology, New Jersey
Medical School, Newark, NJ.
Purpose: Hydrocystoma (also known as sudoriferous cyst) is a
benign cystic proliferation of a sweat gland found commonly on
the eyelid skin of adults; we report three cases of giant orbital
hydrocystoma in children, expanding the clinical and pathological
spectrum of this entity.
Methods: Interventional case series.
Results: Case 1: An 8 year-old-boy presented with a 1 year history
of painless progressive right proptosis. Computed tomographic
and magnetic resonance imaging (MRI) revealed a well-defined,
intraorbital, extraconal cystic lesion in the lateral orbit posterior
to the globe causing bony erosion (Fig 1). Pathologic examination
following total resection via lateral orbitotomy reveals a cystic lesion
with a clear cavity lined by a smooth surface of a double layer of
cuboidal cells, consistent with eccrine hydrocystoma.
Case 2: A 13-year-old girl who had suffered blunt orbital trauma one
year earlier developed a soft, mobile, non-tender subconjunctival
mass in the temporal part of the right upper eyelid. MRI revealed a
large well-defined cystic lesion in the right anterior orbit, which was
later dissected from beneath the conjunctiva. Pathologic examination
reveals a cystic cavity with papillary projections lined by two layers
of cuboidal epithelial cells, with the innermost cells displaying
eosinophilic cytoplasm and apical “snouting” (decapitation),
characteristic of an apocrine hydrocystoma (Fig 2).
Case 3: A two-month-old infant was noted to have a non-tender
subconjunctival orbital growth visible in the medial left palpebral
aperture. Surgical excision revealed a cystic lesion with a smooth
internal surface. Microscopic evaluation revealed characteristics
similar to case 1 and consistent with eccrine hydrocystoma.
Conclusions: Periocular hydrocystoma, which typically presents
on the eyelid skin in adults, only rarely occur beneath the skin or
conjunctiva: only 8 such lesions have previously been reported, 4
of them in the pediatric population. Presumably, the deeper location
of these common skin lesions results either from embryonic rests or
from traumatic implantation of glandular epithelium. All 4 previously
reported pediatric cases were congenital and of apocrine subtype.
Two of the cases in our series were of eccrine subtype, and one was
traumatic in origin, expanding the clinical and pathological spectrum
of this entity. Clinicians should be aware that an orbital cystic lesion
in a child may represent a giant hydrocystoma.
Commercial Relationships: Mehrdad Malihi, None; Roger
Turbin, None; Neena Mirani, None; Paul D. Langer, None
Growth rates of orbital cavernous hemangiomas: A quantitative
analysis
Liza M. Cohen1, Anupam Jayaram1, Gary Lissner1, Achilles
Karagianis2. 1Ophthalmology, Northwestern University, Chicago, IL;
2
Radiology, Northwestern University, Chicago, IL.
Purpose: Cavernous hemangiomas of the orbit are characterized by
their often asymptomatic presentation and slow growth over time.
Our purpose is to calculate the growth rate of orbital cavernous
hemangiomas in order to provide quantitative evidence in counseling
patients regarding the natural progression of these lesions.
Methods: A retrospective chart review from January 1983 to 2013,
searching by radiologic diagnoses consistent with orbital cavernous
hemangioma, identified a total of 57 lesions. Of these 57, 20 had at
least three interval CT and/or MRI scans of the orbit over a period of
at least two years follow-up. Serial imaging studies were reviewed by
a single neuroradiologist, and the size of each lesion was determined
by calculating volume from three-dimensional measurements. Rates
of change in size were computed by constructing best-fit lines for size
of the lesion versus time.
Results: The 20 patients included 13 (65%) females and 7 (35%)
males, with average age at presentation of 56.7 ± 14.9 years. The
average length of follow-up was 5.7 ± 3.3 years. The average number
of serial imaging scans was 5.9 ± 3.8. Of the 20 lesions, 10 (50%)
decreased in size and 10 (50%) increased in size over the course of
the study. Two lesions that increased in size were resected after 2.5
and 3 years. For the 10 lesions that decreased in size, the average
rate of regression was 0.34 ± 0.61 cubic mm/year (range 0.029-2.04),
equating to 2.89 years for the lesion to shrink one cubic mm. For the
eight lesions not requiring surgery that increased in size, the average
rate of growth was 0.25 ± 0.39 cubic mm/year (range 0.029-1.17),
equating to 4.00 years for the lesion to grow one cubic mm. In
total, for all 18 masses not requiring surgery that both increased and
decreased in size, the average of the absolute value of the rate of
change in size was 0.30 ± 0.51 cubic mm/year, equating to an average
3.30 years for the lesion to either grow or shrink one cubic mm. Of
note, one lesion requiring surgery at a size of 22.5 cubic mm grew at
a rate of 3.69 cubic mm/year over three years.
Conclusions: Orbital cavernous hemangiomas, when followed with
serial imaging over an extended period of time, tend to primarily
either increase or decrease in size at a steady slow rate. Quantifying
this rate of growth/regression can aid in confirming a diagnosis of
cavernous hemangioma and in reassuring patients as to the slowly
changing nature of these lesions.
Commercial Relationships: Liza M. Cohen, None; Anupam
Jayaram, None; Gary Lissner, None; Achilles Karagianis, None
Clinicopathologic Correlation of Caruncular Lesions
Sander R. Dubovy1, 2, Antonio J. Bermudez1, 2, Jordan Thompson1,
2 1
. Bascom Palmer Eye Institute, University of Miami, Miami, FL;
2
FLorida Lions Ocular Pathology Laboratory, Miami, FL.
Purpose: Lesions of the caruncle are relatively uncommon. Herein
we report the caruncular lesions seen at the Bascom Palmer Eye
Institute by characterizing the type of lesion, relative frequency and
clinical findings from a single institution.
Methods: The case files of the Florida Lions Ocular Pathology
Laboratory at the Bascom Palmer Eye Institute were reviewed from
1997 to October 2013 searching for lesions that were designated to
have been biopsied from the caruncle. The reports were reviewed and
the histopathologic findings were correlated to the clinical findings.
Results: A total of 198 lesions of the caruncle were found in the
files from January 1997 to October 2013. Thirty different diagnostic
entities were identified. The most common were nevi (n=61, 31%),
followed by non-specific inflammation (n=26,13%), papilloma (n=16,
8%), sebaceous hyperplasia (n=13, 6.6%), oncocytoma (n=10, 5%)
and sebaceous carcinoma (n=8, 4%).
Conclusions: This case series demonstrates that benign nevi are
the most common lesions identified in the caruncle. While more
commonly benign, malignant lesions of the caruncle account for up to
14% of caruncular lesions including sebaceous carcinoma, squamous
cell carcinoma, basal cell carcinoma, lymphoma, melanoma and
intraepithelial carcinoma. A wide variety of lesions may present in
the caruncle and the clinician should be aware of the differential
diagnosis in this anatomic location.
Commercial Relationships: Sander R. Dubovy, None; Antonio J.
Bermudez, None; Jordan Thompson, None
Support: Florida Lions Eye Bank
Immunohistochemical Analysis of Sebaceous Cell Carcinoma in
Comparison to Both Basal Cell Carcinoma and Squamous Cell
Carcinoma
Andre N. Ali-Ridha1, 2, Seymour Brownstein1, 2, Kailun Jiang1, 2,
Tatyana Milman3, Bruce Burns2, Paula Blanco2, James Farmer2.
1
Department of Ophthalmology, University of Ottawa, The Ottawa
Hospital, The Ottawa Hospital Research Institute, Ottawa, ON,
Canada; 2Department of Pathology and Laboratory Medicine,
University of Ottawa, The Ottawa Hospital, Ottawa, ON, Canada;
3
Departments of Ophthalmology and Pathology, New York Eye and
Ear Infirmary, New York, NY.
Purpose: Basal cell carcinoma is the most common malignancy of
the eyelid followed by sebaceous cell and squamous cell carcinoma.
The mortality rate of sebaceous cell carcinoma has been reported
as 9 to 30% and both it and squamous cell carcinoma can develop
metastatic disease. Clinically, sebaceous cell carcinoma frequently
mimics inflammatory conditions and other neoplasms of the eyelid,
including both squamous cell and basal cell carcinoma. Our study
compares the immunostaining profile of sebaceous cell carcinoma to
that of basal cell and squamous cell carcinoma.
Methods: Retrospective and prospective case series. Eight specimens
each of sebaceous cell, basal cell and squamous cell carcinoma of the
eyelid were obtained from the Ottawa Ocular Pathology Laboratory
from 2007 to 2013. We compared the immunohistochemical profile
of these specimens by staining each of them with EMA, BER-EP4,
adipophilin, androgen receptor (AR), P16, BCL-2, CK7, Ki67,
BRST1, BRST2, p53, and CK20. We compared the extent of staining
to that of normal surface and glandular epithelial tissue on each slide
as normal internal controls. The immunoreactivity data was then
analyzed using a 2-tailed Kruskal-Wallis test (p<0.05 is significant)
with a post-hoc analysis using Mann-Whitney tests.
