ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group

Transcription

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Anatomy/Pathology
210 Retinoblastoma: Pre-Clinical Models and Targeted
Therapies
Monday, May 06, 2013 8:30 AM-10:15 AM
608 Paper Session
Program #/Board # Range: 1253-1259
Organizing Section: Anatomy/Pathology
Program Number: 1253
Presentation Time: 8:30 AM - 8:45 AM
In vivo imaging and characterization of an orthotopic
retinoblastoma xenograft model
Timothy W. Corson1, 2, Anna J. Geary3, Andrea Wenzel1, Amanda
Riley4, Brian P. McCarthy4, Barbara Bailey5, Karen E. Pollok5, Paul
R. Territo4, Brian C. Samuels1. 1Ophthalmology, Indiana University
School of Medicine, Indianapolis, IN; 2Biochemistry and Molecular
Biology, Indiana University School of Medicine, Indianapolis, IN;
3
Eastern University, St Davids, PA; 4Radiology & Imaging Sciences,
Indiana University School of Medicine, Indianapolis, IN; 5Pediatrics,
Indiana University School of Medicine, Indianapolis, IN.
Purpose: Xenografts of human retinoblastoma cells are an important
system in which to test novel therapeutics for this pediatric ocular
cancer. One recently developed orthotopic xenograft model involves
injection of a luciferase-expressing Y79 human retinoblastoma cell
line into the vitreous of newborn wild-type rats. Use of neonates
takes advantage of the fact that these animals are naturally
immunonaïve, and also allows for intraocular tumor growth at an age
that is developmentally appropriate for retinoblastoma. We aimed to
better characterize tumor growth in this model by combining both
bioluminescence and intraocular fluorescence imaging of developing
xenografts.
Methods: We engineered Y79 retinoblastoma cells to overexpress a
luciferase-EGFP fusion. We assayed cell line bioluminescence in
vitro using a plate reader and fluorescence with both a plate reader
and a Typhoon laser scanner. PBS vehicle, 1,000, or 10,000 cells
were injected into the vitreous of newborn Sprague-Dawley rats
(N=30). Over a 28 day period, in vivo bioluminescence and
fluorescence imaging was performed using a NightOwl
bioluminescence imager and Phoenix Micron III intraocular imager,
respectively.
Results: We confirmed linearity of detection of both
bioluminescence (luciferase activity) and fluorescence (EGFP) with
increasing cell number in vitro by both plate reader and laser scanner
analysis. Xenografted cells formed rapidly growing tumors in 90% of
animals. In vivo bioluminescence, ex vivo tumor size, and ex vivo
fluorescent signal were all highly correlated. Despite significant
corneal neovascularization, even in vehicle-injected animals,
intraocular brightfield and fluorescence imaging allowed delineation
of tumor growth over time, including small tumors preferentially
sitting atop the optic nerve head, multifocal seeding, and large
vitreous-filling tumors that were highly vascularized.
Conclusions: The combination of non-invasive bioluminescence and
in vivo intraocular brightfield/fluorescence imaging allows both
quantitative and high-resolution spatial analysis of this
retinoblastoma model, and will be applied to other cell lines and
experimental therapeutic trials in future.
Commercial Relationships: Timothy W. Corson, None; Anna J.
Geary, None; Andrea Wenzel, None; Amanda Riley, None; Brian
P. McCarthy, None; Barbara Bailey, None; Karen E. Pollok,
None; Paul R. Territo, None; Brian C. Samuels, Merck & Co., Inc
(F), Merck & Co., Inc (C), ICHE (C)
Support: Indiana CTSI, NCATS TR000006
An Orthotopic Transplantation Model of Retinoblastoma in
Zebrafish: A Novel Gateway for Screening of Anticancer Drugs
Dong Hyun Jo1, Dain Son2, Yirang Na2, Manyoung Jang3, Jae-Hoon
Choi3, Jin Hyoung Kim1, Young S. Yu1, 4, Seung Hyeok Seok2, Jeong
Hun Kim1, 4. 1Fight against Angiogenesis-Related Blindness (FARB)
Laboratory, Clinical Research Institute, Seoul National University
Hospital, Seoul, Republic of Korea; 2Department of Microbiology
and Immunology and Institute of Endemic Disease, College of
Medicine, Seoul National University, Seoul, Republic of Korea;
3
Department of Life Science, College of Natural Sciences, Hanyang
University, Seoul, Republic of Korea; 4Department of
Ophthalmology, College of Medicine, Seoul National University,
Seoul, Republic of Korea.
Purpose: To establish a novel orthotopic transplantation model of
retinoblastoma in zebrafish for efficient screening of anticancer drugs
Methods: We injected differential numbers (20 and 100) of
retinoblastoma cells into the vitreous cavity of zebrafish at 48 hours
after fertilization. Eyeballs of zebrafish were scanned daily under the
confocal laser microscope. Captured images were used for
quantitative analysis with the public image processing program,
ImageJ. Transplanted retinoblastoma cells were isolated to perform
further analyses including Western blotting for glial fibrillary acidic
protein and neuron-specific enolase, reverse transcriptase for cellular
retinaldehyde-binding protein, to confirm that retinoblastoma cells
maintained their characteristics as tumor cells even with
transplantation and isolation. To figure out the potential of this model
for screening of anticancer drugs, zebrafish were treated with
carboplatin and melphalan systemically after the injection of
retinoblastoma cells.
Results: The degree of the tumor population estimated by the mean
intensity of GFP expression was dependent on the number of
retinoblastoma cells injected. The tumor population maintained stably
for at least 4 days after the injection. Transplanted retinoblastoma
cells maintain the proliferative potential and characteristics as
retinoblastoma cells after isolation. Interestingly, systemic
application of carboplatin and melphalan induced significant
reduction in the tumor population, which could be quantitatively
analyzed by the estimation of the mean intensity of GFP expression.
Conclusions: This orthotopic transplantation model of
retinoblastoma in zebrafish is expected to be utilized for the
screening of anticancer drugs for the treatment of retinoblastoma.
Retinoblastoma cells were injected into the vitreous cavity of
zebrafish 48 hours after fertilization. Scale bar = 100 μm.
©2013, 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 arvo@arvo.org.
L1CAM showed little change by qRT-PCR, suggesting posttranscriptional regulation.
Conclusions: Both SSEA-5 and L1CAM are cell surface markers
that show promise as indicators of pluripotency. Stem cell regulatory
mechanisms common to RB cancer stem cells and retinal therapeutic
stem cells may be useful in monitoring and controlling the growth
and differentiation of iPSCs. Further studies may aid in growth
regulatory strategies that are essential to the development of safe
ocular stem cell replacement therapies.
Four days after the injection, the eyeballs of zebrafish were scanned
under the confocal laser microscope for further quantitative analysis.
Scale bar = 100 μm.
Commercial Relationships: Dong Hyun Jo, None; Dain Son,
None; Yirang Na, None; Manyoung Jang, None; Jae-Hoon Choi,
None; Jin Hyoung Kim, None; Young S. Yu, None; Seung Hyeok
Seok, None; Jeong Hun Kim, None
Expression of Pluripotent Markers L1CAM and SSEA-5,
Common to Human Retinoblastomas, Xenografts, Teratomas,
Embryonic Tumors and Induced Pluripotent Stem Cells
Gail M. Seigel1, 6, Meerim Choi1, 6, Rui Chang2, Jason S. Meyer3,
Bruce R. Ksander4, Paraskevi E. Kolovou4, Nadine E. de Waard5, 4,
Linda L. Cassidy1, 6. 1Center for Hearing and Deafness, University at
Buffalo, Buffalo, NY; 2Genetics and Genomic Sciences, Mount Sinai
School of Medicine, New York, NY; 3Biology, Indiana University,
Indianapolis, IN; 4Ophthalmology, Schepens Eye Research Institute,
Boston, MA; 5Ophthalmology, Leiden University, Leiden,
Netherlands; 6SUNY Eye Institute, Buffalo, NY.
Purpose: Cancer stem cells and therapeutic stem cells share common
and divergent pathways involved in cell growth and differentiation.
In this study, we sought to bridge the gap between retinoblastoma
(RB) tumor cells and iPSCs undergoing retinal differentiation by
testing the hypothesis that L1CAM and SSEA-5 could be used to
evaluate pluripotency in both models.
Methods: Magnetically enriched stem-like ABCG2+ and non-stem
like ABCG2- cells from the human RB143 retinoblastoma cell line
underwent microarray analysis using the Agilent platform. We used
ensemble based statistical models to predict differentially expressed
genes of pluripotency. The larger list was condensed into genes with
biological relevance to the behavior of cancer stem cells and iPSCs.
One of these genes was L1CAM, a neural cell adhesion marker. We
included SSEA-5 for analysis based on promising published data as a
novel marker of pluripotency. We used immunohistochemistry to
analyze RB tumors and cell lines, RB xenografts, human embryonic
tumors, iPSC-derived teratomas, as well as iPS cells before and after
previously established retinal differentiation protocols to determine
whether these markers would be useful for monitoring pluripotency.
The expression of L1CAM in these cells was also assessed by qRTPCR.
Results: RB143 cells and xenografts were immunoreactive to
L1CAM and SSEA-5. L1CAM was expressed in most human RB and
human embryonic tumors examined. SSEA-5 was preferentially
expressed in embryonic tumors and some RB tumors. In iPS-induced
teratomas, we saw immunoreactivity to both markers, with some
areas of co-localization. Undifferentiated iPSCs exhibited strong
immunoreactivity to SSEA-5 and L1CAM, with a few positive cells
remaining even after 20 days of retinal differentiating treatment.
Embryonal carcinoma: SSEA-5 (red), L1CAM (green)
Commercial Relationships: Gail M. Seigel, None; Meerim Choi,
None; Rui Chang, None; Jason S. Meyer, None; Bruce R.
Ksander, None; Paraskevi E. Kolovou, None; Nadine E. de
Waard, None; Linda L. Cassidy, None
Support: Cornell Center on the Microenvironment & Metastasis
through Award Number U54CA143876 from the National Cancer
Institute, R21CA127061 and NYSTEM C026412.
Evaluation of response to Carboplatin in putative Cancer Stem
Cells of Retinoblastoma Y79 cell line
Geeta K. Vemuganti1, Rohini M. Nair1, Murali Mohan Sagar Balla2,
Santosh Honavar3, Mohammad J. Ali3, Vijay Anand R. Palkonda4.
1
School of Medical Sciences, University of Hyderabad, Hyderabad,
India; 2Ophthalmic Pathology Laboratory, L.V.Prasad Eye Institute,
Hyderabad, India; 3Ophthalmic and Facial Plastic Surgery, Orbit and
Ocular Oncology, L.V.Prasad Eye Institute, Hyderabad, India;
4
Apollo Cancer Hospital, Hyderabad, India.
Purpose: Evaluation of Cancer Stem Cells (CSCs) in primary tumors
is not only of academic interest but of potential therapeutic
application. Due to the technical challenges in working with primary
cells, this study attempts to evaluate the response to standard Rb
chemotherapeutic agent, Carboplatin in the putative CSC population
of Retinoblastoma Y79 cell line. With the preliminary evidence that
CD133- FSClo/SSClo cells could be putative Cancer stem cells in
Y79 cell line, we evaluated the cytotoxicity of these cells to
Carboplatin.
Methods: Phenotypic characterization and sorting of cultured Y79
cells was done by FACS Aria II using putative CSC marker CD133.
The sorted cells and total cells were analyzed for response to various
doses (0-100μM) of Carboplatin (Alkem Pharmaceuticals) following
48 hr exposure. Controls consisted of untreated cells from CD133±
and total Y79 cells. Cell death was observed under phase contrast
microscope and the wells were treated with MTT(5mg/ml), the
resulting formazan crystals were dissolved with DMSO and
absorbance was read at 570nm. A comparative analysis of percentage
of viability among the three groups- total Y79 cells, CD133+ and
CD133- cells was done using GraphPad Prism .
Results: Retinoblastoma Y79 cell lines when sorted using CD133
revealed 3.8% CD133+ and 16.2% CD133- of total viable Y79 cells.
CD133- cells showed increased resistance and proliferation compared
to CD133+ and unsorted cells (p<0.01) following 48 hr exposure to
higher doses of Carboplatin indicating chemoresistance in this
population. Maximum proliferation (151.57±37.54%) was observed
at 50µM in CD133- cells . At the same dose, the viability of total
Y79 cells and CD133+ cells were 39.66±3.05 and 65.22±10.12
respectively.
Conclusions: The study shows that the Y79 CD133- FSClo/SSClo
cells exhibit resistance to high dose Carboplatin with increased
proliferation as compared to controls. This is in concordance with our
previous findings of quiescence, clonal nature and gene expression
(ARVO abstract no 2643/A450) in putative Cancer Stem Cells of
Retinoblastoma Y79 cell line.
Drug response of CD133 positive, negative and total Y79 cells
treated with various concentrations of Carboplatin for 48 hours by
MTT Assay
Commercial Relationships: Geeta K. Vemuganti, None; Rohini
M. Nair, None; Murali Mohan Sagar Balla, None; Santosh
Honavar, None; Mohammad J. Ali, None; Vijay Anand R.
Palkonda, None
Support: Indian Council of Medical Research, Hyderabad Eye
Research Foundation
High Risk Retinoblastoma: Correlation Between Expression of
Angiogenic Factors and Neovascular Glaucoma
Claudia M. Prospero Ponce1, Dan S. Gombos2, 3, Aravindh S.
Ganapathy1, Patricia Chevez-Barrios1, 3. 1Pathology and Genomic
Medicine, Ocular Pathology, The Methodist Hospital, Houston, TX;
2
Ophthalmology, University of Texas, MD Anderson Cancer Center,
Houston, TX; 3Retinoblastoma Center of Houston, Houston, TX.
Purpose: To analyze the expression of angiogenic factors in the
eye(retina, iris and tumor) in retinoblastoma(Rb) cases with high risk
features(HRF) and Non-HRF. Angiogenesis is essential for tumor
growth and metastasis. In Rb tumors, angiogenesis may complicate
the clinical presentation by inducing glaucoma due to iris
neovascularization(NVI) with secondary peripheral synechiae(PAS)
formation. The expression of angiogenic factors in specific ocular
and tumor cells determines the tumor growth and complications.
Methods: We analyzed 10 Rb eyes with NVI and glaucoma. These
were classified as HRF(4/10) if they showed invasion to choroid
and/or optic nerve post lamina cribrosa. Double staining by
immunohistochemistry was performed with vascular endothelial
growth factor(VEGF)-glial fibrillary acidic protein(GFAP; glial cells)
and VEGFR-2(receptor)-CD105(endoglin; new vessels). Quantitative
analysis per area (μm2) of expression and co-localization of
angiogenic factors with different cell types was performed using
Image-J software(NIH).
Results: VEGF expression: The highest expression is seen in the
tumor of HRF eyes. The iris shows about equal expression in both
groups. The retina however, shows less expression of VEGF in HRF
cases. VEGF-GFAP co-expression: In tumors, the co-expression is
higher in HRF eyes. This co-expression is higher in Non-HRF retina.
VEGFR-2 expression: Tumor and iris in HRF eyes have higher
expression than the Non-HRF. The retina shows lower expression in
HRF. CD105 expression: Tumors demonstrate similar
expression(average in μm2: HRF 144 vs Non-HRF 132). In the iris,
the CD105 is expressed ~60% more than in the Non-HRF. In the
HRF retina, the vessels have lower expression than Non-HRF; The
expression is ~95% lower in HRF than in Non-HRF at the level of
tumor invasion into the retina. CD105-VEGFR-2 co-expression:
There is higher co-expression in HRF eyes in the tumor(average in
μm2: HRF 68 vs Non-HRF 14) and in the iris(average in μm2: HRF
54 vs. Non-HRF 38).
Conclusions: Eyes with HRF show increased expression of VEGF
and its receptor VEGFR-2 in both the tumor and the iris. CD105
expression(newly formed vessels) is elevated in tumors but is
particularly higher in the iris of HRF eyes. Therefore in HRF eyes,
treatments that target cells expressing angiogenic factors and/or the
factors alone may be useful to prevent complications such as
development of neovascular glaucoma.
Commercial Relationships: Claudia M. Prospero Ponce, None;
Dan S. Gombos, None; Aravindh S. Ganapathy, None; Patricia
Chevez-Barrios, None
Support: Glaucoma Research Foundation
Development of sd-rxRNA® for Retinoblastoma Therapy
Michael Byrne1, Hardeep P. Singh2, Donglai Qi2, James Cardia1,
Lakshmipathi Pandarinathan1, Katherine Holton1, Karen Bulock1,
Lyn Libertine1, David Cobrinik2, Pamela A. Pavco1. 1Pharmacology,
RXi Pharmaceuticals, Westborough, MA; 2Memorial Sloan-Kettering
Cancer Center, New York, NY.
Purpose: Retinoblastoma is a cancer that originates in the retina and
primarily affects young children. It is driven by RB mutations as well
as 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, selfdelivering 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 novel retinoblastoma therapeutic targets. Earlier studies of
control sd-rxRNAs in the eye resulted in spontaneous cellular uptake
in the retina as well as statistically significant reduction of target
mRNA levels for up to 21 days. To determine the potential of using
sd-rxRNAs to target genes involved in retinoblastoma, we examined
in vitro mRNA silencing in retinoblastoma cell lines as well as in
vivo uptake by human retinoblastoma cells in a mouse model.
Methods: For in vitro studies, retinoblastoma cell lines RB176,
RB177 and Y79 were treated with sd-rxRNA targeting the PPIB gene
for 48 hours, after which PPIB mRNA levels were analyzed by
qPCR. For in vivo studies, 10 ug of DY547- labeled sd-rxRNA was
administered by intravitreal injection to mouse eyes that were seeded
subretinally 3 weeks earlier with Y79 cells.
Results: In all retinoblastoma cell lines tested, PPIB was silenced in
a dose-dependent manner at 48 hours post administration relative to a
non-targeting control sd-rxRNA. Twenty-four hours post intravitreal
injection in mouse eyes, sd-rxRNA was visible in tumor cells in the
subretinal space and in the vitreous.
Conclusions: sd-rxRNAs reduce targeted mRNA levels in
retinoblastoma cell lines. Additionally, sd-rxRNA is delivered to all
cell layers of the retina and is taken up by tumor cells within 24 hours
of intravitreal administration. These findings, along with our previous
report of specific and extended silencing of retinal genes by sdrxRNA, support the potential use of an sd-rxRNA treatment for
retinoblastoma. Our next step is to design and characterize sd-rxRNA
compounds against novel retinoblastoma targets and to evaluate their
ability to reduce target mRNA levels in human retinoblastoma cells
in vivo using an orthotopic mouse xenograft model.