Results: Our test results were statistically significant (p<0.05) for
positive staining of EMA, adipophilin, P16 and AR in sebaceous cell
carcinoma. This group also showed a higher percentage of positivity
for Ki67. The basal cell tumours exhibited positive staining for
CK7, BCL2, and BER-EP4, while squamous cell carcinoma showed
substantial positivity only for EMA. Adipophilin stained positive
in sebaceous cell carcinoma with intracytoplasmic lipid vesicles
in 94% of the cells in our series as compared to the nonspecific or
minimal staining of granules in basal and squamous carcinoma cells
respectively.
Conclusions: Our study helps clarify much of the controversy in the
literature concerning immunostains which overlap in reactivity for
sebaceous cell carcinoma. We have found that the most informative
panel of immunostains, from our original 12 stains, for differentiating
sebaceous cell carcinoma from other related tumors consists of P16,
adipophilin, EMA, AR, CK7 and Ki67. This diagnostically optimal
panel of immunostains may allow for earlier diagnosis and treatment
of sebaceous cell carcinoma of the eyelid.
Commercial Relationships: Andre N. Ali-Ridha, None; Seymour
Brownstein, None; Kailun Jiang, None; Tatyana Milman, None;
Bruce Burns, None; Paula Blanco, None; James Farmer, None
Sebaceous gland carcinoma of the ocular adnexa – variability in
histological and immunhistochemical appearance
Eva J. Becker1, 2, Martina C. Herwig1, 2, Frank G. Holz1, Hans-Peter
Fischer3, Karin U. Loeffler1, 2. 1Ophthalmology, University Eye
Hospital Bonn, Bonn, Germany; 2Ophthalmic Pathology, University
Eye Hospital Bonn, Bonn, Germany; 3Pathology, University Bonn,
Bonn, Germany.
Purpose: To evaluate the characteristics of sebaceous gland
carcinoma (SGC) of the ocular adnexae which is - due to a high
variability in clinical, histological and immunhistochemical
characteristics – challenging to diagnose.
Methods: Records of 6 patients (1 female, 5 male) with SGC
were reviewed, who underwent surgical excision and who were
histologically diagnosed SGCs. For comparison, specimens from
4 patients with basal cell carcinoma (BCC), 1 with squamous cell
carcinoma (SCC) and 2 with indeterminate lesions were examined.
Histological and immunhistochemical analysis included stains for
HE and PAS, cytokeratins (CKpan, Cam5.2), epithelial membrane
antigen (EMA), androgen receptor (AR441), and adipophilin.
Results: SGCs were located in the upper (n=2) or lower (n=4)
eyelid and were associated with various clinical signs including
chalazion-like lesions with pyogenic granuloma (n=1), papillomatous
conjunctival tumors (n=3), a hyperkeratotic exophytic neoplasm
(n=1) and an ulcerating crusted lesion resembling chronic blepharitis
(n=1). The treatment was tumor resection, followed (if necessary)
by adjuvant therapy with topical Mitomycin C (n=2). Histologic
characteristics included basophilic pleomorphic cells with vacuolated
cytoplasm, prominent nucleoli, mitotic figures and in some cases
pagetoid spread (n=2). CKpan, EMA and Cam5.2 showed a strong
positive immunoreactivity in all specimens (SGC, BCC, SCC).
AR441 positivity was noted with variable intensities in almost all
lesions and in particular in pagetoid spread in contrast to non-tumor
cells. Adipophilin showed an annular staining of lipid granules in
immature sebaceous cells and was mainly found in SGC.
Conclusions: SGCs display a variety of clinical signs and may mimic
many other lesions. Tumor resection, followed by histological and
immunhistochemical analysis, leads to the diagnosis and initiation
of the proper treatment regimen. Herein, immunohistochemistry
showed an unequivocal profile in SGC and did not allow for an
exact differentiation from BCC and SCC by immunohistochemical
means only. An extended evaluation of HE stains remains essential.
However, immunohistochemistry can make relevant contributions to
the diagnosis of SGC, especially in cases of inconclusive histology,
by positive staining for adipophilin in immature sebaceous cells or by
AR441 labeling in cases of pagetoid spread.
Commercial Relationships: Eva J. Becker, None; Martina C.
Herwig, None; Frank G. Holz, None; Hans-Peter Fischer, None;
Karin U. Loeffler, None
Molecular Profiling of Ocular Surface Squamous Neoplasia
Identifies Multiple DNA Copy Number Alterations Including
Recurring 8p11.22 Amplicons
Saeed AlWadani1, 3, Laura Asnaghi1, Hind Alkatan2, Hilal Al
-Hussain2, Deepak Edward1, 2, Charles Eberhart1. 1Ophthalmic
Pathology, Johns Hopkins Hospital, Baltimore, MD; 2King Khaled
Eye Specialist Hospital, Riyadh, Saudi Arabia; 3Ophthalmology, King
Saud University, Riyadh, Saudi Arabia.
Purpose: To uncover novel diagnostic biomarkers and molecular
pathways, which can be targeted using new therapies in Conjunctival
squamous cell carcinoma (cSCC). Very little is known about the
molecular pathways, which drive the formation and growth of ocular
surface squamous neoplasia.
Methods: We analyzed DNA extracted from 14 snap frozen cSCC
tumor specimens using Agilent 180K high density oligonucleotide
array-based Comparative Genomic Hybridization (aCGH), with
12 samples giving high quality hybridizations. 11 cases with
DNA remaining were used to confirm chromosomal alterations by
nanostring analysis.
Results: Of these 12 tumors, the number of clear regions of DNA
loss ranged from 1 to 24 per tumor, while gains ranged from 2 to 14
per tumor. Two of the 12 tumors were recurrent, and these had the
highest number of copy number gains/amplifications 9 and 14, and
8 and 11 losses among the cohort. These recurring aberrations were
observed in chromosome 6, where the region 6p22.1-p21.32 was lost
in 33%, in chromosome 14, where the locus 14q13.2 was lost in 42%,
and in chromosome 22, where 5 samples showed DNA loss and one
DNA gain at 22q11.22. However, the most frequent alteration was
observed in chromosome 8, where the locus 8p11.22 was amplified
in 75% and lost in 25%. This region contains a group of genes coding
for “a disintegrin and metalloprotease” (ADAM) proteins, known
to be involved in the activation of oncogenic receptors and tumor
formation. We observed the most profound DNA alterations in the
region of 8p11.22 which contains part or all of the ADAM1B, 3A,
and 5p genes. We are now investigating mRNA expression at the
8p11.22 loci with PCR.
Conclusions: Recurrent cSCC tumors were found to have significant
chromosomal alteration in region 8p11.22, suggesting that increased
numbers of DNA alterations may be associated with more aggressive
clinical behavior and/or tumor progression. In contrast, case with
fewer gains and losses was somewhat distinct microscopically, and
noted to be relatively undifferentiated with adnexal features. The
ADAM genes discovered in this study could be related to ocular
tumors and previously reported ADAM9 alterations associated with
oral mucosa neoplasia, suggest that ADAM family members may be
involved in the pathogenesis of several types of mucosal squamous
neoplasia, and could be potential therapeutic target.
Commercial Relationships: Saeed AlWadani, King Khaled Eye
Specialist Hospital (F); Laura Asnaghi, King Khaled Eye Specialist
Hospital (F); Hind Alkatan, King Khaled Eye Specialist Hospital
(F); Hilal Al -Hussain, King Khaled Eye Specialist Hospital (F);
Deepak Edward, King Khaled Eye Specialist Hospital (F); Charles
Eberhart, King Khaled Eye Specialist Hospital (F)
Support: King Khaled Eye Specialist Hospital Grant, Riyadh, Saudi
Arabia
Establishment and characterization of squamous cell carcinoma
cells from the human bulbar conjunctiva
Bettina Mueller, Henning Thomasen, Klaus-Peter Steuhl, Daniel
Meller. University Duisburg Essen, Essen, Germany.
Purpose: Until now only limited information about cultivated tumor
initiating cells out of squamous cell carcinoma (SCC) from the
human bulbar conjunctiva are available. Therefore, the establishment
of a model cell line would be a useful tool for further studies.
In particular, the phenotypic and molecular characterization in
comparison to other SCC cells is of high interest. This would enable
the development of new treatment options for clinical application.
Methods: Epithelial cells were isolated from a bulbar conjunctival
SCC obtained from a 74 year old male and were named PeCaUkHb-01. For receiving a pure cell culture, epithelial cells were
harvested by stepwise trypsinisation. Furthermore, mycoplasma
contamination was tested. Cell doubling time and number of
passages were determined. STR (short tandem repeats) and karyotype
analyses were performed to verify the origin of the cells and to
analyze chromosomal heterogeneity. For further characterization
semiquantitative real-time PCR and immunofluorescence staining
were carried out to detect tumor and epithelial progenitor cell
markers. Spheroid and colony forming ability were performed.
Results: The morphology of the new cell line resembled other
epithelial SCC cell lines, furthermore STR experiments confirmed the
origin of the cells from the donor. The cells were free of mycoplasma.
They grew above passage number forty and could be frozen and
recultured. Karyotype analyses revealed a heterogeneous composition
of the cell culture and the karyogram itself showed aberrations and
changes in the chromosome numbers. PeCa-UkHb-01 cells were
able to build colonies and spheroids. Semiquantitative real-time PCR
results displayed an upregulation of SOX2 in comparison with A-431
SCC cells. Furthermore the cells showed expression of the putitative
ocular surface stem cell markers ABCG2, c Myc, Lin28, Oct4, P63,
which indicates stem-cell like characteristics.
Conclusions: PeCa-UkHb-01 cells fulfill the criteria of a cell line
which might display characteristics of cancer stem cells. They
showed similarities to established cell lines from SCCs and other
tumors. Further characterizations are needed to confirm the hint of
stem cell characteristics. This cell line will be a huge advantage for
further basic research and development of therapeutic applications
due to the fact that it is the first of its kind.
Commercial Relationships: Bettina Mueller, None; Henning
Thomasen, None; Klaus-Peter Steuhl, None; Daniel Meller, None
Intraoperative High Dose Rate 32P Brachytherapy for Diffuse
Conjunctival Neoplasms
Brian Marr1, David H. Abramson1, Gil’ad Cohen3, Christopher
Barker2. 1Surgery, Memorial Sloan Kettering, New York, NY;
2
radiation oncology, Memorial Sloan Kettering, New York, NY;
3
medical physics, Memorial Sloan Kettering, New York, NY.
Purpose: : Malignancies occurring near the eye are often effectively
managed with radiation therapy. However, the sensitivity and small
size of the eye often limit options with teletherapy. For this reason,
we have employed a novel brachytherapy system for ophthalmic
malignancies in recent years. This study was conducted to assess the
outcome of treatment with this modality.
Methods: : With permission of the IRB, medical records of patients
treated with 32P brachytherapy for ophthalmic malignancies were
reviewed. Demographic, comorbidity, cancer and treatment related
factors were recorded. Visual acuity, intraocular pressure, grade >2
adverse events (defined and graded per CTCAE) and tumor control
were noted.
Results: : 5 patients (2 women, 3 men) underwent 6 courses of
32P ophthalmic brachytherapy. Median age was 63 (range 51-80).
Median ACE-27 comorbidity score was 1 (range 1-2). Four of 5
patients (80%) had recurrent cancer, which had failed a median of
1 prior therapy (range 1-3), including surgical excision, and topical
chemotherapy and immunotherapy. No patients had evidence of
regional or distant metastases at presentation for brachytherapy.
Doses were prescribed to 1 mm from the surface of the applicator,
and ranged from 5-17 Gy (median 15 Gy), at dose rates of 0.3250.770 Gy/minute (median 0.450), from 32P sources with activities
of 1.35-6.00 mCi (median 5.60), using custom designed applicators
1.0-6.7 cm2 (median 4.5). With a median follow-up of 17 months,
2 patients developed clinical evidence of local recurrence 11 and 4
months after brachytherapy; biopsy confirmed recurrence in only one
patient. One patient required enucleation for extensive local tumor
recurrence. No patient developed regional or distant recurrence, or
has died since brachytherapy. One grade 4 corneal ulcer occurred
1 month after brachytherapy, and one grade 3 cataract occurred 30
months after brachytherapy. Visual acuity >20/200 was preserved in 4
of 5 patients. Glaucoma was not noted in any
Conclusions: : Intraoperative high dose rate 32P brachytherapy
is feasible for ophthalmic malignancies. We have not noted major
complications with doses of ≤15 Gy. Further study with a larger
group of patients will be necessary to validate these preliminary
findings.
Commercial Relationships: Brian Marr, None; David H.
Abramson, None; Gil’ad Cohen, None; Christopher Barker, None
A heterotopic model for tumor-associated (lymph)angiogenesis
in the murine cornea - feasibility of intrastromal tumor cell
injections
Konrad R. Koch, Nasrin Refaian, Deniz Hos, Mario Matthaei, Felix
Bock, Simona L. Schlereth, Martina Becker, Claus Cursiefen, Ludwig
M. Heindl. Department of Ophthalmology, University of Cologne,
Cologne, Germany.
Purpose: The avascular cornea is predestined to study neovascular
responses. First experiences with this model date back to 1972, when
Gimbrone et al heterotopically implanted tumor fragments in rabbit
corneal pockets. Different assays in rabbit, rat, and mouse corneas
have since been published including placement of (anti-)angiogenic
growth factor releasing pellets or proangiogenic corneal sutures.
Using murine corneas is surgically more intricate but advantageous
due to the well-defined genetic background and availability of
genetically modified animals. In mice, so far tumor-associated
angiogenesis has been studied by inserting pre-grown tumor
fragments into corneal pockets. Here we describe an alternative
approach, where a suspension of cultured tumor cells is directly
injected into the corneal stroma.
Methods: One ml of B16F10 melanoma cells suspended in PBS
(100.000 cells/ml) and pre-stained with FITC+ CellTracker Green was
injected into the paracentral corneal stroma of C57Bl/6 mice (n=10,
OD) using a Hamilton 33G microsyringe. After 7 days, eyes were
enucleated and fixated either in aceton for wholemount preparation,
or in formalin for paraffin-embedded sections. LYVE1+ stained
wholemounts were evaluated for the relative corneal area covered by
FITC+ tumor cells (RAT, in relation to the entire corneal area) and
the smallest distance between the tumor cell cluster and the LYVE1+
lymphatic limbus (DTL). Paraffin-embedded sections were stained
for HMB45 or Ki67. The rate of Ki67+ tumor cells was calculated.
Results: Macroscopic evaluation in 9 eyes revealed a smooth
corneal surface with localized pigmented stromal areas. One
mouse was euthanized on post-op day 2 due to corneal perforation/
endophthalmitis. All wholemounts (n=4) showed a paracentral area
of FITC+ tumor cells. RAT was 5,05% (±2.27, mean ±SD). DTL was
1.32mm (±0.34). In paraffin-embedded sections (n=5) pigmented
HMB45+ tumor cells were observed within the corneal stroma. No
tumor cells were found in the adjacent anterior chamber. Thirty-seven
percent of intrastromal tumor cells were Ki67+.
Conclusions: Intrastromal tumor cell injection into the murine cornea
appears feasible, allowing for malignant cell survival and ongoing
proliferation. Further studies are needed to assess tumor growth and
the potential manifestation of associated (lymph)angiogenesis over a
longer time period.
Commercial Relationships: Konrad R. Koch, None; Nasrin
Refaian, None; Deniz Hos, None; Mario Matthaei, None; Felix
Bock, None; Simona L. Schlereth, None; Martina Becker, None;
Claus Cursiefen, None; Ludwig M. Heindl, None
New mouse model for conjunctival melanoma
Simona L. Schlereth1, Sandra Iden2, Melina Mescher2, Konrad
R. Koch1, Claus Cursiefen1, Ludwig M. Heindl1. 1Department of
Ophthalmology, University of Cologne, Cologne, Germany; 2CECAD
Cologne, University of Cologne, Cologne, Germany.
Purpose: Conjunctival melanoma is a rare but potentially aggressive
ocular cancer, affecting about 0.8 patients per million with a mortality
rate of 7-24% in five years. By now, there are no published mouse
models to study this malignancy. In this study we investigate a new
mouse model for conjunctival melanoma.
Methods: Female 6-8 weeks old black six mice were
subconjunctivally injected with different doses of 384-HGF+Cdk4
tumor cells (3 groups - first group: 1x106 cells, second: 5x105, third:
1x105 cells). These tumor cells were isolated from dermal melanoma
of HGF-Cdk4 mice. We tested different doses and documented
findings by a clinical score (including redness, chemosis and tearing
in a grade 0 (not detectable) to 3 (severe) and exophthalmos (yes/
no)). After development of macroscopically detectable conjunctival
tumor, mice were sacrificed and immunohistochemical staining was
performed in liver, lymph nodes, spleen and bulbus including the
conjunctiva for hematoxylin&eosin and different tumor markers
(including Ki67, TRP1). Mice were inspected for macroscopically
visible tumors on the skin and the lung.
Results: Conjunctival melanoma was inducible in all mice receiving
1x106 (group1) or 5x105 HGF+cells (group 2) after only 3 days,
by darkly pigmented conjunctival swelling. Experiments had to be
stopped at day nine due to unilateral exophthalmos in 83% of the
animals. The exophthalmos was induced by complete infiltration
of tumor mass into the retroorbital space. Mice that received 1x105
HGF+cells (group 3) did not develop any clinical alterations of the
eye in an observation period of 40 days.
Immunohistochemistry showed intense Ki67 and TRP1 positivity
within the conjunctival melanoma, even higher than in isolated in
vitro cells. Ki67 expression was elevated in the draining lymphnode.
Conclusions: Injection of HGF+ cutaneous melanoma cells into the
conjunctiva imitates a solid tumor growth within the conjunctiva and
may be used as a new mouse model for a better understanding and
treatment of conjunctival melanoma.