Commercial Relationships: Michael Byrne, RXi Pharmaceuticals
(E); Hardeep P. Singh, None; Donglai Qi, None; James Cardia,
RXi Pharmaceuticals (E); Lakshmipathi Pandarinathan, RXi
Pharmaceuticals (E); Katherine Holton, RXi Pharmaceuticals (E);
Karen Bulock, RXi Pharmaceuticals (E), RXi Pharmaceuticals (P);
Lyn Libertine, RXi Pharmaceuticals (E); David Cobrinik, None;
Pamela A. Pavco, RXi Pharmaceuticals Corp. (E)
KZ-41 prevents melphalan-induced intercellular adhesion
molecule-1 (ICAM-1) upregulation and apoptosis in retinal
endothelial cells
Qiuhua Zhang1, Jordan J. Toutounchian2, Charles R. Yates2,
Matthew W. Wilson1, Jena J. Steinle1, 3. 1Ophthalmology, Univ of
Tennessee Hlth Sci Ctr, Memphis, TN; 2Pharmaceutical Sciences,
University of Tennessee Health Science Center, Memphis, TN;
3
Anatomy and Neurobiology, University of Tennessee Health
Science Center, Memphis, TN.
Purpose: To determine whether a novel NF-κB inhibitor, KZ-41, can
inhibit melphalan’s actions on retinal endothelial cells (REC)
inflammation and apoptosis, without eliminating the
chemotherapeutic efficacy of melphalan on cell death of
retinoblastoma cells (Y79).
Methods: REC were cultured in M131 medium supplemented with
growth factors and antibiotics. Once cells reached confluence, they
were treated with or without 10 μM KZ-41, following treatment with
4 μg/ml melphalan. Cell proteins were extracted and analyzed for
intracellular adhesion molecule 1 (ICAM-1) levels and cell death
ELISA. REC were also transfected with or without NF-κB siRNA
before melphalan treatment to determine the involvement of NF-κB
in REC apoptosis and ICAM-1 levels. Cell lysates were processed for
ICAM-1 levels and cell death ELISA. Western blotting was done to
verify NF-κB knockdown following both NF-κB siRNA transfection
and KZ-41 treatment. Phospho-P38MAPK inhibitor was also treated
before KZ-41 and melphalan treatment and cell lysates were tested
for ICAM-1 levels and REC cell death. We also cultured
retinoblastoma cells (Y79) in RMPI-1640 medium supplemented
with 20% fetal bovine serum and antibiotics and performed a cell
death ELISA after melphalan + KZ-41 treatment to verify that the
treatments did not alter melphalan’s ability to promote cell death of
Y79 cells.
Results: KZ-41 could inhibit melphalan-induced ICAM-1 levels and
REC apoptosis. KZ-41 blocked increased ICAM-1 levels through
p38MAPK activation and NF-κB inhibition; and decreased REC
apoptosis likely through p38MAPK activation. KZ-41 did not alter
melphalan’s effects on Y79 cells.
Conclusions: Use of KZ-41 to block NF-κB and activate P38MAPK
protects REC against melphalan-induced upregulation of ICAM-1
and apoptosis.
Commercial Relationships: Qiuhua Zhang, None; Jordan J.
Toutounchian, None; Charles R. Yates, RxBio, Inc (C), RxBio, Inc
(P), RxBio, Inc (R); Matthew W. Wilson, None; Jena J. Steinle,
None
Support: Knight Templar Eye Foundation (PI: Quihua Zhang); RPB
(PI:Chris Fleming); P30EY013080 (PI: Dianna Johnson)
253 Myopia I, AP
Monday, May 06, 2013 11:00 AM-12:45 PM
Exhibit Hall Poster Session
Program #/Board # Range: 1904-1918/B0271-B0285
Program Number: 1904 Poster Board Number: B0271
Presentation Time: 11:00 AM - 12:45 PM
The effect of horizontal eye movements on changes in central
refraction
Arne Ohlendorf1, Julia Gehrmann2, Rainer E. Sessner1. 1Technology
and Innovation, Carl Zeiss Vision, Aalen, Germany; 2Course of
Optometry, University of Applied Sciences Aalen, Aalen, Germany.
Purpose: The aim of the study was to measure the effect of
horizontal eye movements on changes in central refraction in myopic,
hyperopic and emmetropic subjects.
Methods: The spherical equivalent (SE) of the central refractive
error in 12 myopes (average SE: -2.2 ± 1.45 D; average age: 25 ± 3.8
years); 9 emmetropes (average SE: 0.1 ± 0.13 D; average age: 25 ±
4.23 years) and 5 hyperopes (average SE: 0.8 ± 0.98 D; average age:
28 ± 7.6 years) was measured using infrared eccentric
photorefraction. The room was darkened during the experiments and
the stimulus was a white LED in 1 m distance from the eye of the
subject. To induce horizontal eye movements, the subjects where
instructed to rotate their head left- or rightward and to make
compensatory eye rotations with the measured eye (right or left eye)
to keep the stimulus that was presented centrally in focus. Refraction
measurements were conducted over a range of ±30° (nasal and
temporal visual field) in steps of 5°.
Results: Changes in the spherical equivalent of the central refractive
error occurred in all tested refractive groups. The subjects showed
shifts towards more myopic as well as hyperopic refractions.
Statistically significant changes showed no clinically relevant effect.
Conclusions: Horizontal eye movements have no effect on changes
central refraction. Therefore, there is no evidence that horizontal eye
movements have an effect on the progression of myopia.
Commercial Relationships: Arne Ohlendorf, Carl Zeiss Vision
(E); Julia Gehrmann, None; Rainer E. Sessner, Carl Zeiss Vision
(E)
Prognostic factors associated with pathological myopia in young
patients with high myopia
Tae Yokoi1, 2, Muka Moriyama1, Noriaki Shimada1, 2, Natsuko
Nagaoka1, Kaori Kasahara1, Kosei Shinohara1, Yuichiro Tanaka1,
Yuichiro Kaneko1, Takashi Tokoro1, Kyoko Ohno-Matsui1.
1
Departments of Ophthalmology & Visual Science, Tokyo Medical
and Dental University Graduate School of Medicine and Dental
Sciences, Bunkyou-ku, Japan; 2Department of Ophthalmology,
Kawaguchi Municipal Medical Center, Kawaguchi-shi, Japan.
Purpose: Various kinds of retinochoroidal complications, such as
chorioretinal atrophy and choroidal neovascuralization, are caused by
pathological myopia. Most of these complications occur in middleaged and elderly patients. However, to the extent of our knowledge
there are no predictive factors associated with development of
pathological changes in young patients with high myopia. Thus, the
aim of this study is to find predictive factors for pathological myopia.
Methods: We retrospectively analyzed the medical records of highly
myopic patients who were <30 years old at initial visit with a follow
up period >10 years. According to the age at initial visit, patients
were divided into 2 groups (Group1: <19 years old; Group2: 20-29
years old). Considering fundus photographs at final visit, we also
subdivided each group into 2 subgroups; progressive [presence of
diffuse chorioretinal atrophy (DA), patchy chorioretinal atrophy
(PA), lacquer cracks, or choroidal neovascuralization], nonprogressive [no fundus lesions (N) or tessellated fundus (T) only]. In
addition, patients’ demographics, clinical characteristics and fundus
findings at initial visit were compared between each subgroups.
Results: Finally, 138 eyes of 71 patients were included. In Group1
(74 eyes of 38 patients), 38 eyes (51.4%) were classified as
progressive [DA: 36 eyes (94.7%); PA: 5 eyes (13.2%)], and 36 eyes
(48.7%) were classified as non-progressive [N: 12 eyes (33.3%); T:
24 eyes (66.7%)]. In Group2 (64 eyes of 33 patients), 32 eyes
(50.0%) were classified as progressive [DA: 31 eyes (96.9%); PA: 7
eyes (21.9%)], and 32 eyes (50.0%) were classified as nonprogressive [N: 7 eyes (21.9%); T: 25 eyes (78.1%)]. In both Groups,
spherical equivalents were more myopic (both Groups, P<0.001), and
axial lengths were longer (both Groups, P<0.001) in progressive
subgroups than in non-progressive ones. In Group2, visual acuities
were worse (P=0.021) and optic disc shapes were more oval
(P=0.034) in progressive subgroup than in non-progressive one.
Importantly, 25 eyes (69.4%) in progressive subgroup of Group1 and
30 eyes (93.8%) in progressive subgroup of Group2 showed a DA
mainly around the optic disc, even at initial visit.
Conclusions: Presence of a DA around optic disc at a young age may
be a strong indicator for development of pathological myopia. In
addition, more severe myopia and longer axial length may be related
to pathological changes.
Commercial Relationships: Tae Yokoi, None; Muka Moriyama,
None; Noriaki Shimada, None; Natsuko Nagaoka, None; Kaori
Kasahara, None; Kosei Shinohara, None; Yuichiro Tanaka, None;
Yuichiro Kaneko, None; Takashi Tokoro, None; Kyoko OhnoMatsui, None
Axial Length and its Associations in an Adult Japanese
Population: The Funagata Study
Akira Sugano1, Yusuke Tanabe1, Koko Saito1, Kei Homma1, Ryo
Kawasaki1, 2, Takeo Kato3, Takamasa Kayama4, 5, Hidetoshi
Yamashita1, 5. 1Ophthalmology, Yamagata University Sch of Med,
Yamagata, Japan; 2Department of Public Health, Yamagata
University Sch of Med, Yamagata, Japan; 3Department of Neurology,
Hematology, Metabolism, Yamagata University Sch of Med,
Yamagata, Japan; 4Department of Neurosurgery, Yamagata
University Sch of Med, Yamagata, Japan; 5Global COE Program for
Medical Sciences, Japan Society for the Promotion of Science,
Yamagata, Japan.
Purpose: To describe the distribution of axial length (AxL) and its
associations in an adult Japanese population sample, the Funagata
Study.
Methods: The Funagata study is a population based epidemiologic
study of Japanese aged 35+ years. We used 1219 data from
participants of ophthalmological examination in 2010-2012 (33.19%
of eligible). AxL was obtained using a partial coherence laser
interferometry (OA-1000, TOMEY cooperation, Nagoya, Japan).
Body height and weight were measured while subject were wearing
light clothing without shoes. Body mass index BMI was calculated as
body weight (kg) divided by the square of body height (m). Other
information including physical activity, smoking, frequency of
alcohol consumption, and time spent watching television per day
were collected from a self-reporting questionnaire. Risk associations
of the AxL were determined using multiple linear regression models.
We used logarithm transformed AxL in this analysis, because AxL
was deviated from normal distribution (Kolmogorov-Smirnov test
p<0.01). We first analyze the data for right eyes, and then repeated
the analysis for left eyes to confirm the result from right eyes.
Results: In multivariable adjusted model, younger age (0.002mm per
10 year of age, 95%CI 0.001 to 0.004), taller body height (0.012mm
per 10cm in body height, 95%CI 0.003 to 0.021), and lower level of
physical activity were independently associated with longer axial
length. The body height-specific AxL and 95% range for <149 cm,
150-159 cm, 160-169 cm, and >170 cm were 21.04-25.82 mm,
21.74-26.28 mm, 21.95-26.74 mm, and 23.38-27.61 mm,
respectively.
Conclusions: Younger age, taller body height, and lower physical
activity were associated with longer AxL. Longitudinal study
examining association between lower physical activity and longer
axial length are warranted because this might identify potential
intervention to prevent myopia.
Commercial Relationships: Akira Sugano, None; Yusuke Tanabe,
None; Koko Saito, None; Kei Homma, None; Ryo Kawasaki,
None; Takeo Kato, None; Takamasa Kayama, None; Hidetoshi
Yamashita, Senju (C), Senju (P)
Support: Global COE program of the Japan Society for the
Promotion of Science
Outer nuclear layer and retinal layer ratios in myopic and
emmetropic populations using spectral domain optical coherence
tomography
Christopher A. Clark, Ann E. Elsner, Benjamin Konynenbelt, Joel A.
Papay, Toco Y. Chui. School of Optometry, University of Indiana,
Bloomington, IN.
Purpose: Cone photoreceptors have been shown to vary considerably
between individuals both centrally and peripherally. When
incorporating retinal growth signals such as defocus, this variability
may influence refractive development. The purpose of this study is to
examine differences in photoreceptor density across the retina in
myopic and emmetropic populations.
Methods: 80 subjects right eyes were imaged for this study (age
range: 22 to 34, refractive error: -10 to +5.00.) Thirty degrees OCT
(Spectralis, Heidleberg) images were collected in a radial pattern
along with peripheral refraction (Grand Seiko) and peripheral axial
length measurements (IOLmaster, Zeiss.) OCT images were
segmented with custom software to determine the thickness of the
total retina, ONL, OPL, and INL. Lateral magnification and tilt
effects were corrected. In addition to thickness measurements, a ratio
was calculated for the layers by dividing peripheral thickness by each
subjects central thickness, thereby reducing individual variation.
Statistics were performed using repeat measures ANOVA in SPSS
(IBM.)
Results: Total retinal thickness (TRT) and ONL was relatively
thinner for myopic subjects compared to emmetropic and hyperopic
subjects (P = 0.007.) This effect was larger in the periphery with no
effect centrally. ONL differences between refractive groups were
relatively small (average difference of 21 microns.) Likewise, longer
axial lengths resulted in thinner TRT and ONL in the periphery but
no effect centrally (P = 0.02.) There was a negative correlation
between a subject’s TRT or ONL and the ratio of peripheral to central
TRT or ONL (P < 0.00,) except for the longest eyes (P = 0.56).
Central and peripheral thicknesses were uncorrelated.
Conclusions: Retinal and ONL thickness decrease with axial length
and refractive error. The primary hypothesis for this is due to stretch
factors during elongation though a longitudinal study is needed to
confirm this. An alternative hypothesis would be that the signal
strength from denser vs. sparses photoreceptors and retinal neurons
could influence axial length and refractive development. Further,
ONL and TRT are controlled by at least 2 factors, total numbers and
foveal specialization.
Commercial Relationships: Christopher A. Clark, None; Ann E.
Elsner, Aeon Imaging, LLC (I), Aeon Imaging, LLC (F), Aeon
Imaging, LLC (P); Benjamin Konynenbelt, None; Joel A. Papay,
None; Toco Y. Chui, None
Support: NIH Grant EY007624, T35-EY013937, P30 EY019008,
K23 EYO22064
Extended Clinical Phenotype of Dome-Shaped Macula
Marie-Helene Errera, Michel Michaelides, Pearse A. Keane,
Anthony T. Moore, Jonathan Yeoh, Derek Chan, Catherine A. Egan,
Praveen J. Patel, Adnan Tufail. NIHR Biomedical Research Centre
for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust
and UCL Institute of Ophthalmology., London, United Kingdom.
Purpose: To describe the phenotype, associations and complications
of dome-shaped macula with the use of spectral domain optical
coherence tomography (OCT) scan and B scan ultrasound.
Methods: Fifty-eight eyes of 36 patients were retrospectively
identified as having OCT features of dome-shaped macula (DSM).
All subjects had undergone enhanced depth imaging (EDI) OCT
choroidal imaging as part of the scanning protocol. Fifteen patients
underwent a B scan ultrasound to determine axial length and
posterior coat thickness. Choroidal thickness and retinal thickness
was determined with OCT, with scleral thickness subsequently
calculated by subtraction from the ultrasound derived posterior coat
thickness.
Results: Dome-shaped macula was associated with myopia (81 %).
The underlying clinical diagnosis was variable, including: central
serous chorioretinopathy (CSCR)-like entity (32.7%), chorioretinal
neovascularization (CNV) (20.7%), and inherited retinal disorders
(19%). The subfoveal choroidal thickness of the 9 highly myopic
eyes with a CSCR-like phenotype (239 ± 144µm) was thicker than
the 25 eyes without CSCR (153 ± 161µm) (p=0.169).
Conclusions: This study confirms the previous finding in highly
myopic eyes with dome-macula of the presence of local scleral
thickening and in addition the novel observation of a thickened
choroid if CSCR is present. The observation of the thickened choroid
may have therapeutic implications. We also expand the associations
of dome-shaped macula to include eyes with hypermetropia and
acquired disease, and also inherited retinal disease.
Commercial Relationships: Marie-Helene Errera, None; Michel
Michaelides, Novartis (R); Pearse A. Keane, None; Anthony T.
Moore, None; Jonathan Yeoh, None; Derek Chan, None;
Catherine A. Egan, Bayer (S), Oculogics (S), Novartis (S), Allergan
(S), Novartis (F); Praveen J. Patel, Allergan (R), Bayer (C),
Novartis UK (C), Heidelberg UK (R), Topcon UK (R),
Thrombogenics (C); Adnan Tufail, Allergan (C), Bayer (C), GSK
(C), Oculogics (C), Pfizer (C), Thrombogenics (C), Amakem (C),
Heidelberg Engineering (R), Novarits/Alcon (C), Sanofi/Genzyme
(C)
Thinning of Lens Thickness Might Be Responsible for Myopia
Development
Ji C. He. New England College of Optometry, Boston, MA.
Purpose: Recent myopia research suggests that peripheral hyperopia
is a risk factor for myopia development and progression, but the
sources responsible for the peripheral hyperopic refraction have not
been well explored. In a previous modeling (He, ARVO 2010),
deepening of anterior chamber depth (ACD) was found to make
relative peripheral refraction more hyperopic, and it, therefore, might
be a process leading to myopia development. During the eye
development, however, ACD deepening is often linked to a thinning
process of the lens thickness (LT). The purpose of this study was to
theoretically model the effect of LT thinning on relative peripheral
refraction.
Methods: Ray-tracing on a model eye (Navarro et al. 1985) was
performed by using a self-developed MatLab program to calculated
Zernike aberrations up to 5th order at eccentricities of visual field
across -60 to + 60 deg along the horizontal meridian. LT was
assumed to thin from 4.0mm to 3.5mm in three thinning patterns: (1)
without any ACD deepening; (2) with ACD deepening and posterior
lens surface (PLS) forward moving together; (3) with ACD
deepening but no PLS movement.
Results: LT thinning without any ACD deepening (pattern 1) caused
relative spherical equivalent error at peripheral field become more
myopic. Opposite hyperopic change in relative peripheral refraction
was found if the LT thinning was coupled with same amount of ACD
deepening (pattern 3). Less hyperopic change was found when the LT
was thinned in pattern2. For example, a 0.1mm LT thinning in pattern
1 caused the relative refraction at 40 deg become -0.20D more
myopic. But, 0.1mm LT thinning in pattern 3 resulted in 0.18D
hyperopic shift at 40 deg. The 0.1mm LT thinning made difference
between the two thinning patterns in about 0.38D at 40 deg.