Commercial Relationships: Simona L. Schlereth, None; Sandra
Iden, None; Melina Mescher, None; Konrad R. Koch, None; Claus
Cursiefen, None; Ludwig M. Heindl, None
Support: Cologne GEROK program to SLS; DFG grant to CC: Cu
47/6-1 and LMH: HE 6743/2-1, CIO Cologne-Bonn Special Program
“Ophthalmic Oncology” to CC and LMH
A new conjunctival melanoma model of primary and metastatic
tumor growth
Jinfeng Cao1, Nadine de Waard1, Aat A. Mulder1, Bruce R. Ksander2,
Martine Jager1. 1Ophthalmolgy, Leiden University Medical Center,
Warmond, Netherlands; 2Ophthalmology, Schepens Eye Research
Institute / Mass Eye & Ear, Harvard,, Boston, MA.
Purpose: There is currently no conjunctival melanoma model
available, limiting the ability to study the mechanisms of tumor
progression which would allow the development of more effective
chemotherapy and/or novel therapies, such as immunotherapy.
Methods: In order to develop a xenogeneic model of human
conjunctival melanoma in mice, cell lines derived from human
conjunctival melanoma (CRMM-1, CRMM-2, CM2005.1) were
injected orthotopically (4 x 106 cells/5 μl) into the subconjunctival
space of immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)
mice. Sequential passage of in vivo grown conjunctival melanomas
was achieved by harvesting primary tumors, digestion of tumor
tissue, and injection of tumor cells into the subconjunctival space
of a new naive NSG mouse. Primary tumor growth was assessed by
slit lamp examination, H&E, and immunohistochemical staining of
conjunctival melanoma markers. Metastatic tumors derived from the
cervical lymph nodes were also analyzed.
Results: Xenogeneic human conjunctival melanomas grew
progressively within the subconjunctival space of immunodeficient
NSG mice. Primary tumors formed within two weeks in all mice
(total of 101 mice; CRMM-1 n =33, CRMM-2 n = 34, CM2005.1 n =
35). All three cell lines expressed HMB-45. CRMM-1 and CM2005.1
expressed Melan-A, while S100 was only expressed at high levels in
CM2005.1. On the primary tumors, the melanocyte markers..... were
expressed. Histological analysis demonstrated all three cell lines had
comparable growth and morphological characteristics: tumors grew
along the subconjunctival space and sclera, displayed an epithelioid
cell morphology with large nuclei, prominent nucleoli, and large
cytoplasmic compartments with vacuoles. Surprisingly, initial
primary tumors failed to metastasize in any mice, even after tumors
attained the size requiring euthanasia. However, serial in vivo passage
of primary tumors resulted in consistent metastatic tumor spread to
the cervical lymph nodes draining the tumor-containing eye.
Conclusions: Preventing metastatic spread of conjunctival melanoma
is critical for a positive prognosis. Human xenogeneic conjunctival
melanomas growing in immunodeficient NSG mice display tumor
growth and metastatic progression that is similar to the human
disease, allowing for the analysis of the protective effectiveness of
current and novel therapies.
Commercial Relationships: Jinfeng Cao, None; Nadine de Waard,
None; Aat A. Mulder, None; Bruce R. Ksander, None; Martine
Jager, None
Support: Chinese Scholarship Council
Role of the Renin-Angiotensin System in Human Conjunctival
Lymphoma
Erdal Tan Ishizuka, Atsuhiro Kanda, Satoru Kase, Satoshi Kinoshita,
Saori Takashina, Yoko Dong, Kousuke Noda, Susumu Ishida.
Laboratory of Ocular Cell Biology and Visual Science, Department of
Ophthalmology, Hokkaido University Graduate School of Medicine,
Sapporo, Japan.
Purpose: The renin-angiotensin system (RAS), a known important
controller of systemic blood pressure (circulatory RAS), plays
distinct roles in inflammation and pathological vascular conditions
in various organs including the eye (tissue RAS). Conjunctival
lymphoma is one of the common malignancies found in the ocular
adnexa. In this study, we investigated the role of RAS in extranodal
marginal zone B-cell lymphoma (EMZL) of the conjunctiva.
Methods: Gene expressions of RAS components [prorenin, (pro)
renin receptor ((P)RR), angiotensinogen, angiotensin converting
enzyme, angiotensin II type 1 receptor (AT1R) and AT2R] in EMZL
tissues surgically excised from patients were analyzed using reverse
transcription PCR. Localization of AT1R and (P)RR in the EMZL
tissues were studied by immunofluorescent analyses. Expression
levels of pro-angiogenic and inflammatory genes in human
B-lymphocyte cell lines treated with angiotensin II (Ang II) were
analyzed by real-time PCR.
Results: Gene expressions of RAS components were detected in
EMZL and several human B-lymphocyte cell lines. (P)RR and AT1R
were detected in both vascular endothelial cells and the cytoplasm
of atypical lymphoid cells, co-localized with CD31 (endothelial
tissue marker) and CD20 (B lymphocyte marker), respectively.
Immunofluorescent analyses showed co-localization of prorenin
and angiotensinogen in (P)RR-positive and AT1R-positive cells,
respectively.
Real time-PCR analysis revealed that Ang II significantly upregulated
several mRNA expression levels [e.g. basigin and vascular
endothelial growth factor A], which were suppressed by pre-treatment
of valsartan (AT1R blocker), in human B lymphocyte cell line.
Conclusions: Our data using clinical samples together with in
vitro results provide evidence that tissue RAS is associated with
pathological events in human conjunctival lymphoma.
Commercial Relationships: Erdal Tan Ishizuka, None; Atsuhiro
Kanda, None; Satoru Kase, None; Satoshi Kinoshita, None;
Saori Takashina, None; Yoko Dong, None; Kousuke Noda, None;
Susumu Ishida, None
Support: Creation of Innovation Centers for Advanced
Interdisciplinary Research Areas Program, Takeda Science
Foundation, Mishima Saiichi Memorial Ophthalmic Research Japan
Foundation, and JSPS KAKENHI-24791823
Prognosis of lymphomas developed in the ocular adnexa: A
single-center study
Yoshiko Matsumoto1, Atsushi Azumi2, Azusa Akashi1, Mari Sakamoto1,
Takayuki Nagai1, Makoto Nakamura1. 1Devision of Ophthalmology,
Kobe University Graduate School of Medicine, Kobe, Japan;
2
Ophthalmology, Kobe Kaisei hospital, Kobe, Japan.
Purpose: To demonstrate a prognostic feature of patients suffering
from lymphomas in the ocular adnexa.
Methods: A single-center retrospective study was conducted.
Patients’ information was collected from the medical records of
patients that underwent surgical resection of the lesions developed
in the ocular adnexa, the conjunctiva, lid and orbit, and were
diagnosed with lymphomas by pathological examination from
April 1992 to June 2013. In all cases, Southern blot hybridization
for immunoglobulin-gene rearrangement was performed to exclude
non-lymphoma diseases such as IgG4-related ophthalmic disease.
Flow cytometry analysis and G-banding were also done to make
precise subtype diagnoses in accordance with WHO classification.
For staging classification, systemic examinations were performed to
evaluate metastasis. Kaplan-Meier method was used for survival rate
analyses.
Results: One hundred fifteen cases were diagnosed as lymphomas
developed in the ocular adnexa. The median of patient age was 61
(range 10-87). The most frequent subtype was extranodal marginal
zone B cell lymphoma of mucosa-associated lymphoid tissue type
(MALT lymphoma), seen in 98 cases (85.2%). As for other subtypes
of lymphoma, diffuse large B-cell lymphoma was seen in 6 cases
(5.2%), follicular lymphoma in 5 (4.3%), and mantle cell lymphoma
in 4 (3.5%). Affected sites in the ocular adnexa were orbit; 61 cases
(53.0%), conjunctiva; 52 (45.2%), lid; 2 (1.7%). According to AnnArbor staging system, 75 cases (65.2%) were in stage I, 7 (6.1%) in
stage II, 2 (1.7%) in stage III, and 15 (13.0%) in stage IV.
In most of the cases, the disease was treated by either radiotherapy
or chemotherapy, or both, but some cases were just observed after
diagnosis. Systemic carcinoma development was observed in 14
cases (12.2%).
The 10-year-overall-survival rate of all lymphoma cases was 94.0%
and that of MALT lymphoma cases 94.8%. The cause-specific
survival rates were 98.6% in all cases and 100% in MALT lymphoma
cases. As for 10-year-non-recurrent rates in the cases of stage I or
II, the rates were 64.6% in all cases and 67.6% in MALT lymphoma
cases.
Conclusions: Most common subtype of lymphoma in the ocular
adnexa is MALT lymphoma, and the 10-year-cause-specific survival
rate was 100%. However, the non-recurrent rate was less than
70% and the development of cancer is not rare. The ocular adnexal
lymphomas require long-term systemic follow-up.