Meanwhile, the change in relative peripheral refraction increases with
eccentricity, so that 0.1 mm LT thinning produced difference of
0.70D at 60 deg between the two patterns.
Conclusions: The results suggest that effect of LT thinning on
relative refraction at peripheral field depends on its thinning pattern.
While LT thinning without ACD change could be a protective factor
for maintaining emmetropia, LT thinning with ACD deepening might
a risk factor for myopia development due to the more hyperopic
change in relative peripheral refraction.
Commercial Relationships: Ji C. He, None
Increased Lens Opacities Associated with the Use of Topical
Atropine for Myopia Retardation in Children
Francine P. Yang1, Fong Yee Foo1, Seo Wei Leo1, 2. 1Ophthalmology,
Tan Tock Seng Hospital, Singapore, Singapore; 2Dr Leo Adult and
Paediatric Eye Specialist Pte Ltd, Singapore, Singapore.
Purpose: To describe the clinical effects of atropine eye drops for
myopia retardation in rapid progressors
Methods: Retrospective review of 36 eyes of 18 children who were
treated with atropine for myopia retardation. 28 eyes received topical
atropine 1% while 8 eyes received atropine 0.5%. All children were
prescribed photochromatic progressive addition lenses. Data
collected included demographic data, yearly measurements of axial
length, spherical equivalent based on cycloplegic refraction and the
presence of lens opacities.
Results: The mean age was 8.8 ± SD 2.2 years. 61.1% of the patients
were female and 38.9% were male. Patients were predominantly
Chinese (94.4%), reflecting the demographics of the local population.
66.7% had a positive family history of myopia and 22.2% had a
positive family history of retinal detachment. Before treatment,
baseline myopia progression was -2.04D/year, with a mean axial
length (AL) of 25.12mm and mean spherical equivalent (SE) of 4.77D. After the first year of treatment, mean AL and mean SE were
25.15mm and -4.86D respectively. After 2 years, the mean AL was
25.42mm with a mean SE of -5.4D. Myopia progression was
significantly reduced to -0.09D/year (p<0.001) and -0.43D/year
(p<0.001) after 1 year and 2 years of treatment respectively.
Peripheral lens opacities were present in 5.6% of eyes before
treatment; this significantly increased to 13.9% of eyes after 1 year
(p=0.049), and 22.2% of eyes after 2 years of treatment (p<0.001).
However, none were visually significant. There were no other
reported systemic or local adverse effects such as allergic
conjunctivitis or ocular irritation.
Conclusions: Topical atropine significantly reduced the rate of
progression of myopia but an increase in the presence of peripheral
lens opacities was noted following treatment, highlighting the need
for careful monitoring.
Commercial Relationships: Francine P. Yang, None; Fong Yee
Foo, None; Seo Wei Leo, None
Short-term adaptation of accommodative lag, facility and phoria
in myopes fitted with multifocal contact lenses
Jerome Ozkan1, Ravi C. Bakaraju1, Cathleen Fedtke1, Jiyoon Chung1,
Klaus Ehrmann1, 2, Darrin Falk1, Arthur Ho1, 2, Brien A. Holden1, 2.
1
Brien Holden Vision Institute, Sydney, NSW, Australia; 2School of
Optometry and Vision Science, University of New South Wales,
Sydney, NSW, Australia.
Purpose: Multifocals have been proposed as a method of controlling
myopia progression. Adaptation time to multifocal contact lenses, in
terms of accommodative response, has not been explored in depth. A
study was conducted to investigate if accommodative response
adaptation is dependent on the type of multifocal contact lens.
Methods: Prospective, subject-masked clinical trial in which 40
participants were randomised to two of four lenses of different
multifocal designs (Proclear Multifocal [Distance and Near], Air
Optix Aqua Multifocal, PureVision Multifocal) and a single vision
control lens (Air Optix Aqua) on a bilateral, daily wear basis with a
1-week washout period between lenses. Subjects wore each allocated
lens for a minimum of 8 days (maximum 14) over 4 scheduled visits
(baseline and 3 follow-up visits) with a 1-week washout between lens
types. Accommodative lag was assessed at all visits with the
EyeMapper at +1D, -2D, -3D, -4D and -5D demand settings.
Accommodative facility was assessed by the number of flips of ±2D
per minute and horizontal phoria was assessed with a Howell phoria
card at 33 cm and 300 cm.
Results: A significant difference was found with Proclear Multifocal
Distance lenses between baseline (V1) and the first follow-up visit
(V2) for measurements performed at an accommodative demand of
+2D (-1.66 ± 0.68D vs -2.24 ± 1.02D, p= 0.019, V1 vs V2
respectively). Linear mixed model test showed an increase in
accommodative facility over the 4 visits with the Air Optix Aqua
Multifocal and PureVision Multifocal lenses (p= 0.003). There was
no significant change in distance and near horizontal phoria state over
the 4 visits (p= 0.973).
Conclusions: Adaptation differences were not consistently found in
the static accommodative measures (lag and phoria) but were found
in dynamic measures (facility), the latter may be related to learning
effects. We speculate that accommodative adaptation is unlikely to
occur with long-term multifocal wear.
Commercial Relationships: Jerome Ozkan, None; Ravi C.
Bakaraju, None; Cathleen Fedtke, None; Jiyoon Chung, None;
Klaus Ehrmann, None; Darrin Falk, None; Arthur Ho, None;
Brien A. Holden, Allergan (F), AMO (I)
Clinical Trial: ACTRN12611001004954
Intraocular Pressure in Myopic Eyes during the Water Drinking
Test
Barbara M. Junghans1, 2, Chia-Jung Chang1, Angela Siu1, Natalia C.
Soesanto1, Megan F. Tu1, Melanie J. Murphy2, Sheila G. Crewther2, 1.
1
School of Optometry and Vision Science, Univ of New South
Wales, UNSW Sydney, NSW, Australia; 2School of Psychological
Science, La Trobe University, Melbourne, VIC, Australia.
Purpose: The rapidly increasing prevalence of myopia worldwide
has been linked to increasing complexity of modern urban lifestyles.
In addition, animal as well as pilot human studies have suggested that
stress-induced physiological dysregulation of trans-retinal fluid
outflow may play a role in ocular growth and the enlarged myopic
eye. Thus, this study aimed to investigate differences between
myopes and non-myopes in ocular fluid regulation and cortisol levels
after the rapid ingestion of water.
Methods: Forty-one healthy adults of mean age 22.6±1.6yrs were
randomly assigned either to a control group or to drink 1 liter of
water over 10mins. Refractive error (Shin Nippon open-field
autorefractor), intraocular pressure (IOP) (Tonopen), central corneal
thickness (Sonogage), axial length, anterior chamber depth (IOL
Master) and salivary cortisol (Salivettes) were measured at baseline,
T=20 and T=40mins. Twenty-five subjects were myopes (mean 3.50±2.00DS).
Results: As expected, myopic eyes were significantly longer
(p<0.01). Water loading caused a significant increase in IOP by T=20
(3.9±0.6mmHg) that was similar for both refractive subgroups.
However, IOP returned to within normal range by T=40 in nonmyopes whereas for myopes the increased levels of IOP remained
until at least T=40 (p<0.001). No significant associations were
observed between water loading and central corneal thickness, axial
length, anterior chamber depth or salivary cortisol.
Conclusions: The Water Drinking Test itself was not sufficiently
stressful to show significant change in cortisol levels that may
indirectly affect fluid regulation within the eye. However, myopes
demonstrated altered ocular fluid dynamics which could suggest that
myopic eyes have reduced ocular fluid regulation. This fluid
dysregulation may indirectly influence volumetric enlargement of the
globe.
Commercial Relationships: Barbara M. Junghans, None; ChiaJung Chang, None; Angela Siu, None; Natalia C. Soesanto, None;
Megan F. Tu, None; Melanie J. Murphy, None; Sheila G.
Crewther, None
Optical coherence tomography-based evaluation of peripapillary
tilting in highly myopic eyes
Tomoko Asai1, Yasushi Ikuno1, Masahiro Akiba2, Tsutomu Kikawa2,
Yukari Jo1, Shinichi Usui1, Kohji Nishida1. 1Ophthalmology, Osaka
Univ Grad Sch of Med, Suita, Japan; 2TOPCON Corp., Itabashi,
Japan.
Purpose: Optic disc tilting is reportedly associated with glaucoma in
highly myopic eyes. We attempted to evaluate the peripapillary tilting
in highly myopic eyes using customized software and swept-source
optical coherence tomography (SS-OCT).
Methods: We retrospectively analyzed SS-OCT images of a total of
105 eyes of 105 subjects (32 male and 73 female, mean age of 54.6
years) with high myopia (axial length > 26mm or refractive error < -6
diopter) who visited the Department of Ophthalmology, Osaka
University Hospital. Glaucoma patients were excluded. SS-OCT
images were acquired using 3.4mm diameter peripapillary circle scan
mode. The scan was divided into 24 segments. The segments were
alphabetically named as A to L from temporal 0 degree and then,
superior, and nasal. The opposite segments were named as A’ to L’
similarly. Retinal pigment epitherium (RPE) were automatically
traced and the height was calculated by customized software. The
vertical distance (pixels) between mean RPE height in each sector
and mean RPE height of overall sectors were defined as
perpipapillary tilting index (PTI). Correlation between PTI and
peripapillary choroidal thickness (ChT), size of peripapillary atrophy
area from color fundus photograph, and demographic data were
investigated.
Results: Mean refractive error (RE) was -10.0±5.4D, and mean axial
length (AL) was 28.7±1.8mm. PTI was lowest at K’ (inferotemporal) of -713 pixel. There was significant negative correlation
between PTI and age (P<0.05). PTI did not correlate RE or AL. Age
positively correlated with RE (P<0.01), but did not correlated with
AL (P=0.07). In female, minimum PTI was significantly lower than
male at sector G, H, I, J, K(P=0.05, 0.02, 0.01, 0.01, 0.03). Gender
did not correlate with age, RE or AL (P=0.42, 0.34, 0.23). Minimum
PTI negatively correlated with PPA(P<0.05), and tended to be
coefficient with minimum ChT (P=0.06).
Conclusions: In highly myopic eyes, peripapillary tilting was
remarkable in female, high age and low RE. The more tilt was
remarkably the more choroidal thinning and peripapillary atrophy
were extent.
Commercial Relationships: Tomoko Asai, None; Yasushi Ikuno,
Topcon (F), TOMEY (F); Masahiro Akiba, Topcon Corp. (E);
Tsutomu Kikawa, TOPCON Corp. (E); Yukari Jo, None; Shinichi
Usui, None; Kohji Nishida, Alcon (C), Alcon (F), HOYA (F), Senju
(F), Pfizer (F), Santen (F), Osaka University (P)
Contributions of Eye Power and Optical Eye Length to
Emmetropization during Lens Induced Myopia and Recovery in
the Chick Eye
Zheng Shao1, 2, Kaitlin Bunghardt1, Marsha L. Kisilak1, 3, Elizabeth L.
Irving1, 3, Melanie C. Campbell1, 3. 1Physics and Astronomy,
University of Waterloo, Waterloo, ON, Canada; 2Guelph Waterloo
Physics Institute, Waterloo, ON, Canada; 3School of Optometry and
Vision Science, University of Waterloo, Waterloo, ON, Canada.
Purpose: The refractive error induced by emmetropization to lens
defocus is primarily due to changes in ocular length. However, during
normal growth and diurnal mean ocular refraction (MOR) variation,
we have shown that changes in MOR are due to both changes in
optical length and optical power. We determine the contributions of
eye power and optical length to emmetropization during myopia
induction and recovery from lens induced myopia.
Methods: Two experimental data sets (20 chicks) with eye length
(ultrasound) and MOR (retinoscopy or Hartmann-Shack) were
considered. All were unilaterally treated with a -15 D goggle from the
day of hatching to day 7. The goggle was removed and measurements
continued up to day 15. Calculations of dioptric length and eye power
were made as a function of age and treatment. We tested the
assumption that the power in the goggled eye was the same as in the
control eye.
Results: Changes in optical length and eye power for the control eye
were similar to their behaviour during normal growth (ARVO 2012).
MOR of the goggled eye showed a rapid, almost complete
emmetropization to the -15 D goggle by day 7 primarily due to eye
length changes. However, the power of the goggled eye decreased
faster than the control. The dioptric length for the previously goggled
eye was not significantly different between days 7 and 8 and days 8
and 9, but power decreased until day 8. Both decreased in the control
eye. Beginning between days 7 and 8, the difference in power
between the goggled and control eyes began to decrease significantly.
After goggle removal, recovery from myopia was complete by day 9
with resulting dioptric length and eye power not significantly
different from the control eye (paired t-test). After day 9, the goggled
eye emmetropized similarly to the control eye.
Conclusions: Emmetropization to hyperopic defocus is mainly due to
an increase in optical eye length, but power reduces somewhat. Eye
power and optical length are not significantly different from the
control eye within 2 days of goggle removal. During this recovery
from myopia, optical length remains relatively constant in the
goggled eye while power initially decreases. Differences in both eye
power and optical length between goggled and control eyes decrease.
Emmetropization following goggle removal is more rapid than to the
goggle.
Commercial Relationships: Zheng Shao, None; Kaitlin
Bunghardt, None; Marsha L. Kisilak, None; Elizabeth L. Irving,
None; Melanie C. Campbell, CanCog Technology (F), University of
Waterloo (P)
Support: NSERC, CFI, CRC
Pattern ERG in chicks
Lisa A. Ostrin, Christine F. Wildsoet. School of Optometry,
University of California Berkeley, Berkeley, CA.
Purpose: The pattern (p)ERG is believed to arise from the inner
retinal. The aim of this study was to characterize the chick pERG and
effects of optical defocus, diffusing blur, and optic nerve section
(ONS).
Methods: Monocular pERG was recorded in 18 normal White
Leghorn chickens (ages 2-3 wks) using a checkerboard pattern
stimulus at 50 cm. Chicks were anesthetized, eyelids held open with a
speculum, and DTL fiber electrodes positioned along the lower
cornea. Parameters included 8 spatial frequencies (SFs: 0.1 to 4.4 c/d,
100% contrast), 13 contrast levels (1-100%, 0.2c/d checkerboard),
and 8 temporal reversal frequencies (0.5 to 20 Hz). Effects of optical
defocus (-10, -5, +5 & +10 D lenses) and diffusing blur (0.6, 0.2,
<0.1 & LP Bangerter filters) were also examined. For 4 additional
chicks that underwent monocular ONS, binocular flash and pERG
responses were measured 1 and 3 weeks post surgery, complemented
with SD-OCT imaging to evaluate nerve fiber loss.
Results: The response to 1Hz, 100% contrast stimuli showed
characteristic positive and negative going waveforms at 43 ms (P50)
and 75 ms (N95), respectively, for SFs between 0.1 to 4.4 c/d, with a
maximum P50 amplitude of 21.9±2.5µV (0.13 c/d), and minimum of
6.9±5.2µV (4.4 c/d), which elicited a response in half of the chicks
tested. Amplitudes decreased across all SFs with both optical defocus
and diffusing blur. Contrast levels of 5-10% and higher yielded
measureable P50 responses, with amplitudes plateauing at 50%
contrast. Responses were transient and monophasic for temporal
frequencies from 0.5 to 5 Hz, with higher frequencies (10 & 20 Hz)
yielding steady state responses. While thinning of the nerve fiber
layer was evident by SD-OCT one week after ONS, both flash ERG
and pERG response amplitudes remained near normal three weeks
after the surgery.
Conclusions: Maximum pERG responses in the chick are obtained
with a SF of 0.13 c/d, 50% or great contrast and 1-5 Hz reversal. The
SF curve had a low pass filter shape, with a cut-off ~4.4 c/d. P50
amplitude increased linearly with contrast over the 5-50% range.
Irrespective of its sign, defocus decreased the P50 amplitude, as did
decreases in spatial contrast, with the responses to higher spatial
frequencies being most sensitive. In further ONS experiments, the
possibility that low contrast settings are more sensitive to subtle
changes in retinal integrity will be evaluated and experiments
extended beyond three weeks to look for late changes in function.
Commercial Relationships: Lisa A. Ostrin, None; Christine F.
Wildsoet, None
Support: NIH K08 EY 022696 to LO, NIH R01 EY12392 to CF
Manual segmentation of choroidal volume in emmetropic and
high myopic eyes
Su-Na Lee1, 2, Giulio Barteselli1, Sharif El-Emam1, Huiyuan Hou1,
Dirk-Uwe G. Bartsch1, Lingyun Cheng1, William Freeman1.
1
Ophthalmology, Shiley Eye Center, UCSD, La Jolla, CA;
2
Ophthalmology, Eulji University Hospital, Daejeon, Republic of
Korea.
Purpose: To compare choroidal volume between emmetropic and
high myopic eyes and to assess the variations of choroidal volume
among eyes with non-pathologic high myopia, pathologic high
myopia with vitreoretinal pathology and choroidal neovascularization
(CNV)
Methods: We retrospectively reviewed imaging studies of 137 eyes
of 86 patients who underwent choroidal volume measurement using
enhanced depth imaging (EDI)-OCT. Forty four eyes were
emmetropic (axial length: ≥ 23.5 mm and < 24.5 mm) and 93 eyes
were high myopic (axial length: > 26.5 mm). High myopic eyes were
divided into high myopia without pathology (51 eyes), high myopia
with vitreoretinal pathology (30 eyes), and high myopia with CNV
(12 eyes). The EDI-OCT protocol used to assess the choroidal
volume consisted in 31 high-resolution B-scans centered on the
fovea. The choroid was segmented manually from the hyperreflective line of the retinal pigment epithelium to the chorio-scleral
junction on SD-OCT by two retina specialists. Choroidal volume map
and measurements were obtained applying the 6-mm diameter grid
used by the Early Treatment Diabetic Retinopathy Study (ETDRS).
Results: Mean subfoveal and total choroidal volume were 0.24 ±
0.07 mm3 and 7.67 ± 2.15 mm3 in emmetropic eyes, 0.16 ± 0.06
mm3 and 5.60 ± 1.77 mm3 in high myopic eyes without pathology,
0.08 ± 0.04 mm3 and 3.01 ± 1.26 mm3 in high myopic eyes with
vitreoretinal pathology, 0.09 ± 0.05 mm3 and 3.29 ± 1.41 mm3 in
high myopic eyes with CNV. After adjusting for age and sex,
subfoveal and total choroidal volume were lower in high myopic eyes
without pathology than emmetropic eyes (both P<0.0001). Subfoveal
and total choroidal volume were lower in high myopia with
vitreoretinal pathology and high myopia with CNV than high myopia
without pathology (both P<0.01) after adjusting for age, sex, and
axial length. Among the 4 ETDRS quadrants, superior quadrant had
the greatest choroidal volume in emmetropia (2.07 ± 0.71 mm3) and
high myopia without pathology (1.54 ± 0.62 mm3), while the nasal
quadrant had the lowest choroidal volume in emmetropia (1.62 ± 0.69
mm3) and high myopia without pathology (1.08 ± 0.59 mm3).
Conclusions: We concluded that the choroid is thinner in high
myopic eyes than emmetropic eyes, but eyes with coexisting myopic
pathology have more severe thinning.
Commercial Relationships: Su-Na Lee, None; Giulio Barteselli,
None; Sharif El-Emam, None; Huiyuan Hou, None; Dirk-Uwe G.
Bartsch, None; Lingyun Cheng, Spinnaker Biosciences (C);
William Freeman, OD-OS, Inc. (C)
Strain differences in mouse lens refractive indices when
measured with OCT
Christopher C. Tan1, Han na Park1, Jacob G. Light1, Kip Lacy2,
Machelle T. Pardue1, 2. 1Department of Ophthalmology, Emory Univ
School of Med, Atlanta, GA; 2Rehab Center of Excellence, Atlanta
VA Medical Center, Atlanta, GA.
Purpose: Many labs use optical coherence tomography (OCT) to
determine ocular parameters in the mouse eye when studying
refractive development. The optical linear distance obtained with the
OCT is converted to geometrical linear distance using the refractive
index of the tissue. Since the optical power of the nocturnal mouse
eye is largely dependent on the large ocular lens, many published
studies have used the refractive index value of 1.433 for C57BL/6J
mouse lenses reported by Remtulla and Hallett (Vision Res 25:21-31;
1985) using the Abbé refractometer. Calculations from the mouse
paraxial schematic eye model suggest that refractive index increased
from 1.568 to 1.605 between 22 and 100 days in C57BL/6J mice
(Schmucker & Schaeffel, Vision Res 44:1857-67; 2004 ). The
purpose of this study was to measure the refractive index of isolated
mouse lenses across age and in different strains using OCT.
Methods: Lenses were carefully dissected from C57BL/6J wild-type
(C57) and nob mice eyes at 4, 10, and 16 weeks of age. Nob mice
have an ON pathway defect caused by nyx mutation and have
previously been shown to be more susceptible to form deprivation
myopia (Pardue et al., IOVS 49:706-12; 2008). Lens thickness (LT)
and refractive index (RI) was measured using OCT, as reported by
Uhlorn et al. for human ocular lenses (Vision Res 48:2732-8; 2008).
Briefly, RI was calculated from optical length differences in
Dulbecco’s medium with and without the mouse lens present.
Results: RI of the mouse lens did not change across age in either
strain. However, nob mice had overall higher lens RI than C57 mice
(Ave ±SEM: 1.433 ±0.004 vs 1.420 ±0.003, ANOVA main effect,
p=0.011). LT increased with age in both strains from 1.253 ±0.01 to
1.471 ±0.01 mm (ANOVA main effect, p<0.001) but was not
significantly different between strains.
Conclusions: C57 mice had lower ocular lens RIs than that reported
previously for the mouse eye using refractometry and both strains had
lower RIs than that calculated with the schematic eye model.
Contrary to the schematic eye calculations, mouse lens RI was stable
across age. As expected, lens thickness increased with age; however,
no change was detectable between nob and C57 mice. These data
suggest that mouse lens RI may need to be more carefully determined
in order to accurately measure ocular parameters with OCT
techniques and make comparisons between strains.
Commercial Relationships: Christopher C. Tan, None; Han na
Park, None; Jacob G. Light, None; Kip Lacy, None; Machelle T.
Pardue, None
Support: NIH EY016435 (MTP), NIH P30 EY006360,
Departmental grant from Research to Prevent Blindness, and the
Department of Veterans Affairs
Isoflurane and lid retractors affect the optics of the chick eye
Marsha L. Kisilak1, 2, Kaitlin Bunghardt1, Vivian Choh2, Elizabeth L.
Irving2, Melanie C. Campbell1, 2. 1Physics & Astronomy, University
of Waterloo, Waterloo, ON, Canada; 2School of Optometry and
Vision Science, University of Waterloo, Waterloo, ON, Canada.
Purpose: Isoflurane affects blood flow and repeated use in chicks
slows growth of the eye. Lid retractors, used with anesthetic, may
deform the cornea. We explored effects of isoflurane and lid
retractors on the eye’s optics.
Methods: 23 chicks were obtained on the day of hatching. Hartmann
Shack measurements were taken on all eyes. Axial length was
measured with A scan ultrasound. Refractive error and higher-order
aberrations (HORMS) were analyzed at constant pupils. On day 0, 11
chicks were measured sequentially: 1) awake without lid retractors,
2) awake after recovery (1 min) from brief exposure to isoflurane (2
min), 3) with lid retractors under isoflurane. Differences between
conditions were detected using paired t tests or Wilcoxon Signed
Rank tests for non-normal data. Of the remaining chicks, 6 awake
and 6 chicks with lid retractors under isoflurane were measured.
Then, right eyes of all 12 birds were goggled with a -15D lens.
Measurements were repeated on days 2, 4, 7. Data were examined for
differences in awake vs under isoflurane with lid retractors, lenses
worn and time (ANOVA).
Results: On day 0, in awake chicks, brief exposure to isoflurane gave
significantly increased pupil size and HORMS. There were
significant increases in pupil size, JCC45, HORMS, 3rd and 4th order
RMS and cylinder between those under anesthesia with lid retractors
and awake birds (either prior to or post anaesthesia). Mean ocular
refraction (MOR) decreased significantly in birds with lid retractors
under anesthesia vs awake birds pre-anaesthesia. There was no
significant difference in axial length in any condition.
Prior to goggling, cylinder and 4th order RMS were significantly
higher with lid retractors under isoflurane vs awake chicks. Post
goggling, with lid retractors under isoflurane, pupil size, JCC0,
HORMS, 3rd and 4th order RMS and cylinder increased significantly
across all eyes. JCC45 increased only in control eyes. HORMS
effects depended on the eye and day. For control eyes, pupil size
effects were dependent on day.
Conclusions: Isoflurane alone and with lid retractors change the
optical properties of the chick eye. This includes MOR, pupil size,
cylinder, its components and HORMS. Effects on HORMS depended
on age and effects on HORMS and JCC45 interacted with goggling.
Overall, lid retractors with isoflurane decreased MOR and image
quality worsened (from HORMS, JCC0 and JCC45).
Commercial Relationships: Marsha L. Kisilak, None; Kaitlin
Bunghardt, None; Vivian Choh, None; Elizabeth L. Irving, None;
Melanie C. Campbell, CanCog Technology (F), University of
Waterloo (P)
Support: NSERC Canada, CFI, CRC Canada
321 Anatomy
Tuesday, May 07, 2013 8:30 AM-10:15 AM
Program #/Board # Range: 3031-3065/C0194-C0228
Clinical and Histopathologic Characteristics in Floppy Eyelid
Syndrome
Karin U. Loeffler1, Nina Stratmann2, Teresa Maeueler2, Frank G.
Holz2, Martina C. Herwig1. 1Ophthalmology, Ophthalmic Pathology,
University of Bonn, Bonn, Germany; 2Ophthalmology, University of
Bonn, Bonn, Germany.
Purpose: Floppy eyelid syndrome (FES) is a serious cause of
therapy-resistent keratoconjunctivitis sicca. Nevertheless, it is a
frequently overlooked entity. During the last 2 years, we have treated
8 patients successfully by upper eyelid wedge resection and have
examined the excised tissue for specific histopathologic alterations.
Methods: Nine eyes from 8 patients were clincally diagnosed with
typical floppy eyelid. Clinical data were collected, and all affected
eyelids were - after exclusion of infectious or other causes and
intensive treatment with artificial tears - treated surgically with upper
eyelid wedge resection. - The eyelid wedge was submitted for
ophthalmopathologic evaluation and embedded in paraffin. In
addition to regular H&E and PAS stains, immunohistochemistry was
performed using an antibody to collagen type V (CollV; BioLogo,
1:200 ), to macrophage marker CD68 (DAKO, 1:50) and to vimentin
(DAKO, 1:500) and desmin (DAKO, 1:200). The immunoreaction
product was visualized using AEC as chromogen.
All sections were evaluated by 2 independent observers.
Results: All patients were male with a mean age of 50 years at
presentation (age range 39 to 69). Most were hypertensive, and in
several patients sleep apnea was known. Symptoms had usually been
present for some years, causing marked superficial keratitis despite
intense lubrication. After surgery, all of our patients described almost
immediate relief. With continuing use of lubricants, all but one
patient were actually free of symptoms. - Histologic findings revealed
tarsal atrophy (4/9), marked subepithelial inflammation (5/9),
intraepithelial inflammation (2/9), epithelial metaplasia with
elongated crypts and “buried” goblet cells (4/9), complete squamous
metaplasia with no residual goblet cells (1/9) and eosinophilic
infiltrates in a patient with atopic disease (2/9).
Immunohistochemistry did not show significant distinctive findings
when compared to normal controls.
Conclusions: Surgical intervention (upper eyelid wedge resection)
was confirmed as a very rewarding therapeutical procedure.
Histopathologic findings in floppy eyelid reveal some variation but
loss of (exposed) goblet cells and inflammation are the most
consistent findings. It remains unclear why FES is significantly
associated with sleep apnea and why mostly male patients are
affected.
Commercial Relationships: Karin U. Loeffler, None; Nina
Stratmann, None; Teresa Maeueler, None; Frank G. Holz,
Acucela (C), Allergan (C), Genentech (F), Heidelberg Engineering
(F), Zeiss (F), Novartis (F), Novartis (C), Optos (F), Merz (C), Bayer
(F), Bayer (C), Boehringer Ingelheim (C); Martina C. Herwig, None
Program Number: 3032 Poster Board Number: C0195
Spatial Relationship between Bruch’s Membrane Opening and
Lamina Cribrosa Determines Optic Disc Tilting
Pat-Michael Palmiero1, Sung Chul Park1, 2, Yiyi Liu1, 3, Camila F.
Netto1, Rafael L. Furlanetto1, Mohammed Al-Jumayli1, Jeffrey M.
Liebmann1, 4, Robert Ritch1, 2. 1Moise and Chella Safra Advanced
Ocular Imaging Laboratory, Einhorn Clinical Research Center, New
York Eye and Ear Infirmary, New York, NY; 2Department of
Ophthalmology, New York Medical College, Valhalla, NY; 3New
York Medical College, Valhalla, NY; 4Department of
Ophthalmology, New York University School of Medicine, New
York, NY.
Purpose: To investigate the association between tilted disc geometry
and location of the lamina cribrosa (LC) relative to Bruch’s
membrane opening (BMO).
Methods: Normal subjects with axial length less than 26.5 mm were
recruited. Serial horizontal and vertical enhanced depth imaging
optical coherence tomography (EDI OCT) B-scans of the optic nerve
head (interval between scans, ~30 μm) were obtained for one eye of
normal subjects. After reconstructing a 3-dimensional image of BMO
and the anterior LC insertion points, the distance between the
centroids of BMO and the LC was measured (Fig A). The direction
of lateral LC displacement from BMO was measured as an angle (θ1)
between the line connecting the two centroids and temporal
horizontal line (Fig B). Disc ovality index (longest disc
diameter/shortest disc diameter) was measured using infrared disc
photographs. The direction of disc tilting was measured as an angle
(θ2) between the axis of shortest disc diameter and the temporal
horizontal line (Fig C).
Results: 23 normal eyes (23 subjects) were included (mean age,
42±15 years). Mean distance between the centroids of BMO and the
LC was 222±93 (range, 70 to 368) µm. Mean disc ovality index was
1.14±0.08 (range: 1.00 to 1.28) mm. The distance between the two
centroids was significantly correlated with the disc ovality index
(p<0.001, R=0.739; Fig D). The direction of lateral LC displacement
from BMO (θ1) was also significantly correlated with the direction of
disc tilting (θ2) (p<0.001, R=0.896; Fig E).
Conclusions: Lateral displacement of the LC from BMO underlies
optic disc tilting. The severity and direction of lateral LC
displacement from BMO determines the severity and direction of
optic disc tilting.
(A and B) Green and blue dotted circles = anterior lamina cribrosa
(LC) insertion points and Bruch’ membrane opening (BMO),
respectively. Green and blue dots = centroids of the LC and BMO,
respectively. (A) Red double arrow = distance between the two
centroids. (B) Angle θ1 = direction of lateral LC displacement from
BMO. (C) Angle θ2 = direction of disc tilting; yellow dotted lines =
longest and shortest disc diameters.
Commercial Relationships: Pat-Michael Palmiero, None; Sung
Chul Park, None; Yiyi Liu, None; Camila F. Netto, None; Rafael
L. Furlanetto, None; Mohammed Al-Jumayli, None; Jeffrey M.
Liebmann, Alcon Laboratories, Inc. (C), Allergan, Inc. (C),
Allergan, Inc. (F), Carl Zeiss Meditech, Inc (F), Heidelberg
Engineering, GmbH (F), Topcon Medical Systems, Inc. (F), National
Eye Institute (F), New York Glaucoma Research Institute (F), SOLX,
Inc. (C), Bausch & Lomb, Inc (C), Diopsys, Inc. (C), Diopsys, Inc.
(F), Merz, Inc. (C), Glaukos, Inc. (C), Quark, Inc. (C); Robert Ritch,
None
Support: Joseph M. Cohen Research Fund of the New York
Glaucoma Research Institute, New York, NY; James Cox Chambers
Research Fund of the New York Eye and Ear Infirmary, New York,
NY; American Glaucoma Society MAPS Award; Peter Crowley
Research Fund of the New York Eye and Ear Infirmary, New York,
NY
Endothelial Glycocalyx Layer in the Aqueous Outflow Pathway
of Bovine Eyes
Chen-Yuan C. Yang1, 2, Tiffany Huynh2, Mark Johnson3, Haiyan
Gong2, 1. 1Anatomy and Neurobiology, Boston University, Boston,
MA; 2Ophthalmology, Boston University, Boston, MA; 3Biomedical
Engineering, Northwestern University, Evanston, IL.
Purpose: The glycocalyx on the vascular endothelium is known to
have an important role as a transport barrier and in the
mechanotransduction of fluid shear stress. The glycocalyx in the
aqueous humor outflow pathway has not yet been reported. The
purpose of this study is to determine whether this layer exists in the
aqueous outflow pathway and its distribution therein.
Methods: Enucleated bovine eyes were obtained within 6 hours postmortem. The eyes were either perfusion fixed (N=2) or immersion
fixed (N=2) with 1% glutaraldehyde and 4% paraformaldehyde in
PBS containing 30 mmol/L MgCl2 and 0.05% (w/v) Alcian Blue
8GX. All eyes were cut into small tissue segments and post-fixed in
1% aqueous osmium tetroxide and 1% lanthanum nitrate followed by
staining with uranyl acetate and processed for transmission electron
microscopy. The distribution of glycocalyx was examined and the
height of glycocalyx (in those regions where it was seen) was
measured on the trabecular beams, and in the aqueous plexus (AP),
and collector channels (CCs).
Results: The glycocalyx, which appears as a layer of hair-like
brushes, was most prominent in the CCs but was also found on the
endothelial surfaces of the AP and the trabecular beams. The
glycocalyx was relatively uniform in the CCs (Fig. 1A) and had a
mean height of 98±27 nm (Mean±SD; n=397 measurements of
glycocalyx). In the AP (Fig. 1B) and trabecular beams (Fig. 1C), the
glycocalyx was much more variable, with some regions showing little
or no glycocalyx; in those regions with glycocalyx, its mean height
was 99±37 nm (n=156) in the AP and 101±37 nm (n=154) on the
trabecular beams.
Conclusions: A non-uniform glycocalyx was found coating the
surface of the endothelium of the aqueous outflow pathway of bovine
eyes. The uneven distribution in the trabecular meshwork and AP is
perhaps due to the segmental flow of the aqueous humor through the
outflow pathways. Further studies are needed to determine the
function of glycocalyx in the aqueous outflow pathway and whether
it contributes to the aqueous outflow resistance.
arteriosclerosis. In Timbo’s left eye, we found a significant area of
geographic atrophy in the central macula that corresponded to the
area that had been noted clinically on pre-operative exams during the
planning for cataract surgery in 2009. This area appeared grossly
much like the geographic atrophy seen in humans with age-related
macular degeneration.
One of Timbo’s eyes had peripheral optic nerve atrophy of unknown
etiology that may be associated with the peripheral retinal
degeneration seen greater temporally in that eye than in other
peripheral quadrants. We also found corpora amylacea in periodic
acid-Schiff (PAS) stains of the optic nerve and surrounding
structures.
Conclusions: Lens capsule and supporting lens structures are similar
to humans except that the posterior capsule is two to three times
thicker, which would theoretically make cataract surgery on this
subspecies somewhat more forgiving. Age-related retinal and optic
nerve changes are remarkably similar to those found in humans.
Commercial Relationships: Clay Holley, None; Nick Hogan, None
Figure 1: The glycocalyx (black arrows) was relatively uniformly
distributed on the endothelium surfaces of the collector channel (CC)
(A), and non-uniformly distributed on the endothelium surfaces of the
aqueous plexus (AP) (B) and trabecular beam (C). ITS:
intertrabecular space; Red arrows: area without glycocalyx.
Commercial Relationships: Chen-Yuan C. Yang, None; Tiffany
Huynh, None; Mark Johnson, None; Haiyan Gong, None
Support: NIH EY019696, NIH EY022634, The Massachusetts Lions
Eye Research Foundation, Inc.
Comparative Ocular Anatomy & Age-Related Ocular Changes of
the Western Lowland Gorilla
Clay Holley, Nick Hogan. Ophthalmology, UT-Southwestern
Medical Center, Dallas, TX.
Purpose: To evaluate age-related ocular changes of the Western
lowland gorilla and how those compare to the human eye.
To our knowledge, this is the first report of age-related ocular
changes in this species.
Methods: Timbo, a 49 year old female Western lowland gorilla, died
from anesthesia complications in August 2011. Her eyes were placed
in formalin within 15 hours of her death. We took a standard
horizontal approach to the left eye and a modified ora serrata
approach to the right eye when dissecting the eyes. After taking
photos of the gross specimens, we sent tissue for histological stains
and reviewed these to complete our report of the findings.
Results: Globe dimensions, extraocular muscle insertions, tendon
widths, and corneal and scleral thicknesses are similar to humans.
Timbo had previously had cataract surgery in both eyes, so the
anterior portion of the lens capsule could not be evaluated. However,
the posterior lens capsule is thicker in Western lowland gorillas (10
micrometers) than in humans (average of 4 micrometers).