Commercial Relationships: Yoshiko Matsumoto, None; Atsushi
Azumi, None; Azusa Akashi, None; Mari Sakamoto, None;
Takayuki Nagai, None; Makoto Nakamura, None
Prognostication in Ocular Adnexal Lymphoma – Feasibility of
the Latest Tumour, Tode, Metastasis Staging System
Peter Rasmussen2, 1, Sarah E. Coupland3, Paul T. Finger4, Enrique
O. Graue4, Hans E. Grossniklaus5, Penny McKelvie7, Kaustubh
Mulay6, Jan Ulrik Prause2, Elisabeth Ralfkiaer8, Steffen Heegaard2,
1 1
. Department of Ophthalmology, Glostrup Hospital, University
of Copenhagen, Glostrup, Denmark; 2Eye Pathology Institute,
University of Copenhagen, Copenhagen, Denmark; 3Department
of Cellular and Molecular Pathology, University of Liverpool,
Liverpool, Liverpool, United Kingdom; 4Department of Ocular
Oncology, The New York Eye Cancer Center, New York City, NY;
5
Emory Eye Center, Section of Ocular Oncology, Emery University,
Atlanta, GA; 6Prasat Eye Institute, Kallam Anji Reddy Campus,
Hyderabad, India; 7The Orbital Plastic and Lacrimal Clinic, The
Royal Victorian Eye and Ear Hospital, Melbourne, VIC, Australia;
8
Department of Pathology, Rigshospitalet, University of Copenhagen,
Copenhagen, Denmark.
Purpose: To evaluate the prognostic utility of the tumour, node,
metastasis (TNM)-based staging system for primary ocular adnexal
lymphomas (i.e. lymphomas arising in the orbit, the eyelids, the
conjunctiva, the lacrimal gland and the lacrimal sac) proposed by the
American Joint Committee on Cancer.
Methods: Retrospective, multicenter study of patients with primary
ocular adnexal lymphoma collected from six eye-cancer centers from
January 1st 1980 through December 31st 2010.
Results: A total of 578 eligible patients were included in the study,
293 (51%) were females. The median age was 61 years (range 3 –
97 years). The most frequent lymphoma subtypes were: extranodal
marginal zone lymphoma (397/578, 69%), follicular lymphoma
(63/578, 11%), and diffuse large B-cell lymphoma (52/578, 9%).
The TNM-stages were: T1N0M0/conjunctiva (23%), T2N0M0/
orbit including the lacrimal gland (67%), T3N0M0/eyelid (6%) and
T4N0M0/extension beyond the orbit (2%).
For the entire study group the 5-year survival was 73%. The group
of patients with diffuse large B-cell lymphoma had a significantly
poorer survival compared with extranodal marginal zone lymphoma
and follicular lymphoma (5-year survival, 31% vs. 79% vs. 81%,
respectively) (log-rank p < 0.01). The survival was not associated
with the extension or site-specific location of the lesions as depicted
by the TNM-based stages in any of the lymphoma subtypes.
Conclusions: Lymphoma arising in the ocular adnexal region
is mainly prevalent in elderly patients. The survival is primarily
determined by histopathology rather tumour size or site-specific
location.
Commercial Relationships: Peter Rasmussen, None; Sarah E.
Coupland, None; Paul T. Finger, None; Enrique O. Graue, None;
Hans E. Grossniklaus, None; Penny McKelvie, None; Kaustubh
Mulay, None; Jan Ulrik Prause, None; Elisabeth Ralfkiaer, None;
Steffen Heegaard, None
Support: Fight for Sight Denmark, the Danish Cancer Society,
the Danish Eye Research Foundation, Synoptik Foundation, the
Danish Foundation for Cancer Research, Engineer Lars Andersens
Foundation, the A.P. Møller Foundation for the Advancement of
Medical Science and the Merchant Kjaer and Wife Kjaer, born la
Cour-Holmens Foundation, and The Eye Cancer Foundation, Inc.
IgG4 Immunostaining in Patients with Orbital Sarcoidosis: A
Pilot Study
Kateki Vinod1, Jordan Spindle2, Tatyana Milman1, Roman Shinder2.
1
Ophthalmology, New York Eye and Ear Infirmary, New York, NY;
2
Ophthalmology, SUNY Downstate Medical Center, New York, NY.
Purpose: IgG4-related disease (IgG4-RD) is an emerging entity
characterized histopathologically by a lymphoplasmacytic infiltrate
with increased numbers of IgG4-positive plasma cells (IgG4+ PC).
In the last decade, IgG4-RD has been described in multiple organs,
including the lung and orbit. Several case reports have appeared in
recent pulmonary literature demonstrating evidence of concurrent
sarcoidosis and IgG4-RD. The purpose of this study is to determine
the strength of association between IgG4-RD and clinically
documented and orbital biopsy-confirmed sarcoidosis.
Methods: The databases of orbital biopsies performed at the
New York Eye and Ear Infirmary (NYEEI) and the SUNY
Downstate Medical Center between 1990 and 2013 were searched
for “sarcoidosis” and/or “granulomatous inflammation” and/or
“granuloma.” Patients with orbital biopsies suggestive of sarcoidosis
(i.e. non-caseating granulomas), who also had clinical, serologic,
and/or radiographic evidence of systemic sarcoidosis, were included.
Paraffin-embedded tissue blocks were sectioned and immunostained
with IgG4 and IgG antibodies. Histopathology and the degree of
IgG4+ PC infiltration were evaluated by 1 ophthalmic pathologist.
This study is NYEEI Institutional Review Board approved.
Results: Nine patients with orbital biopsies consistent with
sarcoidosis and clinical diagnosis of sarcoidosis were identified.
Histopathologic findings included discrete non-necrotizing
granulomas in 9/9 (100%) patients, mild focal non-storiform
fibrosis in 5/9 (56%), moderate focal non-storiform fibrosis in 4/9
(44%), obliterative phlebitis in 1/9 (11%), and mild to moderate
lymphoplasmacytic infiltrate in 8/9 (89%). One patient had moderate
to severe lymphoplasmacytic infiltrate with elevated IgG4+ PC count
>50 per high power field (HPF), IgG4:IgG ratio >90%, elevated
total serum IgG and IgG1, 2, and 3 subsets, but normal IgG4. The
remaining patients had an average IgG4+ PC count of 5.3 per HPF
(range 0-21), and IgG4:IgG ratio of 6.6% (range 0-22%).
Conclusions: These results confirm prior data indicating an
infrequent association of sarcoidosis with IgG4-RD. These
observations are limited by the small sample size, lack of reliable
diagnostic criteria for orbital IgG4-RD, and lack of consensus on
IgG+ and IgG4+ PC quantification algorithms. Further research is
needed to elucidate whether elevated tissue and serum levels of IgG4
in patients with sarcoidosis have clinical significance.
Commercial Relationships: Kateki Vinod, None; Jordan Spindle,
None; Tatyana Milman, None; Roman Shinder, None
IgG4-related ophthalmic disease: A clinicopathological study of
40 cases
Shunichiro Ueda1, 2, Hiroshi Goto1, Yoshihiko Usui1, Keisuke Kimura1,
Kazuhiko Umazume1, Jun Matsubayashi2, Toshitaka Nagao2.
1
Ophthalmology, Tokyo Medical University, Tokyo, Japan; 2Anatomic
Pathology, Tokyo Medical University, Tokyo, Japan.
Purpose: The aim of this study is to clarify the clinical and
histopathological features of IgG4-related ophthalmic disease (IgG4ROD).
Methods: We reviewed the medical records of patients with IgG4ROD diagnosed at Tokyo Medical University Hospital between
2002 and 2013. The age and sex of the patients as well as the IgG4
serum levels and the findings of imaging studies, histopathological
assessments, flow cytometric studies, and immunoglobulin heavy
chain rearrangement assessments were investigated. IgG4-ROD was
diagnosed based on (1) the presence of a swelling, enlargement, or
mass in any ocular adnexal tissue detected by imaging studies and (2)
IgG4 serum levels > 135 mg/dl or (3) >40% of IgG-positive plasma
cells being IgG4-positive and >50 cells/field when a biopsy sample
was observed using a high-powered microscope.
Results: Based on the above criteria, 40 patients were diagnosed
with IgG4-ROD, of which 20 (50%) were women. The mean age
of the patients was 58.4 years (age range, 27–81 years). Imaging
studies showed infiltrative lesions in both (n = 30) or one (n = 3)
lacrimal gland, eyelid and extraocular muscles on both sides (n =
2), extraocular muscles on both sides (n = 2), the caruncles on both
sides (n = 1), and as a unilateral orbital mass (n = 2). The average
IgG4 serum levels were 613 mg/dL (range, 57–1,920 mg/dL).
Twenty-seven patients underwent biopsy. Although histological
examination revealed lymphoid follicles and mild fibrosis in many
cases, strong fibrosis and unclear lymphoid follicles were also
observed in a few cases. Immunohistochemically, average 61.5%
of the IgG-positive plasma cells were IgG4-positive. No evidence
of light chain restriction was observed in the flow cytometric study.
Immunoglobulin heavy chain rearrangement was not detected but one
patient.