Timbo had several chorioretinal scars in both eyes, which could be
age-related. The age-related changes found were very similar to those
that are often found in human eyes. Notably, Timbo was found to
have lattice degeneration, peripheral retinal atropy and microcystoid
degeneration, cobblestone degeneration, and mild retinal vascular
Assessment of ciliary muscle morphology using a new Fourier
domain swept source anterior chamber optical coherence
tomographer
Phillip Buckhurst, Hetal Buckhurst, Catriona Hamer. School of
Health Professions, Plymouth University, Plymouth, United
Kingdom.
Purpose: Despite the growing interest in understanding the ciliary
muscle (CM) morphology, there are no standardized ciliary muscle
parameters. Uni-dimensional measures of ciliary muscle thickness
(CMT) via anterior segment optical coherent tomography (AS-OCT)
suggest the temporal CMT to be thicker than the nasal CMT. Such
measures of thickness are limited by the accuracy in locating the
scleral spur and errors introduced by using straight-line calipers along
curved scleral wall. In order to overcome these limitations the study
utilizes a new fourier domain swept source anterior chamber optical
coherence tomographer (SSOCT) to examine measures of temporal
and nasal CM thickness, cross-sectional area and their changes with
accommodation.
Methods: Twenty young phakic subjects aged between 18 - 30 years
were recruited with a range of refractive error (MSE (D) -1.64 ± 2.64;
range -9.50 to +1.25). Using SSOCT (Casia SS-1000, Tomey)
temporal and nasal ciliary muscle cross-sections were imaged in the
relaxed state and at stimulus vergence levels of -2.5D and -10D.
Using Casia software, manual measures of nasal and temporal CMT
(NCMT and TCMT respectively) were taken in successive posterior
steps from the scleral spur at 1 mm (CMT1), 2 mm (CMT2) and 3
mm (CMT3). For each accommodative target, ciliary muscle area
was calculated for the nasal and temporal cross sections. Noncycloplegic refractive error measurements were taken with a open
field autorefractor (Grand Seiko Co. Ltd). Statistical comparisons and
analyses were conducted using mixed repeated measures ANOVAs
and Pearson’s correlation coefficient.
Results: CMT1, CMT2 and CMT3 were thickest on the temporal
side for all accommodative stimuli (p<0.05). The ciliary muscle area
was greatest on the temporal side in comparison to the nasal side in
the relaxed state (p<0.001) and with the -2.5D vergence (p=0.025)
and -10D vergence (p=0.026).
Conclusions: The CM area and thickness is significantly greater on
the temporal side in comparison to the nasal side. The high resolution
cross sectional images of the CM provided by the Casia enables
measurement of cross sectional area. This additional parameter of the
CM morphology may assist with modeling the accommodative
process and understanding the potential biomechanical role of the
ciliary muscle in myopigenesis.
Commercial Relationships: Phillip Buckhurst, None; Hetal
Buckhurst, None; Catriona Hamer, Bausch and Lomb (F)
Comparative Anatomy of Trabeculum and Smooth Zone
(Schwalbe's Line) Widths of Various Primate and non-Primate
Eyes by Scanning Electron Microscopy (SEM)
Ann E. Barker-Griffith1, Jerrold L. Abraham2, Hengsheng Fang2,
Mark P. Breazzano1. 1Ophthalmology, SUNY Upstate Med Univ,
Syracuse, NY; 2Pathology, SUNY Upstate Med Univ, Syracuse, NY.
Purpose: Animal eyes are often used in glaucoma research, but the
anatomy of the irido-corneal angle has not been well defined across
all species. A remaining question is which species best represents the
human eye structurally. We studied the width of the smooth zone and
trabecular meshwork in several commonly used animals and human,
by SEM, to test the hypothesis of whether an animal model has
trabeculum and smooth zone widths similar to the human eye.
Methods: Corneal-scleral rings were extracted from various
formalin-fixed animal eyes, dehydrated in ethanol, and critical point
dried. Scanning electron microscopy generated images of the corneal
endothelial smooth zone, if present, and trabeculum from which each
quadrant was measured and width calculated. The animals studied to
date include: monkey (2), dog (2), pig (9), cow (6), rabbit (8), and
human (26).
Results: Preliminary studies showed absence of smooth zone in the
dogs, monkeys, rabbits, and pigs studied. Furthermore, these species
showed uniform width of the trabecular meshwork in all quadrants,
whereas the bovine eyes showed varying trabecular widths and
presence of a smooth zone (248 ± 53 µm). We have previously
shown that human eyes have a wider smooth zone (103 ± 8 µm) in
the superior quadrant.
Conclusions: The bovine eye possesses a smooth zone (Schwalbe's
line) and variable quadrant width of trabeculum as in human eyes.
Therefore, the cow eye may serve as a more appropriate candidate for
in vivo studies. The monkey, dog, rabbit and pig eyes have dissimilar
trabeculum and smooth zone anatomy from the human eye; raising
questions on the suitability of these species in modeling certain
human eye diseases.
Commercial Relationships: Ann E. Barker-Griffith, Allergan
Sales, LLC (F); Jerrold L. Abraham, None; Hengsheng Fang,
Allergan (C); Mark P. Breazzano, None
Support: Allergan Sales, LLC; Research to Prevent Blindness, New
York, NY; Lions District 20-Y1, Syracuse, NY, the Department of
Pathology at SUNY Upstate Medical University, and NYSTAR.
Cell-ECM interactions during formation of the zebrafish hyaloid
vasculature
Andrea E. Hartsock, Jeffrey M. Gross. Cell, Mol, and Dev Biology,
University of Texas, Austin, Austin, TX.
Purpose: Vasculature formation requires an orchestrated series of
morphogenetic changes to generate an integrated vessel system.
Virtually nothing is known about how hyaloid vasculature
morphogenesis occurs, despite its importance in the developing eye.
Thus, the purpose of this study was to identify the cellular
underpinnings of hyaloid morphogenesis and test the hypothesis that
cell-ECM interactions play an integral role in building the hyaloid.
Methods: Fixed sample and in vivo time lapse imaging of fli1a:GFP
and flk:mCherry transgenics were utilized to image and quantify
dynamic aspects of hyaloid morphogenesis in wild-type, fibronectin1
(fn1) and integrin-α5 (itga5) mutant embryos. Lens transplantation
was utilized to determine if lens-derived Fn was required for hyaloid
formation.
Results: Hyaloid formation can be divided into five distinct
morphogenetic stages: During Stage I (48hpf) vascular precursor
cells arrive at the posterior of the lens. Throughout Stage II (4872hpf) hyaloid precursor cells increase in number, forming a
meshwork that covers the posterior lens. During Stage III (72-96hpf),
remodeling of this meshwork initiates; here, Stage II hyaloid cells
reorganize into a highly branched web that surrounds the lens. An
anterior ring is also observed at this time. At Stage IV (96hpf), the
anterior ring is no longer present. Throughout Stage V (108-120hpf),
disorganized hyaloid precursor cells mature into connected lumenal
vessels. The hyaloid in fn1 mutants possessed “clumps” of vascular
precursor cells on their lenses that did not reorganize into mature
vessels. The hyaloid in itga5 mutants extends anteriorly, but vessel
thickening, maturation, and organization are disrupted.
Conclusions: Fixed sample and in vivo time-lapse imaging of
hyaloid formation revealed that hyaloid morphogenesis involves a
series of dynamic cell migration events and morphogenetic changes
in order to build the functional hyaloid. Analyses of two mutations
that affect cell-ECM interactions, fn1 and itga5, reveal specific
requirements for these factors during hyaloid development. Analyses
of the roles of additional cell-ECM proteins, as well as other
morphogenetic regulators, during hyaloid formation will generate a
comprehensive understanding of the cellular underpinnings of
hyaloid morphogenesis during embryonic eye development.
Commercial Relationships: Andrea E. Hartsock, None; Jeffrey
M. Gross, None
Support: NIH RO1 EY18005-04S1
Dimensional Variation of the Orbicularis Oculi Muscle in Nonpreserved, Fresh Frozen Human Cadavers
Bryan R. Costin1, Natta Sakolsatayadorn1, Stephen A. McNutt1, Tal
Rubinstein1, George Trichonas1, Jennifer M. McBride2, Julian D.
Perry1. 1Cole Eye Institute, Cleveland Clinic, Cleveland, OH;
2
Department of Anatomy, Cleveland Clinic Lerner College of
Medicine, Cleveland, OH.
Purpose: To investigate polymorphic variation in the dimensions of
the orbicularis oculi muscle (OOM) through anatomic dissection of
fresh-frozen human cadavers.
Methods: Skin incisions were created along a line 1 cm lateral and
parallel to a line connecting the supraorbital notch (SON) and the
infraorbital foramen (IOF) and from the lateral canthus to the
superior border of the tragus. The OOM was isolated using a
combination of sharp and blunt dissection until each of its distal
borders were identified. A metric ruler measured the superior,
inferior, and lateral dimensions of the OOM from the orbital rim.
Data collection included age at time of death, gender, and race.
Results: A total of 12 hemifaces from 6 fresh frozen human cadavers
were dissected. All specimens were male. Cadavers were of
Caucasian (5) and African (1) decent. Average age at time of death
was 65.7 years (36 - 84 years). Mean lateral OOM dimension was 2.8
cm (1.7 - 3.3 cm) on the right and 3.1 cm (1.9 - 3.8 cm) on the left.
Mean superior dimension was 1.9 cm (1.5 - 2.5 cm) on the right and
1.7 cm (1.1 - 2.2 cm) on the left. Mean inferior dimension was 1.6 cm
(0.8 - 2.4 cm) on the right and 1.5 cm (1 - 2 cm) on the left. The
average distance from the lateral orbital rim to the superior border of
the tragus was 8.1 cm (7.7 - 8.4 cm) on the right and 8.2 cm (7.9 - 8.7
cm) on the left.
Conclusions: In unpreserved, fresh frozen cadavers, the OOM
extends approximately 1.8 cm superiorly from the orbital rim 1 cm
lateral to the SON, approximately 1.6 cm inferiorly from the orbital
rim 1 cm lateral to the ION, and extends approximately 2.9 cm
laterally from the orbital rim at the level of the lateral canthus.
Increased knowledge of these dimensions has significant clinical and
surgical implications, including more nuanced neurotoxin
administration and surgical applications.
Commercial Relationships: Bryan R. Costin, None; Natta
Sakolsatayadorn, None; Stephen A. McNutt, None; Tal
Rubinstein, None; George Trichonas, MEEI-Patent Application
U.S. Serial No. 61/327,476 “Combination Therapy for Preserving
photoreceptor cell viability following retinal detachment” (P);
Jennifer M. McBride, None; Julian D. Perry, None
Ocular Effects of Blood Collection Techniques in Rabbits
Shwu-Fei Lee1, Steven D. Sorden1, Dale G. Dunn1, Peter J.
Sonnentag1, Alexander J. Dwyer2. 1Nonclinical Safety Assessment,
Covance Laboratories Inc., Madison, WI; 2College of Veterinary
Medicine, Iowa State University, Ames, IA.
Purpose: Rabbits are commonly used in nonclinical safety evaluation
of ophthalmic drugs. To reduce the number of animals, blood
samples for toxicokinetic (TK) and toxicity tests are often taken from
the same animals. Medial auricular artery catheterization is common
when >4 TK timepoints are needed in one day. Potential
catheterization-related ocular lesions occurred in previous rabbit
studies. This study evaluated ocular effects of various blood
collection techniques in rabbits.
Methods: 50 female Hra:(NZW)SPF rabbits were assigned to 5
groups (10 animals/group). On Days 1 and 7, blood was collected at 7
timepoints in an 8-hour interval via repeated hypodermic needle
puncture of a medial auricular artery (Group 1), a jugular vein (Group
2), or a combination of medial auricular artery and a jugular vein
(Group 3); or via a medial auricular artery catheter (Group 4). Group
5 had a catheter without a needle taped to the surface of one pinna
(control for Group 4, no blood collected). Clinical signs, dermal
irritation at collection sites, body weight, and anatomic pathology
were assessed.
Results: Skin lesions or irritation at the collection/sham sites
occurred in all groups. Jugular venipuncture sites had the most severe
skin irritation and resulted in the fewest useable samples. Ear artery
catheterization yielded the most useable samples and caused only
minor skin lesions but was associated with multiple foci of
necrosis/acute inflammation or subacute inflammation in Harderian
and lacrimal glands. Findings correlated with catheter site, i.e., only
animals with a catheter in the right ear had findings in right Harderian
and/or lacrimal gland. Unilateral multifocal necrosis/acute
inflammation in the mandibular salivary gland and focal
degeneration/necrosis in the brain were also restricted to catheterized
animals. Groups 1 and 3 only had microscopic lesions at intraartery
sites. Group 2 had no technique-related microscopic lesions.
Conclusions: Repeated blood collection via ear artery catheter can
result in foci of necrosis and inflammation in paraocular and
mandibular salivary glands. Paraocular gland lesions could confound
microscopic assessment of ophthalmic drugs. A combination of
jugular vein and ear artery collections is a reasonable alternative with
minimal samples missed. However, a satellite group for TK sample
collections is recommended for ocular toxicity studies in rabbits.
Commercial Relationships: Shwu-Fei Lee, Covance Laboratories
Inc. (E); Steven D. Sorden, Covance Laboratories, Inc. (E); Dale G.
Dunn, Covance Laboratories Inc. (E); Peter J. Sonnentag, Covance
Laboratories Inc. (E); Alexander J. Dwyer, Covance Inc (E),
Covance Inc (I)
The Use of Commercially Available Grafts in Lower Eyelid
Reconstruction
Neel Kumar1, 2, Shalin Shah1, 2, Adham B. al-Hariri1.
1
Ophthalmology, Ochsner Clinic Foundation, New Orleans, LA;
2
Ophthalmology, Louisiana State University Health Science Center,
New Orleans, LA.
Purpose: The use of supportive and spacer grafts is often necessary
in eyelid reconstruction. While the use of autologous grafts
minimizes tissue rejection and restores structural integrity, the size of
harvested grafts and secondary wound morbidity can be limiting
factors. Although commercial grafts have become increasingly
available over the past decade, it is not clear which graft(s) are best to
restore the eyelid’s complex anatomy and function. Furthermore,
there is little information documenting the use of these grafts in
combination.
Methods: A retrospective chart review was performed and 6 patients
were identified with cicatricial lower eyelid retraction requiring
allogeneic, xenographic, or bioengineered grafts. We examined the
preoperative anatomical considerations, types of procedures
performed, postoperative outcomes, and complications.
Results: Multiple commercially available grafts were utilized
depending on the level of injury within the eyelid to restore anatomic
and functional properties in all 6 patients. The most complex
reconstruction was for a patient who had an iatrogenic cicatricial
entropion and eyelid retraction with hardware adhesion to an
extruding orbital implant. The patient suffered from epiphora, inferior
scleral show, and lagophthalmos with exposure keratopathy. An
allogeneic dermal graft (Alloderm©,LifeCell, Branchburg, NJ) was
used to cover the titanium plates and prevent future adhesions. A
bioengineered eyelid spacer graft (tarSys™, IOP Ophthalmics, Costa
Mesa, CA) was used to reconstruct the lower eyelid posterior lamella
(FIGURE 1). Finally, an allogeneic amniotic membrane (Ambio5®,
IOP Ophthalmics, Costa Mesa, CA) was used to reconstruct the
palpebral conjunctiva. The other patients received either a porcinederived acellular dermis (ENDURAGen©, Stryker, Newnan,GA) or
tarSys™. None of the patients developed an infection or graft
rejection.
Conclusions: Commercially available grafts are establishing their
role in lower eyelid reconstruction. While the goal in tissue selection
usually focuses on restoring form, the dynamic properties of the
lower eyelid should also dictate the choice of graft. Depending on the
layer(s) of the eyelid involved, this is currently achievable with an
array of grafts used alone or in combination. The complex anatomy
and function of the lower eyelid can be safely simulated without
donor site morbidity or significant complications.
FIGURE 1: Implanted tarSys™ graft
Commercial Relationships: Neel Kumar, None; Shalin Shah,
None; Adham B. al-Hariri, None
Outcomes of surgery for removal of visually significant
hyperplastic persistent pupillary membranes
Courtney L. Kraus, Gregg T. Lueder. Ophthalmology & Vision
Sciences, Washington University, Saint Louis, MO.
Purpose: Hyperplastic persistent pupillary membranes (PPM)
obstructing the visual axis are rare among children. Among those
born with the anomaly, most regress by age one. However, persistent
clinically significant obstruction of the visual axis places a child at
risk of amblyopia. Therefore, medical and surgical interventions are
implemented to ensure clearing of the visual axis and optimal visual
development.
There are many different approaches to surgical removal of PPM. We
describe our specific surgical technique and the long-term visual and
cosmetic results following PPM removal.
Methods: We retrospectively analyzed 10 cases of PPM removal in 6
patients. Each PPM was felt to be clinically significant based on poor
visual acuity, poor retinoscope reflex, or inability to visualize the
fundus. Cases of bilateral PPM underwent sequential surgery with the
more visually significant membrane removed first.
Surgical technique employed a clear corneal incision. A viscoelastic
agent was injected beneath the pupillary strands bowing them
forward. Residual synechiae were peeled from the anterior lens
capsule using a Sinskey hook. To complete the optical iridectomy,
additional viscoelastic was injected beneath iris strands, which were
cut using intraocular scissors. The pupillary membrane was removed
with McPherson forceps. Miosis was pharmacologically induced to
assess symmetric pupillary constriction.
Post-operative vision and cosmesis were recorded on follow-up.
Results: The PPMs were bilateral in 4 patients and unilateral in 2
patients. Age at time of surgery ranged from 2.5 months to 2.5 years
(mean 14 months). Mean postoperative follow-up was 4.4 years
(range 2-8 years). Visual acuity improved in all patients, ranging
from 20/20 to 20/70. One patient was treated for anisometropic
amblyopia. No operative complications occurred.
Conclusions: Several techniques have emerged for management of
clinically significant PPM including nonsurgical (e.g. neodymiumYAG laser) and surgical removal. This retrospective review
demonstrates excellent outcomes with our technique.
Potential complications of PPM removal include iris atrophy,
iridodialysis, and damage to lens. We did not encounter any of these
complications in our cohort. The described technique for the surgical
removal of PPM represents a safe and effective treatment. We
observed excellent post-operative visual acuities and cosmetic
outcomes.
Commercial Relationships: Courtney L. Kraus, None; Gregg T.
Lueder, None
Anatomical and Histologic Evaluation of the Frontalis Muscle in
Non-Preserved, Fresh-Frozen Cadavers
Stephen A. McNutt, Bryan R. Costin, Thomas Plesec, Natta
Sakolsatayadorn, Tal Rubinstein, George Trichonas, Jennifer M.
McBride, Julian D. Perry. Ophthalmology, Cole Eye Institute,
Cleveland, OH.
Purpose: To characterize the macroscopic and microscopic structure
of the frontalis muscle in fresh-frozen cadavers, and specifically the
frontalis dehiscence.
Methods: Three fresh-frozen cadavers were marked in preparation
for removal of tissue from the forehead for microscopic evaluation.
Three blocks of tissue were removed at inferior, middle, and superior
locations of the forehead. Dimensions of tissue blocks were 5mm x
3cm in height and width, respectively, with depth from skin to frontal
periosteum.
The first block of tissue was removed 1cm superior to a horizontal
line drawn through the right and left supraorbital notches. The second
block was subsequently removed starting 1cm above the superior
aspect of the first block and the third block removed in similar
fashion 1cm from the superior aspect of the second block (Fig.1). All
blocks were marked with suture to define laterality and were
submitted to pathology in formalin.
Dissection was subsequently performed to expose the frontalis
muscle. Inferior incisions were made at the upper eyelids with lateral
incisions made lateral to the temporal fossa so as not to disturb the
frontalis muscle during reflection and removal of skin.
Results: The level of frontalis dehiscence was measured at 2 cm and
3 cm in two of the three cadavers. The third cadaver was Cushingoid
due to high-dose steroids and lacked macroscopically identifiable
frontalis muscle. Microscopic evaluation showed a lack of muscle
fibers in the midline of tissue blocks. A microscopic area of
“dehiscence,” or lack of muscle units, in all three cadavers was
found, which corresponded to the level of dehiscence in the two
macroscopically identifiable regions of dehiscence.
Conclusions: Dehiscence of the frontalis muscle observed
macroscopically during gross dissection of fresh-frozen cadaveric
specimens correlates with areas devoid of muscle fiber by
microscopic evaluation of these same tissue samples. Clinical
implications of these findings include administration of neurotoxin in
the forehead, as administration in the midline forehead may not be
efficacious.
Commercial Relationships: Stephen A. McNutt, None; Bryan R.
Costin, None; Thomas Plesec, None; Natta Sakolsatayadorn,
None; Tal Rubinstein, None; George Trichonas, MEEI-Patent
Application U.S. Serial No. 61/327,476 “Combination Therapy for
Preserving photoreceptor cell viability following retinal detachment”
(P); Jennifer M. McBride, None; Julian D. Perry, None
Primary Blast-Induced Ocular Trauma Modulated by Peak
Pressure
Daniel Sherwood1, Brian Lund2, Randolph D. Glickman3, Walter
Gray4, Richard Watson1, Kimberly Thoe5, William E. Sponsel5, 1,
Matthew A. Reilly1. 1Biomedical Engineering, University of Texas at
San Antonio, San Antonio, TX; 2Institute of Surgical Research, San
Antonio, TX; 3University of Texas Health Science Center at San
Antonio, San Antonio, TX; 4Geological Sciences, University of
Texas at San Antonio, San Antonio, TX; 5WESMDPA, San Antonio,
TX.
Purpose: In proportion to surface area, battlefield ocular trauma is 20
to 50 times more likely than other exposed areas (Sobaci et al., Am J
Ophthalmol 2000). Although the ability of secondary-blast effects
(fragments, shrapnel) to cause ocular trauma is well known, it is not
yet known whether ocular trauma may be induced by primary blast
effects (i.e., those due solely to the shock-wave). We hypothesized
that primary, sub-lethal blast alone is sufficient to induce ocular
trauma with the corollary that the level of trauma is correlated with
the peak overpressure. We tested this hypothesis using a shock-tube
capable of replicating physically accurate shock-waves within a
controlled environment.
Methods: Porcine eyes were enucleated and set in a rigid mimic of
the orbit filled with gelatin (Sponsel et al., IOVS 2011). The porcine
eyes were imaged via UBM and B-scan ultrasound along 12 vectors
both before and after blast. Each eye was re-pressurized via
intracameral injection using a 30 gauge needle with balanced salt
solution and placed in a shock-tube (Fig. 1). The eyes were then
exposed to blasts of 2 ms duration with peak pressures ranging from
7-22 psi. After post-blast imaging, eyes were fixed in formalin for
later dissection or histopathology.
Results: We observed corneal abrasion and fissuring, angle
recession, circumferential iris plication into the zonule, uveal pigment
forced between the axonal fasciculi of the optic nerve (Fig. 2a). Mid
peripheral chorioretinal detachments, radial peripapillary retinal
detachments (Fig 2b), and intrinsic internal scleral delamination (Fig
2c) were also observed. In an extreme case, an incident pressure of 22
psi caused axial-posterior displacement of 8 mm within 67 ms and
eventual evulsion of the eye from its orbit mimic on rebound.
Conclusions: Primary blast in the absence of particle insult was
sufficient to cause a range of significant ocular trauma. The
magnitude of the incident peak-pressure was correlated with the
probability of trauma occurrence and severity of damage. The ability
of primary blast overpressure to produce severe eye damage
underscores the necessity of developing protective eyewear
specifically designed to shield the eye and orbital structures from this
source of trauma.
Commercial Relationships: Daniel Sherwood, None; Brian Lund,
None; Randolph D. Glickman, None; Walter Gray, None; Richard
Watson, None; Kimberly Thoe, None; William E. Sponsel, New
World Medical (P); Matthew A. Reilly, None
Support: DOD VRP Grant W81XWH1220055
Positional Relationship Between the Ethmoidal Foramina and the
Optic Canal
Eric S. Ahn2, 1, Milap Mehta2, Julian D. Perry2. 1Johns Hopkins
Hospital Wilmer Eye Institute, Baltimore, MD; 2Cleveland Clinic
Cole Eye Institute, Cleveland, OH.
Purpose: To determine the position of a ray comprised of the
anterior and posterior ethmoidal foramina as it bisects the optic canal.
Methods: A prospective case series evaluating human skulls was
performed between May 2012 and June 2012 at the Cleveland
Museum of Natural History. Data collected included skull
demographics if available and color photographs of orbits. A ray was
added to the orbital photo using Adobe Photoshop, beginning at the
anterior ethmoidal foramen and extending through the posterior
ethmoidal foramen and optic canal. If the anterior-posterior
ethmoidal ray (APER) entered the middle third of the optic canal it
was described as “middle,” and if entering in the superior or inferior
third of the canal, it was described as “superior” or “inferior”
respectively. Skulls were excluded if there was significant damage to
the orbits or wear precluding the ability to easily identify the anterior
and posterior ethmoidal foramina and the optic canal.
Results: A total of 5 skulls (10 orbits) were available for review.
There were 5 male and 0 female skulls with an average age of 48
years (range, 24-69 years). The APER was considered in a middle
position in three orbits, superior in five orbits, and inferior in two
orbits.
Conclusions: The anterior and posterior ethmoidal foramina serve as
critical landmarks often used when navigating towards the posterior
orbit through a medial approach. Surgeons have been taught that a
ray connecting these two points will lead directly to the center of
optic canal. Although the APER can be a useful guide, its
relationship with the optic canal varies considerably. Therefore
surgeons should proceed with gentle dissection and caution when
advancing towards the optic nerve using the APER alone.
Commercial Relationships: Eric S. Ahn, None; Milap Mehta,
None; Julian D. Perry, None
A Computational Model for Investigation of Ocular Trauma Due
to Primary Blast
Walter Gray1, Richard Watson2, Matthew A. Reilly2, Brian Lund4,
Rick E. Sponsel5, Randolph D. Glickman3. 1Geological Sciences,
University of Texas at San Antonio, San Antonio, TX; 2Biomedical
Engineering, University of Texas at San Antonio, San Antonio, TX;
3
Ophthalmology, University of Texas Health Science Center-SA, San
Antonio, TX; 4Ocular Trauma, U.S. Army Institute of Surgical
Research, Fort Sam Houston, TX; 5Sponsel Professional Association,
San Antonio, TX.
Purpose: Ocular trauma due to blast has increased dramatically in
the Iraq and Afghanistan conflicts. Eye trauma has been observed in
approximately one-quarter of battlefield injuries. However, trauma
from blast pressure alone, or primary blast, is not well documented as
pressure is commonly accompanied by debris and acceleration of the
body. Some researchers argue that primary blast cannot create
significant ocular trauma, instead serious injury results from the
secondary debris or body acceleration. Recent experiments have
suggested the potential for injury from high levels of blast pressure.
To further evaluate this hypothesis and investigate the experimental
results, high-fidelity computational models of the eye were developed
and exercised. Modeling confirmed the reality of primary blast injury
and allowed for identification of the physical mechanisms
responsible.
Methods: Numerical simulations were run in conjunction with shock
tube experiments. Sub-lethal blast levels (7-22 psi; 2 ms) were used
in the experiments and modeling. Previous experiments focused on
globe rupture, but our interest was characterizing lower but
potentially serious internal injuries. Two different numerical models
were used: (1) a Lagrangian Finite Element Analysis (FEA) model
with fluid/structure interaction, and (2) a Eulerian finite volume
hydrocode. High-fidelity geometrical and tissue constitutive
representations were developed and implemented in both models.
Results: The computational models successfully predicted the types
of injury observed in the physical experiments. Observed injuries
included retinal detachment, angle recession, lens displacement, and
injury to the iris and zonules. Higher levels of blast pressure were
associated with higher levels of injury. However, some injury types
could not be duplicated with the models. Specifically smearing of
pigment into the optic nerve was observed in the experiments, but
could not be duplicated with the current models.
Conclusions: The numerical models confirmed the potential for
ocular injury due to primary blast. Computational results were well
correlated with the physical experiments and provided invaluable
insight into the mechanisms responsible for injury. The models can
therefore be used in future efforts to evaluate protection schemes
against primary blast.
Crosssectional view of the eye model used to evaluate primary blast
injury
Commercial Relationships: Walter Gray, None; Richard Watson,
None; Matthew A. Reilly, None; Brian Lund, None; Rick E.
Sponsel, New World Medical (P); Randolph D. Glickman, None
Support: U.S. Army Medical Research and Material Command
W81XWH-12-2-0055
Endonasal mitomicyn-C during diode laser transcanalicular
dacryocystorhinostomy
Jorge E. Peraza Nieves, Johnny Javier Castellar Cerpa, Paula
Arribas Pardo, Paula Bañeros, Juan Antonio Troyano Rivas, Angel
Romo Lopez. ophthalmology, HOSPITAL CLINICO SAN CARLOS,
Madrid, Spain.
Purpose: To evaluate the benefit of endonasal topical application of
mitomycin-C in patients undergoing transcanalicular diode laser
dacryocystorhinostomy (DL-DCR).
Methods: We carried out a retrospective descriptive study of 400
patients who underwent transcanalicular DL-DCR. The procedure
was performed under local anesthesia. In 30 patients, endonasal
mitomycin-C (0.4 mgr/0.2 ml) was administered during the surgical
intervention and in 370 any anti-cicatricial agent was used. We
compared the lacrimal permeability by syringing lacrimal pathway,
after 12 months of performing surgery. The results were compared
using the Chi-squared test with the Yates correction.
Results: The average follow-up was 12 months (range 6 to 18
months). Of the 370 patients who underwent DCR without
mitomycin-C, 34 failed after a year of surgery. Only one failed of the
30 patients treated with mitomycin-C. We found a statistically
significant association between mitomycin-C used procedure and
lacrimal permeability after 12 months of performing surgery
(p<0.05).
Conclusions: In our study, the administration of topical endonasal
mitomycin-C during transcanalicular diode laser
dacryocystorhinostomy is associated to permeability of nasolacrimal
duct . It is a minimally invasive quick procedure yielding a high
success rate. We did not find secondary effects due to application of
mitomcyn-C.
Commercial Relationships: Jorge E. Peraza Nieves, None;
Johnny Javier Castellar Cerpa, None; Paula Arribas Pardo,
None; Paula Bañeros, None; Juan Antonio Troyano Rivas, None;
Angel Romo Lopez, None
Effects of Oxygen Level on In Vitro Culture of Human Choroidal
Melanocytes
Solange Landreville1, 2, Juliane Guay1, 2, Florence Pagé-Larivière1, 2.
1
Ophtalmologie-ORL, Université Laval, Québec, QC, Canada;
2
LOEX/CUO-recherche, Centre de recherche du CHU de Québec,
Québec, QC, Canada.
Purpose: Most cells in the human body are exposed to oxygen levels
varying from 2-14% (physiological oxygen levels). The choroidal
cells for instance, are exposed to 5% oxygen. Traditional cell culture
at 21% oxygen thus induces an hyperoxic state in these cells, which
reduces the validity of the in vitro models. Our goal was to study the
differentiation and proliferation of choroidal melanocytes grown
under physiological (5%) and atmospheric (21%) oxygen levels.
Methods: Melanocytes were isolated from human choroids by
successive digestions in trypsin and collagenase, then the cells were
exposed to 5% or 21% oxygen. Melanocyte morphology and
pigmentation were assessed by phase-contrast microscopy. Culture
purity was verified by immunostaining with melanocyte and retinal
pigment epithelium (RPE) markers. Proliferation was measured using
MTS assay.
Results: Reduced oxygen conditions did not change the morphology
and pigmentation of melanocytes. Compared to the melanocytes
grown in 5% oxygen, the cultures exposed to 21% oxygen contained
contaminant RPE cells, as confirmed by HMB45, ZO-1 and
cytokeratins 8/18 immunostaining. In addition, an increased
percentage of proliferation was measured in the 5% oxygen group
(+30-64%).
Conclusions: This study demonstrated that pure long-term cultures
of choroidal melanocytes could be established using physiological
oxygen conditions that more closely replicates the native tissue
environment.
Commercial Relationships: Solange Landreville, None; Juliane
Guay, None; Florence Pagé-Larivière, None
Support: Research scholar career award from the CRCHU de
Québec Foundation (SL), Summer student research grant from the
Mach-Gaensslen Foundation (JG); Funding from CRCHU de Québec
Foundation, Université Laval Foundation, Eye Disease Foundation,
and FRQS Vision Research Network
Diurnal Variation of Macular Choroidal Volume in Healthy
Volunteers
Natalia Alpizar-Alvarez, Erick Hernandez-Bogantes, Lihteh Wu.
Instituto de Cirugía Ocular, San Jose, Costa Rica.
Purpose: The chorioscleral interface may be irregular in different
areas of the same eye. Single point choroidal thickness measurements
may be misleading. The purpose of this study is to report the pattern
and magnitude of diurnal variation of macular choroidal volume
(MCV) measured by spectral domain optical coherence tomography
(SD-OCT) in normal subjects.
Methods: SD-OCT with enhanced depth imaging (EDI) and image
tracking (at least 49 average images) using a standarized protocol was
performed in 14 healthy volunteers at 2 hr intervals starting at 8 am
and finishing at 6 pm. Twenty five choroidal scans centered at the
fovea were performed using a raster protocol. The choroidal
thickness was segmented manually for each choroidal scan. The
retinal boundary reference lines placed by the built in automated
segmentation software were moved to the choroidal boundaries. A
standarized grid centered on the fovea was positioned automatically
by the SD-OCT software. This grid divided the macula into 3 circles
of 1 mm (central), 3 mm (inner) and 6 mm (outer) diameter. The
automated software calculated the volume in each of these circles.
Intraocular pressures (IOP), systolic (SBP) and diastolic (DBP) blood
pressures were also measured at the same time points.
Results: The mean age of the study subjects was 34.4 years (range,
23 to 59 years, SD ± 10.8) with 4 males and 10 females. The mean
spherical equivalences (SE) in the OD and OS were -0.64 and -0.56
D respectively. The mean axial length (AL) was 23.52 mm (range,
22.06 to 25.67 mm, SD ± 1.02) in the OD and 23.52 mm (range,
21.02 to 26.05 mm, SD ± 1.20) in the OS. The mean MCV in the OD
and OS fluctuated during the day from 8.95 to 9.70 µm3 (p=0.3577)
and from 8.23 to 9 µm3 (p=0.1651) respectively. The largest
fluctuation in MCV in the OD was 1.68 µm3 and the minimal
fluctuation was 0.27 µm3 compared to 1.75 µm3 and 0.33 µm3 in the
OS. There was a highly significant difference between the maximal
and minimal MCV in both eyes (p<0.0001 and p=0.0001). The mean
diurnal variation of MCV were 0.74 (7.8%) and 0.78 (8.6%) µm3 OD
and OS respectively. The amplitude of MCV variation did not
correlate with age, AL, SE, SBP, DBP or IOP.
Conclusions: The MCV fluctuates significantly during the day in
healthy subjects. Factors that influence MCV fluctuation remain to be
identified.
Commercial Relationships: Natalia Alpizar-Alvarez, None; Erick
Hernandez-Bogantes, None; Lihteh Wu, Heidelberg Engineering
(R)
Evaluating Choroidal Microvascular Angiogenesis by Choroid
Sprouting Assay
Zhuo Shao1, Mollie Friedlander1, Christian G. Hurst1, Zhenghao
Cui1, Lucy Evans1, Jing Chen1, Przemyslaw Sapieha2, Sylvain
Chemtob3, 2, Jean-Sebastien Joyal1, Lois E. Smith1. 1Ophthalmology,
Harvard Medical School, Boston Children's Hospital, Boston, MA;
2
Ophthalmology, Research Centers of Hôpital MaisonneuveRosemont, University of Montreal, Montreal, QC, Canada;
3
Pediatrics Ophthalmology and Pharmacology, Research Centers of
CHU Sainte-Justine, Montreal, QC, Canada.
Purpose: Age-related macular degeneration (AMD) is a major cause
of blindness. The disease is associated with choroidal vascular
dropout (dry AMD) or choroidal neovascularization (wet AMD). The
mechanism that causes abnormal choroidal angiogenesis is not
completely understood, partially due to the lack of an assay of
choroidal vascular growth. Here we characterized and optimized an
easily isolated, robust, quantitative and reproducible organotypic
microvascular model from the choroid.
Methods: Choroid tissue from Sprague Dawley rats, C57BL/6J and
129S6/SvEvTac mice was isolated and incubated in MatrigelTM.
After 3-7 days of incubation, the area covered by tube-like sprouts
was quantified by ImageJ software and the cell types of the sprouts
were analyzed by fluorescence-activated cell sorting (FACS). The
normalization and quantification methods for calculating the
sprouting area have been standardized, and the impact of retinal
pigment epithelial (RPE) cells, age of the animals, the type of media
used for incubation and the responses of the assay to pharmacological
stimulation has been characterized.
Results: The variation of vascular sprouting area is comparable
between choroid samples obtained within or between animals. The
sprouting area is highly reproducible between batches when
normalized to controls. A semi-automated quantification macro has
been developed for efficient quantification. The removal of RPE from
the choroid and aging of choroid reduce the sprouting rate.