Conclusions: The findings of the present study suggest that IgG4ROD occurs in various regions of the ocular adnexa.
Commercial Relationships: Shunichiro Ueda, None; Hiroshi
Goto, None; Yoshihiko Usui, None; Keisuke Kimura, None;
Kazuhiko Umazume, None; Jun Matsubayashi, None; Toshitaka
Nagao, None
Primary Epithelial Malignancies of the Lacrimal Gland: Trends
in Survival
Maxwell Elia1, Charles Tuggle2, Javier Servat1, Flora Levin1.
1
Ophthalmology, Yale University School of Medicine, New Haven,
CT; 2Plastic and Reconstructive Surgery, Yale University School of
Medicine, New Haven, CT.
Purpose: To determine the trends in incidence, treatment, and
survival of primary epithelial malignancies of the lacrimal gland
in the United States from 1988 to 2010 using a systemic review of
the National Cancer Institute Surveillance, Epidemiology and End
Results (SEER) database.
Methods: One hundred and thirty-two cases of primary epithelial
malignancy of the lacrimal gland were identified in the Surveillance,
Epidemiology, and End Results (SEER) program database in the
United States from 1988 to 2010. Survival rates were calculated by
the Kaplan-Meier method and significance was determined using
chi-squared testing.
Results: There were 132 cases of primary epithelial lacrimal gland
tumors with histopathologic confirmation in the SEER database. The
most common tumor types were adenoid cystic carcinoma (51.5%),
mucoepidermoid carcinoma (17.4%), adenocarcinoma in situ (15.1%
). The majority of tumors (61%) presented as locally invasive disease.
The remainder, were confined to a tumor capsule (35%) or metastatic
at presentation (4%). Most tumors were 2-4 cm in size at diagnosis
(68%), with 19% less than 2cm and 13% greater than 4cm (13%).
Surgery was the treatment of choice in 90% of patients, with 58%
receiving radiation therapy (RT). There was a statistically significant
improvement in survival among patients undergoing surgery versus
those without surgical treatment (8.24 years vs. 1.0 year; p< 0.0001).
Those undergoing radiation therapy fared poorer (2.25 years vs. 8.94
years; p = 0.02). Patients with advanced disease were significantly
more likely to be treated with radiation therapy alone (70% regionally
invasive or metastatic vs. 45% locally invasive; p<0.01). Patients
with a history of prior malignancy had worse survival (5.3 years vs.
9.6 year; p = 0.014). There was no significant gender, age, or race
predilection. There has been no significant improvement in survival
between the 1988-1997 group and the 2004-2010 group (9.6 years vs.
8.36 years, p=0.77).
Conclusions: Surgery confers a statistically significant improvement
in survival among patients diagnosed with primary epithelial tumors
of the lacrimal gland and should remain an important tool in disease
management. The SEER data demonstrates that there has been no
significant improvement in survival among patients diagnosed with
primary epithelial malignancies of the lacrimal gland during the
period of data collection.
Commercial Relationships: Maxwell Elia, None; Charles Tuggle,
None; Javier Servat, None; Flora Levin, None
Selective Uptake of Vital Stains in Orbital and Periorbital Tissues
Nicole Nikolic1, 2, James Qiao1, Ping Bu1, 2, David K. Yoo1, 2.
1
Ophthalmology, Loyola University Stritch School of Medicine,
Chicago, IL; 2Ophthalmology, Hines Veterans Affairs Hospital,
Hines, IL.
Purpose: Fluorescein-guided resection of tumors is a relatively
new modality being used in neurosurgery and urology, with recent
literature describing the ability to obtain clearer margins. However,
current knowledge on direct staining characteristics in various tissue
types is limited at best. Herein, we investigate the staining properties
of vital dyes to advance our knowledge for potential use during in
vivo resection of tumors.
Methods: 4 vital dyes were prepared into aqueous solutions of
2 concentrations: either 1 mg/ml and 0.5 mg/ml concentrations
(fluorescein, lissamine green, and rose bengal) or 0.1% and 0.06%
(trypan blue.) Sprague-Dawley rats were used to harvest periorbital
tissues of interest: muscle, adipose, optic nerve, brain and meninges.
Tissue samples were stained with the dyes for 2 minutes then washed
with 3 saline baths (3 x 5 mins) to determine the amount of residual
staining. Color photographs were taken prior to staining, after
staining, and after saline washes.
Results: Muscle, adipose, nerve and meninges appeared to stain
relatively strongly with each dye, with visibly significant retention
of stain after saline washing. Brain had variable but often minimal
preliminary staining with the dyes; however, it appeared to have
little to no retention of stain. All of the vital dyes appeared to stain
meninges quite strongly, with little appreciable loss of dye. Of the
4 vital stains, lissamine green was the poorest at both initial and
retained staining. Rose bengal appeared to be the most resilient stain
of the 4, with minimal dissipation of dye after washing of the tissues.
Conclusions: With respect to vital dye utility for in vivo tumor
resection, the most promising finding of our investigation is the
visible disparity in staining between brain and meningeal tissue, with
minimal uptake of the vital dyes in brain tissue. This predilection for
staining in meningeal tissue would be of most interest for resection
of meningiomas, potentially offering clear delineation between brain
and meningioma, allowing for maximal visualization of tumor and
ideally greater preservation of healthy brain during surgery.
Brain and dura prior to staining with vital dye, after staining, and
after saline washes.
Above: 0.5 mg/ml fluorescein
Below: 0.5 mg/ml lissamine green
Brain and dura prior to staining with vital dye, after staining, and
after saline washes.
Above: 0.5 mg/ml rose bengal
Below: 0.06% trypan blue
Commercial Relationships: Nicole Nikolic, None; James Qiao,
None; Ping Bu, None; David K. Yoo, None
Support: Illinois Society for the Prevention of Blindness - AU
514230
534 Ocular Tumors - Management and prognosis
Thursday, May 08, 2014 12:00 PM–1:45 PM
(Neonatal) Retinoblastoma in the First Month of Life:
Comparison of the Pre- and Post-Intraarterial Chemotherapy
Era
Talia R. Kaden1, Jasmine H. Francis1, Brian Marr1, Scott E. Brodie3,
Y. Pierre Gobin2, David H. Abramson1. 1Memorial Sloan Kettering
Cancer Center, New York, NY; 2NewYork-Presbyterian Hospital/
Weill Cornell Medical Center, New York, NY; 3The Mount Sinai
Medical Center, New York, NY.
Purpose: This project evaluated the clinical features, treatment and
outcomes of children diagnosed with retinoblastoma (Rb) before 1
month of age. We assessed how intra-arterial chemotherapy (IAC)
impacted outcomes when compared with a similar, pre-IAC cohort in
the literature.
Methods: This is a retrospective review of patients diagnosed with
Rb before 31 days between 2002 and 2013. We recorded patients’
sex, family history, age at diagnosis, manifesting symptom, laterality,
stage of ocular disease at diagnosis, number of and location of
tumors, electroretinograms pre-and post treatment, development of
new ocular or extraocular tumors, treatment, length of follow-up,
ocular and patient survival. Each eye was classified at diagnosis
by Reese-Ellsworth (RE) and International Classification (ICRB)
systems.
Results: We identified 22 patients with a median follow-up of 4.3
years. The patients in the pre- and post-IAC era were comparable
in mean age at diagnosis, gender distribution, reason for diagnosis,
laterality on presentation, location of presenting and recurrent tumors
and initial tumor classification.
Only 1 patient in our post-IAC cohort received external beam
radiotherapy (EBRT) as compared to 21 of 46 patients in the pre-IAC
cohort (p<0.05). All eyes were treated with laser therapy and 70%
of our patients underwent IAC after receiving bridge intravenous
chemotherapy. 30-Hz flicker ERG responses remained stable or
improved by 6 months after the first IAC.
Most notable were the increased patient and ocular survival in our
cohort. 100% of our patients are alive while 17% of the pre-IAC
cohort died during follow-up (p<0.05). We retained all but one
RE Va/ICRB D eye. In contrast, 10 out of 11 stage V eyes were
enucleated in the pre-IAC era (p<0.05). 4 patients developed second
nonocular cancers within 5 years of diagnosis in the pre-IAC cohort
while we have no extraocular Rb or second tumors in our cohort to
date.
Conclusions: Our study is consistent with previous literature in terms
of demographics, but our data differs substantially from previous
reports in ocular survival, patient survival and incidence of nonocular
tumors. These results suggest that the use of IAC in patients with
very early diagnoses of Rb has meaningful benefits for patients,
particularly for those who present initially with extensive intraocular
disease.
Commercial Relationships: Talia R. Kaden, None; Jasmine H.
Francis, None; Brian Marr, None; Scott E. Brodie, None; Y.