Furthermore, endothelial selective medium CSC and EGM-2 provide
a better growth condition for the choroid explants compared to nonselective medium DMEM. Vascular endothelium growth factor
(VEGF) promotes choroidal sprouting where 4-HDHA (4-hydroxydocosahexaenoic acid), an anti-angiogenic metabolite of omega-3
polyunsaturated fatty acid, reduces choroid sprouting dose
dependently.
Conclusions: This study defined optimal conditions for a highly
reproducible, efficient and quantifiable choroid microvascular ex
vivo sprouting assay. This method provides a new experimental tool
not only for AMD studies, but also for physiological and
pharmacological research related to microvascular diseases in
general.
Commercial Relationships: Zhuo Shao, None; Mollie
Friedlander, None; Christian G. Hurst, None; Zhenghao Cui,
None; Lucy Evans, None; Jing Chen, None; Przemyslaw Sapieha,
None; Sylvain Chemtob, None; Jean-Sebastien Joyal, None; Lois
E. Smith, None
Support: NIH (EY017017-04S1, EY017017-05), V. Kann
Rasmussen Foundation, Boston Children's Hospital Mental
Retardation and Developmental Disabilities Research Center
PO1HD18655, Research to Prevent Blindness Senior Investigator
Award, Alcon Research Institute Award and Lowy Medical
Foundation (LEHS). ZS is supported by CIHR System Biology
Training Scholarship and Travel Grant. JC is supported by Boston
Children’s Hospital Ophthalmology Foundation, Charles H. Hood
Foundation Child Heath Research Award, and Blind Children’s
Center (to JC). PS holds a Canada Research Chair tier II. SC holds a
Canada Research chair tier I (Vision science) and the Leopoldine
Wolfe Chair in translational research in macular degeneration (U
Montreal).
Distribution, morphology, and dynamic behavior of macrophages
in the adult mouse choroid
Anil Kumar1, Lian Zhao1, Minhua Wang1, Robert Fariss2, Wai T.
Wong1. 1Unit on Neuron-Glia Interactions in Retinal Disease,
National Eye Institute, National Institute of Health, Bethesda, MD;
2
NEI Biological Imaging Core, National Eye Institute,National
Institute of Health, Bethesda, MD.
Purpose: Choroidal macrophages are resident ocular immune cells
capable of influencing the inflammatory environment of the outer
retina and play a role in AMD pathogenesis. However, choroidal
macrophages have not been characterized in detail in terms of their
anatomy, distribution, behavior, and possible endogenous functions.
Methods: Albino, transgenic CX3CR1GFP/+ mice in which
choroidal macrophages are labelled with green fluorescent protein
(GFP) were used to study macrophage morphology, distribution, and
vascular relationships. Live-imaging of GFP+ cells in sclerochoroidal
explants was performed to examine the dynamic behavior of
choroidal macrophages. The structure of the choroidal vasculature
was simultanously visualized by the intravascular perfusion of DiI.
Results: GFP+ macrophages are present throughout the choroid but
vary in their morphology, distribution, and vascular associations at
each level of the choroidal vascular tree. Perivascular macrophages
associated with large choroidal arteries and primary arterioles have
elongated, spindle-shaped morphologies aligned with the long axes of
vessels, with secondary dendritic processes projecting into the
perivascular space. Macrophages associated with smaller terminal
arterioles demonstrate a more symmetric, ramified morphology and
are mainly positioned within interstitial spaces between vessels and at
vessel branch points. Macrophages associated with the
choriocapillaris are located only on the scleral, but not the vitreal,
choriocapillaris surface and show a flattened morphology with
symmetrically ramified processes. Live imaging experiments reveal
that choroidal macrophages demonstrate dynamic surveying in their
processes but exhibit limited cellular migration. In addition to this
predominant population of ramified macrophages which are CD11b+,
Iba1+, CD68+, a smaller subpopulation of GFP+ cells with rounded
morphologies and a CD11blow/-, Iba1 low/-, CD68 low/immunophenotype was also observed.
Conclusions: Choroidal macrophages demonstrate morphological
diversity and varied associations with the choroidal vasculature at
each level. Their dynamic process behavior and close vascular
associations suggest that they may play functional roles in
vasoregulation and vascular surveillance in the choroid.
Commercial Relationships: Anil Kumar, None; Lian Zhao, None;
Minhua Wang, None; Robert Fariss, None; Wai T. Wong, None
Support: NIH/NEI intramural research program
Mast Cell Degranulation in AMD Choroid
Gerard A. Lutty1, Imran A. Bhutto1, Johanna M. Seddon2, D. S.
McLeod1. 1Wilmer Eye Inst, Johns Hopkins Univ Sch of Med,
Baltimore, MD; 2Tufts Medical Center, Tufts U School of Medicine,
Boston, MA.
Purpose: Mast cells (MCs) are effector cells of the innate immune
system and are inhabitants of most mammalian choroids. They are
activated by complement (C5a), IgE, and many microorganisms.
When activated, they degranulate, releasing a plethora of proteases,
growth factors, endoglycosidases, histamine, and lipid metabolites.
The purpose of this study was to determine MC numbers and their
degranulation in age-related macular degeneration (AMD) compared
to aged choroidal tissues.
Methods: Human postmortem eyes were obtained within 24 hours
post mortem. Retina was excised from the eye cup and then the
choroid dissected intact. RPE was left on the choroid so the area of
RPE atrophy could be mapped in geographic atrophy eyes (GA). The
choroid was incubated for alkaline phosphatase enzyme activity
yielding a blue formazan reaction product in viable blood vessels.
After washing, the tissue was also incubated for nonspecific esterase
activity, which labels MCs and granulocytes as published previously
(Lutty et al, A J Path 1997). The choroid was then partially bleached
and counts of mast cells were made in at least three fields in each
area of choroid.
Results: Human choroid had MCs randomly dispersed. The number
of nondegranulated MCs in submacular choroid was greater in all
groups than temporal equatorial choroid. Although the number of
nondegranulated MCs was similar in submacular choroid between the
GA subjects and controls (mean mean 97.7 +/- 45.8/mm2 vs 90.3 +/94.9/mm2), the number of degranulated MCs was significantly
greater in the GA submacular choroid (76.9 +/- 45.2 vs 2.8 +/10/mm2, p<0.0001 using unpaired t test with Welch corrections).
Also, the GA subject had significantly less degranulated MCs in
temporal equatorial choroid than submacular (76.9 +/- 45.2 vs 6.9 +/9.2/mm2, p<0.0001). One control eye was note worthy: this had the
highest mast cell counts of all eyes (215.7/mm2 in macula and
83/mm2 at equator) and the subject had fibromyalgia, which
increases mast cell numbers in skin. However, none of the mast cells
were degranulated in this subject.
Conclusions: The significantly higher number of degranulated mast
cells in GA submacular choroid suggests that mast cell degranulation
may contribute to the pathologic decline of this tissue in GA. The
proteases released in degranulation could degrade the choroidal
stroma and Bruchs membrane and cause death of RPE and
endothelial cells, while the cytokines released can attract
macrophages.
Commercial Relationships: Gerard A. Lutty, None; Imran A.
Bhutto, None; Johanna M. Seddon, Genentech (F), Tufts Medical
Center (P); D. S. McLeod, None
Support: EY09357 (GL), EY016151 (GL), EY01765 (Wilmer),
EY011309 (JMS), RPB unrestricted funds, Lions, and the Beckman
Foundation
The Adult DCX-dsRed Transgenic Rat Retina: Characterization
of DsRed Positive Cells
Andrea Trost1, Falk Schroedl1, 2, Barbara Bogner1, Clemens
Strohmaier1, Christian Runge1, Guenther Grabner1, Ludwig Aigner3,
Herbert A. Reitsamer1. 1Ophthalmology/Optometry, Paracelsus
Medical University, Salzburg, Austria; 2Anatomy, Paracelsus
Medical University, Salzburg, Salzburg, Austria; 3Molecular
Regenerative Medicine, Paracelsus Medical University, Salzburg,
Salzburg, Austria.
Purpose: The doublecortin-dsRed transgenic reporter rat was
designed to analyze neurogenesis in the aged brain. Doublecortin
(DCX) is specifically and transient active in neuronal precursors and
young neurons. The aim of this study was to characterize possible
DCX-dsRed positive cells in the adult rat retina and to analyze
whether the DCX-dsRed rat might represent an appropriate model to
study neuronal de- and regeneration in the rat eye in different
pathological situations (e.g., ocular hypertension, optic nerve
transection, diabetes).
Methods: Whole mounts and sections of adult DCX-dsRed rat
retinas were prepared for immunohistochemistry and tested for the
neuronal markers DCX, NF200, Brn3a1, SOX2, calbindin, calretinin,
PKCa, ChAT as well as the glial markers GFAP and CRALBP. Colocalization with dsRed positive cells was analyzed via confocal
laser-scanning microscopy.
Results: In adult DCX-dsRed transgenic rats, dsRed-positive cells
were detected in the inner nuclear layer (INL), the ganglion cell layer
(GCL) and in perivascular cells.
DsRed positive cells in the INL showed co-localization with the
horizontal cell marker calbindin and were also positive for NF200
and DCX, but were lacking the Müller glia marker CRALBP, the rod
bipolar marker PKCa, the amacrine cell marker ChAT, and the
progenitor marker SOX2. About half of all dsRed positive cells in the
INL were lacking the above mentioned markers and could not be
classified yet.
In the GCL the majority of dsRed positive cells showed colocalization with the ganglion cell marker Brn3a, while a minority
displayed immunoreactivity for calbindin only (probably representing
displaced amacrine cells). A subpopulation was enmeshed by GFAP
positive filaments and showed perivascular localization.
Conclusions: In adult rat retina, DCX-dsRed cells in the GCL were
identified as retinal ganglion cells, amacrine cells and perivascular
cells, while in the INL part of dsRed positive cells represented
horizontal cells. Although the DCX-dsRed staining in the retina do
not seem to mark retinal neuronal progenitor cells specifically, as
seen in the brain, this model represents an useful tool to study retinal
ganglion cells in pathological conditions, such as glaucoma.
Commercial Relationships: Andrea Trost, None; Falk Schroedl,
None; Barbara Bogner, None; Clemens Strohmaier, None;
Christian Runge, None; Guenther Grabner, AcuFocus (F),
Polytech (C), AMO (F); Ludwig Aigner, None; Herbert A.
Reitsamer, None
Support: PMU-FFF, Fuchs Endowment, Lotte Schwarz Endowment,
Adele Rabenstein Endowment
Lipid Peroxidation in the Rat Retina after Elevated Intraocular
Pressure
Karen M. Joos1, Raymond Mernaugh2, Ratna Prasad1, Pengcheng
Lu3, Lyman Roberts4. 1Vanderbilt Eye Institute, Vanderbilt
University, Nashville, TN; 2Biochemistry, Vanderbilt University,
Nashville, TN; 3Biostatistics, Vanderbilt University, Nashville, TN;
4
Clinical Pharmacology Division/Pharmacology, Vanderbilt
University, Nashville, TN.
Purpose: Evidence of oxidative stress has been demonstrated in
tissues with glaucoma damage. Oxidative stress may lead to lipid
peroxidation including formation of highly-reactive γ-ketoaldehydes
(γ-KA) which rapidly form covalent adducts to proteins,
phospholipids, or DNA. In the current study, we determined whether
there is increased γ-KA protein adducts within the eye following
early moderate intraocular pressure (IOP) elevation.
Methods: The study was approved by the Vanderbilt IACUC and
conducted in compliance with the ARVO Statement for the Use of
Animals in Ophthalmic and Visual Research. IOP was transiently
elevated for 1 hour with an adjustable lasso around the right topically
anesthetized eye of Sprague-Dawley rats. IOP was measured before,
immediately after, at the end of 1 hour treatment, and 1 hour after rest
using TonoLab tonometry (ICare, Finland). Rats were euthanized and
retinas immediately harvested for SDS-PAGE/Western blot analysis,
or rats were euthanized and perfused transcardially with phosphate
buffered saline followed by 3% formaldehyde, 0.1% glutaraldehyde
(v/v) and 0.2% saturated picric acid (v/v), in 0.1M phosphate buffer.
Ocular sagittal sections were prepared for immunohistochemistry
(IHC) with a single-chain antibody that recognizes γ-KA protein
adducts on all proteins. Intensity of immunoreactivity was assessed
semi-quantitatively using the MetaMorph® Microscopy Automation
& Image Analysis 7.6 Software (Molecular Services, Sunnyvale,
CA).
Results: Mean baseline IOP of 19.6 ± 1.4 mm Hg increased to 43.6 ±
1.1 mm Hg (P < 0.01) during 1-hour treatments and returned to 20.4
± 2.3 mm Hg (P = 0.75) 1 hour after completion. The final IOP also
was not significantly different from the final control eye IOP of 20.4
± 0.9 mm Hg (P = 0.75). Elevated γ-KA protein adducts were found
in the Western Blot retinal lysate, and in the inner plexiform layer of
the IHC experimental eyes compared to the paired contralateral
control eyes (n =10) with an OD/OS ratio = 1.55 ± 0.23, (P = 0.006,
Wilcoxon Rank Sum Test).
Conclusions: One hour of moderate elevation in IOP increased the
presence of γ-KA adducts within the inner retina in the rat model.
Lipid peroxidation appears present within the eye following early
IOP elevation.
Commercial Relationships: Karen M. Joos, Vanderbilt University
(P); Raymond Mernaugh, None; Ratna Prasad, None; Pengcheng
Lu, None; Lyman Roberts, None
Support: Joseph Ellis Family and William Black Glaucoma
Research Funds, NEI Core Grant 5P30 EY08126, Unrestricted
Departmental Grant from Research to Prevent Blindness, Inc., NY.
Detection of the neuroregulatory peptide alarin in cranial
autonomic ganglia of the rat
Falk Schroedl1, 2, Alexandra Kaser-Eichberger2, Andrea Trost2,
Clemens Strohmaier2, Barbara Bogner2, Christian Runge2, Barbara
Kofler3, Herbert A. Reitsamer2. 1Ophthalmology and Anatomy,
Paracelsus University Salzburg, Salzburg, Austria; 2Ophthalmology,
Paracelsus University Salzburg, Salzburg, Austria; 3Pediatrics, LauraBassi Centre of Expertise, THERAPEP, Paracelsus University
Salzburg, Salzburg, Austria.
Purpose: Alarin is a recently discovered neuroregulatory peptide
with vasoconstrictive activity in murine skin. It is expressed in
various regions of the brain and was lately also detected in retinal
neurons of rat and mouse and in humans in intrinsic choroidal
neurons. Autonomic innervation is essential for many aspects of
ocular homeostasis, and alarin might be involved in this autonomic
control. Here we ask if alarin is present in the various autonomic
ganglia supplying the eye and explore its impact in ocular
innervation.
Methods: Cranial autonomic ganglia of the rat (i.e. superior cervical,
SCG; ciliary, CIL; pterygopalatine, PPG; trigeminal, TRI) were
prepared for immunohistochemistry against alarin using affinity
purified antibodies and respective established ganglionic markers
(SCG: TH; PPG and CIL: ChAT; TRI: SP). For documentation,
confocal laser scanning microscopy was used. Presence of alarin was
quantified in ten non-consecutive serial sections of each ganglion and
quantitative real-time PCR was applied to detect alarin mRNA
expression in corresponding ganglia.
Results: Weak alarin-like immunoreactivity was detected in neurons
of all cranial autonomic ganglia. Quantitative evaluation revealed that
those represent only a minority of the overall cell-population in the
ganglia investigated: TRI: 8/772; PPG: 4/940; SCG: 18/903;
CIL:11/315. Quantitative real-time PCR was not able to detect a
stable alarin mRNA signal in any of the (pooled, n= 4) ganglia.
Conclusions: Alarin has been described in various regions of the
CNS and eye. Since it is only present in a minority of neurons of rat
cranial autonomic ganglia, and since we were not able to detect alarin
mRNA, we consider it of low impact on ocular autonomic
innervation, at least under physiological conditions. Further
investigations in other species are needed to clarify the role of alarin
function in the eye.
Commercial Relationships: Falk Schroedl, None; Alexandra
Kaser-Eichberger, None; Andrea Trost, None; Clemens
Strohmaier, None; Barbara Bogner, None; Christian Runge,
None; Barbara Kofler, None; Herbert A. Reitsamer, None
Support: PMU FFF (E-11713/068-SRO), The Fuchs Foundation,
Adele Rabensteiner Foundation, Lotte Schwarz Endowment
Immunohistochemical Features of Encapsulated Blebs Following
Ahmed Glaucoma Valve Implantation
fatima fikri1, Deepak P. Edward1, 2, Sami A. Al Shahwan1, Khitam Al
Hati1, Ibrahim Al Jadaan1. 1King Khaled Eye Specialist Hospital,
Riyadh, Saudi Arabia; 2The Wilmer Eye Institute/Johns Hopkins
Hospital, Baltimore, MD.
Purpose: We hypothesized that the extracellular matrix (EM) of
encapsulated
bleb played an important role in the hydraulic resistance of
encapsulated
blebs. To test this hypothesis we used immunohistochemistry to label
the EM and
proliferating fibroblasts in excised encapsulated blebs following
Ahmed
Glaucoma valve (AGV) implantation and compared them with
control Tenons
tissue.
Methods: Excised encapsulated blebs (n=14) and normal Tenons
tissue (n=4)
were studied. Using indirect immunohistochemistry, the tissue was
labeled
with antibodies against collagen III, heparan sulfate, TGF β and
PCNA. The
staining intensity was assessed in a semiquantitative fashion by two
masked
observers and graded from 0-+4 staining. The clinical features of the
patients were reviewed retrospectively.
Results: The mean age of patients was 15 ±16.3 years. The diagnosis
included congenital glaucoma (50%), secondary glaucoma (35.7%),
primary
angle closure glaucoma and primary open angle glaucoma (7.1%
each). AGV,
model S2 was inserted in 85.7%, while 14.3% received the S1 model.
The mean
IOP prior to AGV revision was 38.6 ±11.6 mmHg. The mean interval
between AGV
insertion and valve revision was 37.3 ±51.8 month. There were no
significant difference in the staining intensity of collagen III and TGF
β between the cases and controls (p=0.72 and p=0.87 respectively).
Heparan staining was more intense in the blebs compared to controls,
however it was not statistically significant (p=0.19).
EM labeling was patchy with variable intensity in the excised tissue.
The
PCNA cell count did not differ significantly from control (p=0.32).
There was a weak positive correlation between IOP at time of
revision and the degree of collagen III and PCNA staining. A weak
negative correlation was noted between IOP and revision
time, and between Collagen III, heparan and PCNA label and age.