Pierre Gobin, None; David H. Abramson, None
Support: The Fund for Ophthalmic Knowledge, Inc
Intravitreal chemotherapy virtually eradicates indication to
external beam irradiation for vitreous seeding in retinoblastoma
Francis L. Munier1, Marie-Claire Gaillard1, Aubin Balmer1, Susan
Houghton1, Maja Beck Popovic2. 1Ophthalmolgy, Jules-Gonin
Eye Hospital, Lausanne, Switzerland; 2Unité d’hémato-oncologie
pédiatrique, Centre Hospitalier Universitaire Vaudois, Lausanne,
Switzerland.
Purpose: Vitreous seeding is the worst prognostic factor for eye
survival and still represents the most common indication for external
beam irradiation and/or enucleation. Here we present the long term
follow-up on a published series (Munier et al.Brit J Ophthalmol
2012) treated by intravitreal chemotherapy (IVC) and describe the
results of a newly recruited cohort of patients presenting with active
vitreous seeding.
Methods: Fifty nine consecutive heavily pretreated patients (63
eyes) eligible for IVC were reviewed, including the first series of
23 eyes with globe retention achieved in 20 eyes. The combined
63 eyes received a total 358 IVC (20 to 40 micrograms melphalan)
given every 7 to 10 days. Ocular status was monitored under
anesthesia with fundus photography, fluorescein angiography, and
optic coherence tomography. Additional treatments, including
chemohyperthermia, hyperthermia, photocoagulation, periocular
topotecan, intra-arterial melphalan, and brachytherapy were applied
as necessary to control the retinal tumors.
Results: The follow-up of our initial cohort of 23 patients (20
conserved eyes) is marred by the loss of 2 children. One child had
undergone enucleation but was disease-free until lost to follow-up
in the referral country. The other child showed no ocular recurrence
after IVC with survival of 30 months, but was recently diagnosed
with uncurable pinealoblastoma. The follow-up for the remaining 19
conserved eyes is extended with mean tumor-free survival of 34.8
months (range 17-49) and no recurrence observed. In the second
cohort complete vitreous response is achieved in 37 of the 40 eyes
(92.5%, with 3 cases still ongoing), with no evidence of tumor spread
and only infraclinical retinal toxicity. Mean follow-up is 12 months
(range 1-30). Additional focal treatments were required to achieve
complete inactivation of retinal and subretinal tumors in 88% of the
eyes.
Conclusions: In conclusion, not only can IVC reverse the negative
prognostic value of vitreous seeding, but has virtually eradicated
the indication to external beam radiotherapy. However it should be
emphasized that full tumor control is only rarely achieved by IVC
alone without concomitant treatment of the retinal source of the
seeding as well as all other active retinal tumors.
Commercial Relationships: Francis L. Munier, None; MarieClaire Gaillard, None; Aubin Balmer, None; Susan Houghton,
None; Maja Beck Popovic, None
A Prospective Single Institution Trial Using Toptecan Based
Chemotherapy for the Treatment of Bilateral Intraocular
Retinoblastoma: 5 Year Results.
Matthew W. Wilson1, 2, Rachel Brennan1, 3, Shenghua Mao4, Ibrahim
Qaddoumi3, Carlos Rodriguez-Galindo5. 1Ophthal/Hamilton Eye Int,
Univ of Tennessee Health Sci Ctr, Memphis, TN; 2Surgery, St Jude
Children’s Research Hospital, Memphis, TN; 3Oncology, St Jude
Children’s Reseach Hospital, Memphis, TN; 4Biostatics, St Jude
Children’s Reseach Hospital, Memphis, TN; 5Pediatric Oncology,
Dana Farber Cancer Institute, Boston, MA.
Purpose: Purpose: To evaluate efficacy of systemic chemo-reduction
using topotecan for advanced intraocular retinoblastoma.
Methods: Methods: 27 newly diagnosed bilateral retinoblastoma
patients (14 males, median age 7.9 months), worse eye ReeseEllsworth (RE) Group IV-V, were treated with 11 cycles of
chemotherapy: topotecan and vincristine (TV) x 2 followed by 3
alternating courses of carboplatin and vincristine x 2 and TV x 1.
Intensive focal therapy was applied after the first 2 cycles. Event free
survival (EFS) was defined as avoidance of external beam radiation
(EBRT) and enucleation.
Results: Results: Of 54 eyes, 42 were RE IV-V and 36 were
International Classification C-E. 25 patients completed all prescribed
chemotherapy; one was removed due to persistent viral infection and
one had bilateral progressive disease requiring EBRT. 11 eyes were
enucleated: 1 at diagnosis, 9 with progressive disease including 3
treated with EBRT, and 1 which developed neovascular glaucoma.
At 8 years, cumulative incidence of EBRT was 2.4% (SE±2.4%),
EFS for patients was 66.7% (SE± 38.5%), and ocular survival for RE
IV-V eyes was 76.2% (SE±26.3). Hematologic toxicities included
febrile neutropenia (29 episodes in 275 courses) and 41 episodes of
grade 4 thrombocytopenia (1.6 platelet transfusions/patient). Other
patient specific toxicities included infection (n=15, 40% viral), grade
3 diarrhea (n=9) and carboplatin reaction (n=1). All patients are alive
with median follow up was 4.9 years.
Conclusions: Conclusions: Topotecan combined with vincristine and
carboplatin and aggressive focal therapies is and effective regimen
for treatment of retinoblastoma. Toxicities were anticipated and
managed with appropriate supportive care.
Commercial Relationships: Matthew W. Wilson, None; Rachel
Brennan, None; Shenghua Mao, None; Ibrahim Qaddoumi, None;
Carlos Rodriguez-Galindo, None
Support: Research to Prevent Blidness, St Jude Children’s Research
Hospital Cancer Center Support (CORE) Grant No. CA21765
An Update on the Development of sd-rxRNA for Retinoblastoma
Therapy
Michael Byrne1, Donglai Qi2, Kevin Stachelek2, Hardeep Singh2,
James Cardia1, Lakshmipathi Panderarinathan1, Katherine Holton1,
Karen Bulock1, David Cobrinik2, Pamela A. Pavco1. 1Pharmacology,
RXi Pharmaceuticals, Westborough, MA; 2Children’s Hospital Los
Angeles, Los Angeles, CA.
Purpose: Retinoblastoma is a cancer that originates in the retina
and primarily affects young children. It is driven by inactivating
RB1 mutations as well as by the expression of genes involved in
the “normal” signaling circuitry of retinal cells, particularly that
of cone precursors. Some of these genes have been found to be
critical to retinoblastoma cell growth and survival, suggesting that
they may be effective therapeutic targets. We have developed a new
class of stable, self-delivering RNAi compounds (sd-rxRNA®) that
incorporate features of RNAi and antisense and results in spontaneous
cellular uptake. Our goal is to use the sd-rxRNA platform to develop
compounds against retinoblastoma therapeutic targets. Initial studies
examined in vitro mRNA silencing in retinoblastoma cell lines as
well as in vivo uptake by human retinoblastoma cells in a mouse
model following treatment with control sd-rxRNA compounds. Here
we designed and examined the efficacy of therapeutically relevant
sd-rxRNAs specific for retinoblastoma in vitro.
Methods: Twenty-five sd-rxRNAs were designed, synthesized and
screened in 3-point dose response studies in RB177 cells. Based on
targeted mRNA reduction, potential hits were selected and further
evaluated in 6-point dose response studies in RB177 and RB176
cells.
Results: Multiple sd-rxRNAs resulted in > 50% reduction of the
targeted mRNA in studies carried out in RB177 cultured cells.
Four potential hits were selected and further evaluated in 6-point
dose response studies in RB177 and RB176 cells. Two candidate
compounds that demonstrated > 50% mRNA target reduction at < 0.5
uM in both cell lines were selected (a lead and a backup) for in vitro
evaluation of tumor cell viability post treatment and in vivo analysis
in an orthotopic mouse xenograft model.
Conclusions: Therapeutically-relevant, target-specific sd-rxRNAs
were identified and evaluated in vitro. Lead candidates were selected
based on their ability to reduce the targeted mRNA in human
retinoblastoma cell lines in vitro. Our next step is to evaluate if
treatment with sd-rxRNAs results in reduced tumor cell viability in
vitro and reduced target mRNA levels in human retinoblastoma cells
in vivo in an orthotopic mouse xenograft model. These findings,
along with our previous report of specific and extended silencing of
retinal genes by sd-rxRNA, support the potential use of sd-rxRNA for
retinoblastoma therapy.
Commercial Relationships: Michael Byrne, RXi Pharmaceuticals
(E); Donglai Qi, None; Kevin Stachelek, None; Hardeep Singh,
None; James Cardia, RXi Pharmaceuticals (E); Lakshmipathi
Panderarinathan, RXi Pharmaceuticals (E); Katherine Holton,
RXi Pharmaceuticals (E); Karen Bulock, RXi Pharmaceuticals (E);
David Cobrinik, None; Pamela A. Pavco, RXi (E)
Support: NIH SBIR Grant number: 1R43CA165899-01A1
The 2014 American Brachytherapy Society (ABS) Guidelines for
Plaque Brachytherapy of Uveal Melanoma and Retinoblastoma
Paul T. Finger. Ophthalmic Oncology, New York Eye Cancer Center,
New York, NY.
Purpose: To present the current, updated American Brachytherapy
Society (ABS) concenus guidelines for plaque brachytherapy of
choroidal melanoma and retinoblastoma to the community of eye
care professionals.
Methods: An international, multicenter Ophthalmic Oncology Task
Force (OOTF) was assembled to include 47 radiation oncologists,
medical physicists and ophthalmic oncologists from 13 centers in
10 countries. Thus, the committee included specialists from Europe
(Sweden, Finland, United Kingdom, Germany, France), Russia,
Japan, India, The United States of America (New York, Tennessee,
Georgia, Rhode Island) and Toronto- Ontario-Canada.
Thus, the ABS-OOTF produced universally acceptable collaborative
guidelines, based on their eye cancer specific clinical experience
and knowledge of the literature. Further, these resultant guidelines
were staged by levels of consensus. Once completed, this work was
reviewed and approved by the 10 person American Brachytherapy
Society Board of Directors as well as 4 external reviewers.
Results: 1) The ABS-OOTF reached consensus that ophthalmic
plaque radiation therapy is best performed in subspecialty
brachytherapy centers, experienced with plaque therapy.
2) Quality assurance, methods of plaque construction/dosimetry
should be consistent with the 2012 joint guidelines of the American
Association of Physicists in Medicine and ABS.
3) Though there exist select restrictions related to tumor-size, location
and vision status; the ABS-OOTF agreed that most uveal melanomas
(iris, ciliary body and choroid) could be treated with plaque
brachytherapy. Specifically, the ABS-OOTF reached consensus that
tumors with gross orbital extension, blind painful eyes and those with
no light perception vision are unsuitable for brachytherapy.
4) Only select retinoblastomas were considered eligible for plaque
brachytherapy.
5) Additional subjects addressed include prescription doses, dose
rates, treatment durations and clinical methods.
Conclusions: Plaque brachytherapy is an effective, eye and visionsparing method to treat patients with intraocular tumors. Eye cancer
specialists (radiation oncologists, medical physicists and ophthalmic
oncologists) are encouraged to use both the AAPM-ABS guidelines
for plaque dosimetry and quality assurance as well as the ABS-OOTF
clinical guidelines to enhance their practice.
Commercial Relationships: Paul T. Finger, None
Support: The Eye Cancer Foundation, Inc and The American
Brachytherapy Society
Comparison of Uveal Melanoma Cytopathologic Sample
Retrieval in Transscleral Versus Vitrectomy-Assisted Transvitreal
Fine Needle Aspiration Biopsy
Melinda Chang, Ben J. Glasgow, Tara A. McCannel. Ophthalmology,
Jules Stein Eye Institute, Los Angeles, CA.
Purpose: Fine needle aspiration biopsy has become a standard
technique to obtain material from primary uveal melanoma for
cytopathology, prognostication and research. The purpose of this
investigation was to compare sample retrieval for cytopathology
between transscleral and vitrectomy-assisted transvitreal biopsy in
the same tumor in patients who underwent iodine-125 brachytherapy
in combination with vitrectomy.
Methods: All clinical records of patients who underwent iodine-125
brachytherapy for the treatment of uveal melanoma in conjunction
with pars plana vitrectomy from whom transscleral and vitrectomyassisted transvitreal fine needle biopsies were obtained, were
reviewed. Transscleral and transvitreal biopsies were performed using
a 30-gauge and 27-gauge needle, respectively. Baseline patient and
tumor characteristics, biopsy sample yield for cytopathology, and
surgical outcomes were reviewed.
Results: Thirty-eight patients with a mean follow-up time of 4.3
months were included. The mean tumor height was 4.8 mm (range
1.74 to 13.27 mm) and the mean tumor largest basal diameter was
10.7 mm (range 5.47 to 15.9 mm). Overall, 71.1% of transvitreal
biopsies and 65.8% of transscleral biopsies yielded sufficient material
for cytopathologic analysis (p = 0.62). Greater tumor height and
largest basal diameter were significantly associated with increased
cytopathologic yield in both transvitreal (p = 0.03 and p = 0.02,
respectively) and transscleral biopsies (p= 0.015 and p = 0.007,
respectively). Tumor location was not significantly associated with
cytopathologic yield for transvitreal (p = 0.26) and transscleral (p =
0.63) biopsies. Fifteen eyes (39.4%) developed transient localized
vitreous hemorrhage. No eye developed diffuse or non-clearing
vitreous hemorrhage, rhegmatogenous retinal detachment, local
treatment failure or metastatic disease during follow-up interval.
Conclusions: Both transscleral and vitrectomy-assisted transvitreal
fine needle aspiration biopsy resulted in similar tissue yield for
cytopathologic analysis when compared during biopsy of the same
uveal melanoma. As molecular prognostication becomes the standard
of care for obtaining patient information and directing clinical
management, further investigation of biopsy techniques is warranted.
Commercial Relationships: Melinda Chang, None; Ben J.
Glasgow, None; Tara A. McCannel, None
Support: Supported by an unrestricted grant from Research to
Prevent Blindness and the George E. and Ruth Moss Trust
Primary Oculo-Cerebral lymphoma (POCL): Efficacy
of intravenous high-dose methotrexate (MTX) based
polychemotherapy without radiotherapy on intraocular disease
control
Diem Trang Nguyen1, Nathalie Cassoux2, Sylvain Choquet3,
Carole Soussain4, Chloé Le Cossec5, Antonio Omuro6, Phuc
Lehoang1, Bahram Bodaghi1, Khe Hoang Xuan6, Valerie Touitou1.
1
Ophthalmology, DHU View Maintain, Pitié Salpêtriere Hospital,
Paris, France; 2Ophthalmologic oncology, Insitut Curie, Paris,
France; 3Clinical Hematology, Pitie Salpetriere hospital, Paris,
France; 4Oncology, Institut René Huguenin, Saint Cloud, France;
5
Biostatistics, medical informatic, Pitie Salpetriere Hospital, Paris,
France; 6Neuro-Oncology, Pitie Salpetriere Hospital, Paris, France.
Purpose: Chemotherapy with radiotherapy (RT) is the current
standard treatment of primary oculo-cerebral lymphoma (POCL)
but exposes to a high risk of radiation-induced neurotoxicity,
especially in the elderly. We recently reported encouraging results of
2 intravenous (IV) high-dose (HD) MTX based polychemotherapy
regimens without RT in a randomized multicenter phase II trial
(“CNS trial”) for primary central nervous system lymphoma patients
over 60 years old (MPVA: HD-MTX, procarbazine, vincristine,
and cytarabine versus HD-MTX-Tmz: MTX, temozolomide).
The objective was to evaluate the efficacy of IV MTX-based
chemotherapy on intraocular lymphoma (IOL) in POCL without RT.
Methods: Patients with pathologically proven POCL randomized
in the CNS trial, with baseline and follow-up ocular evaluations
available were retrospectively analysed. The primary study endpoints were the ocular response and event-free survival. Secondary
outcome measures were: cerebral event-free survival and overall
survival (OS) of both treatments.
Results: Thirteen patients out of 98 (9 males / 4 females, median age
63 years old) met the inclusion criteria. Six patients were randomized
into the MPVA group and 7 into the MTX-Tmz group. Overall
ocular complete response rate was 58% (MPVA: 67%, MTX-Tmz:
50% - p=0.99). Disease progressed in 17% of patients (MPVA: 17%,
MTX-Tmz: 17%) and remained stable in 25% (MPVA: 17%, MTXTmz: 33%). With a median follow-up of 32 months, ocular relapse
after an initial complete response was observed in 42% of patients
(MPVA: 75%, MTX-Tmz: 67% - p=0.71). The median ocular eventfree survival was 15 months (0-43) in the MPVA group, and 16.5
months (5-69) in the MTX-Tmz group (p=0.997). The median OS
for the entire population was 32 months (6-69) without significant
difference between the two groups (p=0.481), neither in the cerebral
event-free survival (p=0.572). Systemic toxicities occurred in both
groups (p>0.05) but were managable. Neurocognitve functions were
well preserved.
Conclusions: IV HD MTX based polychemotherapy as first line
treatment is active in IOL in the setting of POCL. MPVA and MTXTmz demonstrate similar efficacy. Despite a good response rate,
relapses remain frequent and improvement of intra-ocular disease
control is needed. Future chemotherapy regimens and treatment
strategies have to focus on this issue.
Commercial Relationships: Diem Trang Nguyen, None; Nathalie
Cassoux, None; Sylvain Choquet, Shering-Plough/Merck (F);
Carole Soussain, Shering-Plough/Merck (F); Chloé Le Cossec,
None; Antonio Omuro, Shering-Plough/Merck (F); Phuc Lehoang,
None; Bahram Bodaghi, None; Khe Hoang Xuan, Shering-Plough/
Merck (F); Valerie Touitou, None
Support: Study sponsored by APHP – PHRC AOM 06 175 /P06239,
Temozolomide and partial financial support provided by ScheringPlough/ Merck, Aknowledgement to the French LOC network (INCa)

ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group

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