Conclusions: The EM markers used in this study did not show
differences in
immunolabeling between excised capsules and control tissue. It is
possible
that a) EM markers other than those studied played an important role
in
increased hydraulic resistance or b) EM density as reflected by the
patchy
distribution of EM marker label rather than averaging
immunohistochemical
label maybe important in predicting the hydraulic resistance of the
capsule.
Commercial Relationships: fatima fikri, None; Deepak P.
Edward, None; Sami A. Al Shahwan, None; Khitam Al Hati,
None; Ibrahim Al Jadaan, None
Functional interactions between Mmp-2 and Mmp-14a during
axonal innervation in the developing and regenerating zebrafish
optic tectum
Inge Van Hove, Els Janssens, Djoere Gaublomme, Kim Lemmens,
Lieve K. Moons. KU Leuven, Leuven, Belgium.
Purpose: Matrix metalloproteinases (MMPs) cleave structural
elements of the extracellular matrix and many molecules involved in
signal transduction. Although an involvement of MMPs in the proper
development and regeneration of the optic circuit has been
sporadically reported, their role in pathfinding of retinal ganglion cell
(RGCs) axons and in tectal (re)innervation are still largely unknown.
Therefore, elucidating the contribution of Mmps in formation of
retinotectal projections in the developing and regenerating visual
system of zebrafish might unravel novel molecules, able to support
mammalian CNS (re)innervation.
Methods: In situ hybridization (ISH), immunohistochemistry (IHC)
and/or Western blotting were used to investigate Mmp-2 and Mmp14 expression in the retina and optic tectum (OT) during tectal
(re)innervation in zebrafish embryos and in regenerating adult
zebrafish after optic nerve crush (ONC). Antisense morpholinos for
both MMPs were applied to unravel their functions and Western
blotting was used to investigate possible interactions between both
Mmps in retinotectal development.
Results: In developing zebrafish, ISH and IHC revealed expression
of Mmp-14 in RGC axons and in the neuropil of the OT. Mmp-2
mRNA and protein were also found in the OT, more specifically in
interneurons of the stratum periventriculare (SPV) and in the stratum
fibrosum et grisum superficiale (SFGS) of the tectal neuropil.
Knockdown of Mmp-14a with specific ATG or splice morpholinos
(MOs) resulted in a reduced RGC axon innervation of the OT. Mmp2 and Mmp-14a double knockdown, performed with suboptimal
doses of ATG MOs, showed a significantly decreased tectal area, as
compared to embryos after single Mmp-14a knockdown. Importantly,
active Mmp-2 levels were reduced in Mmp14a knockdown embryos,
indicating that, also in zebrafish, Mmp14a contributes to Mmp2
activation. After ONC in adult zebrafish, preliminary data revealed
Mmp-14 downregulation during the axon outgrowth phase. However,
when tectal reinnervation occurs, Mmp-14 levels were found to be
upregulated in RGC axons. Currently, we are further investigating
whether both Mmps are involved in tectal reinnervation after ONC.
Conclusions: Overall, these findings suggest a functional link or coinvolvement between Mmp-2 and Mmp-14 in RGC axonal
(re)innervation of the OT in the developing and regenerating
zebrafish brain.
Commercial Relationships: Inge Van Hove, None; Els Janssens,
None; Djoere Gaublomme, None; Kim Lemmens, None; Lieve K.
Moons, None
Dissecting the primary site of pathogenesis in COL4A1 related
anterior segment dysgenesis
Mao Mao, Douglas B. Gould. Ophthalmology, Univ of California, SF
Sch of Med, San Francisco, CA.
Purpose: Anterior segment dysgenesis (ASD) is a spectrum of
disorders affecting the development of anterior structures of the eye,
leading to vision loss including glaucoma. Mutations in a basement
membrane component, collagen type IV alpha1 (COL4A1) have been
recently identified to cause ASD in mice and humans. As COL4A1 is
present in all ocular basement membranes, and multiple tissues are
affected in ASD, dissecting the primary site of pathogenesis can be
difficult. Here, we assessed the primary location of insult in COL4A1
mediated ASD by utilizing conditional expression of mutant
COL4A1.
Methods: Previously we characterized a Col4a1 mutant with a splice
site mutation resulting in skipping of exon 41 (Col4a1 Δex41). To
recreate the Col4a1 Δex41 allele, we developed a conditional allele
with LoxP sites flanking exon 41 of Col4a1 (Col4a1 flox41). Three
tissue-specific CRE recombinase lines were crossed with the Col4a1
+/flox41
mice to generate corresponding tissue-specific mutants.
MLR10-Cre mice express CRE in whole lens at E10.5. Wnt1-Cre
mice express CRE as early as E8.5 in neural crest derived tissues
including periocular mesenchyme that give rise to multiple cell
lineages of the anterior segment. In addition, as Col4a1 +/Δex41 mice
have abnormal iris vasculature and anterior hyphema, Tie2-Cre mice
expressing CRE in vascular endothelial cells were also included. Slitlamp examination was performed to assess the extent of ASD. As
Col4a1 +/Δex41 mice also develop optic nerve hypoplasia, histological
analysis of the optic nerves was performed to assess if Col4a1
mutations also affect optic nerve development.
Results: All three tissue-specific mutants developed ASD; however,
the disease severity differed. While Col4a1 +/flox41; MLR10-Cre and
Col4a1 +/flox41; Wnt1-Cre mice both had mild ASD that was
characterized by abnormal iris vasculature and cataract, ASD in
Col4a1 +/flox41; Tie2-Cre mice are more severe. In addition to
abnormal iris vasculature and cataract, Col4a1 +/flox41; Tie2-Cre mice
often had anterior synechia and enlarged anterior chamber. Moreover,
Col4a1 +/flox41; Tie2-Cre had mild optic nerve hypoplasia while the
other two mutants did not.
Conclusions: Our results suggest that ASD in Col4a1 mutant mice
largely results from a primary insult from ocular vasculature. The
mechanism of how abnormal ocular vasculature mutants lead to
abnormal anterior segment development remains to be determined.
Commercial Relationships: Mao Mao, None; Douglas B. Gould,
None
Support: NEI Grant 5R01EY019887
Effects of Retinopathy of Prematurity (ROP) on Intraocular
Structures
James D. Akula1, 2, Robert J. Munro1, Anne Moskowitz1, 2, Ronald M.
Hansen1, 2, Toco Y. Chui3, Sanjay P. Prabhu4, 5, Anne B. Fulton1, 2.
1
Ophthalmology, Boston Children's Hospital, Boston, MA;
2
Ophthalmology, Harvard Medical School, Boston, MA; 3Optometry,
Indiana University, Bloomington, IN; 4Radiology, Boston Children's
Hospital, Boston, MA; 5Radiology, Harvard Medical School, Boston,
MA.
Purpose: Measure the effects of ROP on intraocular structures.
Methods: We reviewed extant magnetic resonance images (MRIs)
obtained at Boston Children’s Hospital Department of Radiology
from term- and preterm-born patients for images suitable for
generation of high-resolution, coronal, pupil-optic-nerve sections.
Selected subjects (n=161) were binned twice: 1) Into four
postmenstrual age (in weeks) at birth and ROP status bins, ‘Group’
(‘Term’ ≥37; ‘Intermediate’ >32 to <37; ‘Premature’ ≤32 with no
ROP, ‘ROP’ ≤32 with ROP), and 2) into four age (in years) at
imaging bins, ‘Test Bin’ (<1; 1-3; 3-10; >10). Using software
modified from our study of the rat eye (Chui et al., J Opthalmol
2012), on images of both eyes of every subject, we measured the
axial positions of the anterior and posterior corneal surfaces (AC,
PC), the anterior and posterior lens surfaces (AL, PL), and the inner
retinal surface (Ret), among other features (see below). We fit growth
curves, L=Lm●Agen/(Age½n+Agen), to anterior chamber depth (ACD),
posterior chamber depth (PCD), and axial length (AxL) of each
Group to derive the age at which each reached half its adult length;
lens thickness (LT) did not increase with age. For every eye, an
‘abnormality’ score was calculated for ACD, PCD and AxL by
subtracting the value predicted by the Term subjects’ growth curve
from the measured value. Finally, we derived anterior segment length
(ASL) and respectively computed the ratios of ACD and ASL to
PCD. All data were analyzed by ANOVA (Group×Test Bin×Eye)
followed by Tukey’s HSD.
Results: For ACD, LT, PCD, and AxL, Term and Premature subjects
did not significantly differ, but Term and ROP subjects did;
importantly, ACD, PCD and AxL were lower in ROP eyes but LT
was higher. Consequently, ACD/PCD did not differ within levels of
Group but ASL/PCD was significantly higher in ROP subjects. Age½
was later for ACD, AxL, and especially PCD in ROP compared to
Term. 'Abnormality' in ACD, PCD and AxL were statistically
indistinguishable in Term, Intermediate, and Premature but higher in
ROP subjects.
Conclusions: Ocular abnormalities are increased more in ROP than
in preterm birth alone.
Commercial Relationships: C Denwood, None; Nisha Kheradiya,
None; Na Luo, None; Michael Conwell, None; Ryan M. Anderson,
None; Yang Sun, NIH (F), American Glaucoma Society (R), Knights
Templar Eye Foundation (R), Reeves Foundation (R)
Support: NIH EY-K08022058, Knights Templar Eye foundation,
American Glaucoma Society
The eye (green shaded region) and lens (blue shaded region) were
segmented individually and the AC, PC, AL, PL and Ret surfaces
detected (red lines). From these surfaces, the ACD and PCD (orange
arrows), ASL (green arrow) and AxL (blue arrow) were measured.
Commercial Relationships: James D. Akula, None; Robert J.
Munro, None; Anne Moskowitz, None; Ronald M. Hansen, None;
Toco Y. Chui, None; Sanjay P. Prabhu, None; Anne B. Fulton,
None
Support: Children's Hospital Boston Ophthalmology Foundation
PTF1a in anterior eye development in zebrafish
C Denwood, Nisha Kheradiya, Na Luo, Michael Conwell, Ryan M.
Anderson, Yang Sun. Ophthalmology, Indiana University Glick Eye
Institute, Indianapolis, IN.
Purpose: Pancreas transcription factor 1 alpha (Ptf1a) is involved in
the differentiation of pancreas islet cells and may be involved in the
differentation of cell types in other organ systems. In particular it has
been shown to be expressed in the horizontal and amacrine cells
during retinal zebrafish development. Our purpose is to determine the
presence of anterior segment tissues of zebrafish during
embryogenesis.
Methods: Transgenic Ptf1a:green fluorescent protein (GFP) strain
zebrafish were acquired and allowed to develop for 5 days. Fish was
embedded in Agarose gel and in-vivo imaging was performed. The
zebrafish were scanned with 0.5 micron slices, using Zeiss confocal
camera and Z-stack images were obtained.
Results: Ptf1a is transiently expressed early in the anterior segment
development of zebrafish, as well as in the horizontal and amacrine
cells of retina. Protein expression was evaluated and distribution of
protein expression was confirmed by immunoblot analysis and
immunohistochemistry.
Conclusions: In this study we show Ptf1a expression in the anterior
segment during early development in the zebrafish. Future steps
involve determining differentiation pathways and resulting cell types
with particular attention to the inner non-pigmented ciliary
epithelium and possible implications for aqueous production.
In-vivo imaging of Ptf1a expression in the anterior segment of
developing transgenic Ptf1α:GFP zebrafish
Pathologic features, expression and activity of MMPs in sclera
from patients with nanophthalmos
Jing Tao, Ningli Wang. Beijing Tongren Eye Center, Beijing Tongren
Hospital,Capital Medical University, Beijing, China.
Purpose: Nanophthalmos is the leading cause of blindness in
hereditary eye diseases, characterized by abnormal remodelling of
scleral stroma. The purpose of this study was to investigate the
mechanism of nanophthalmos by assessing the pathologic features
and the role of matrix metalloproteins (MMPs) in sclera.
Methods: 80 eyes of 40 patients (15 patients from 8 pedigrees and 25
sporadic patients) with nanophthalmos were studied between 1995
and 2012. 51 nanophthalmos eyes with complications during the
progressive stage were treated by improved sclerectomy and
sclerotomy. Sclera samples were collected from 14 patients with
nanophthalmos during sclerectomy, and sclera samples from eye
bank eyes were collected as normal control. Pathologic characters of
the excised sclera were analyzed by immunohistochemical and
electron microscopic examinations. The protein levels of MMP-1,
MMP-2, MMP-3 and TIMP-1, TIMP-2 in sclera were analyzed by
western-blot, and the activity level of MMP-2 was analyzed by
gelatin enzymography.
Results: Nanophthalmos is characterized with short ocular axial
length (15.95±0.76mm), thick sclera (0.917±0.119mm) and ciliary
body, and crowded anterior chamber, complicated with uveal
effusion or/and angle-closure glaucoma during the progressive stage.
The collagen fiber bundles are irregularly arranged and separated into
small fibrils, and glycogen granules were found accumulated between
the twisting or fraying collagen fibrils in sclera of nanophthalmos.
The proteins of MMP-1, MMP-2, MMP-3 and TIMP-1, TIMP-2 were
detected both in nanophthalmos and normal sclera. There was no
statistically difference in the protein and activity levels between the
two groups (P>0.05).
Conclusions: The abnormal remodelling of scleral stroma played
important roles in the occurrence and development of nanophthalmos
complication. No abnormal expression of MMP-1, MMP-2, MMP-3
and TIMP-1, TIMP-2 was detected in sclera from patients with
nanophthalmos, according to this study.
Commercial Relationships: Jing Tao, None; Ningli Wang, None
Support: National Natural Science Foundation of China (No.
30700920),Beijing Nova Program (No. 2005B50)
Development, Composition, and Architecture of the Mouse
Ciliary Zonule
Steven Bassnett, Alicia De Maria, Yanrong Shi. Ophthal & Vis
Science, Washington Univ Sch of Med, Saint Louis, MO.
Purpose: Ocular manifestations of Marfan syndrome include myopia
and ectopia lentis. Marfan syndrome is caused by mutations in
fibrillin1, a glycoprotein enriched in force-bearing structures such as
ciliary zonule. The use of mouse Marfan models for ocular studies
has been limited because little is known about the nature of the
murine zonule. The present study was designed to help fill this
knowledge gap.
Methods: Oligonucleotide probes were raised against fbn1 and fbn2
and used for in situ hybridization. Antibodies against fibrillin
isoforms and microfibril-associated glycoprotein-1 (Magp1) were
used for immunofluorescence applications, in conjunction with
volume-rendering techniques.
Results: Fbn2 was the dominant fibrillin expressed in the embryonic
eye. Fbn2 was transiently expressed in the vascular tunic, where it
was found associated with Magp1 in microfibrils. By E16.5, Fbn2
was expressed strongly by non-pigmented ciliary epithelial (NPCE)
cells. Zonular fibers were evident by P1, after which Fbn2 expression
declined and Fbn1 expression increased. By P30, a well-organized
ciliary zonule was present. Zonular fibers projected from the
posterior portion of the pars plicata to anterior, posterior, and
equatorial termination points on the lens capsule. The posterior fibers
attached to a dense meshwork of radially-oriented microfibrils on the
capsular surface. The microfibrils formed a 100 micrometer-wide
band encircling the lens. 3D-reconstructions revealed that this
“fibrillar girdle” was situated above the transition zone, a region of
the epithelium where cells commit to terminal differentiation.
Conclusions: The organization/composition of the mouse ciliary
zonule was grossly similar to that of humans, suggesting that mouse
Marfan models may provide useful insights into human ocular
disease. A spatial relationship between the zonular attachment and
the transition zone of the lens epithelium was noted. In view of the
known ability of microfibrils to modulate BMP signaling, the zonule
could thus serve both a structural role and a role in lens growth.
Finally, our data suggest a model for how the complex architecture of
the zonule may arise. Connections between the ciliary epithelium and
the lens capsule form during embryogenesis, when the two tissues are
in intimate contact. The characteristic fanning of zonular fibers may
be the consequence of differential growth rates in lens and NPCE
surface areas.
Commercial Relationships: Steven Bassnett, None; Alicia De
Maria, None; Yanrong Shi, None
Support: NIH Grant R01 EY09852
Closing the gap: development of a novel zebrafish-based tool to
assess optic fissure closure
Pamela R. Pretorius1, Yusuf Agamawi1, Julia M. Hatler1, Stephanie
L. Lerach1, Fei Qi2, Bo Zhang2, Brent R. Bill3, Shuo Lin4, Lisa A.
Schimmenti1. 1Department of Pediatrics, University of Minnesota,
Minneapolis, MN; 2College of Life Sciences, Peking University,
Beijing, China; 3Semel Institute for Neuroscience and Human
Biology, UCLA, Los Angeles, CA; 4Department of Molecular, Cell
and Developmental Biology, UCLA, Los Angeles, CA.
Purpose: Proper closure of the optic fissure during early
embryogenesis is critical for normal eye formation. Failure of optic
fissure closure results in coloboma and related ocular defects.
Mutations in several developmentally important genes are known to
cause colobomas; however, the genetic etiology for most patients
remains unknown. As a means to characterize novel genes and
pathways that contribute to coloboma formation, we developed a
method to evaluate optic fissure closure in zebrafish.
Methods: The genetic and developmental similarities to the
mammalian eye make zebrafish (Danio rerio) an ideal model to study
early vertebrate eye development. To evaluate optic fissure closure,
we assayed for changes in Pax2a expression, a gene transiently
expressed in the ventral optic cup prior to optic fissure closure. The
quantity of green fluorescent protein (GFP) in the enhancer trap
mp204a:GFP transgenic zebrafish line was used as a proxy for Pax2a
expression. As proof of principle, we tested whether sema3e, a gene
expressed in ventral mesenchyme that exists between the edges of the
optic fissure, affected GFP levels in the enhancer trap mp204a:GFP
transgenic line. As a second test of optic fissure closure, we evaluated
basement membrane dissolution by observing changes in laminin
expression at the edges of the closing optic fissure.
Results: Transgenic expression of GFP in the mp204a:GFP enhancer
trap line recapitulates endogenous Pax2a expression in the eye field,
midbrain-hindbrain boundary, otic placode and pronephric
mesoderm. Quantitative three-dimensional analysis using ImageJ
software revealed increased expression of Pax2a in sema3e
knockdown embryos compared to uninjected controls at 48 hours
post fertilization (hpf), indicating aberrant optic fissure closure.
Moreover, a reduction in overall eye size was observed at 48 hpf. We
also observed that in sema3e knockdown zebrafish, laminin
expression was increased compared to uninjected wild-type and the
optic fissure edges exhibited delayed closure.
Conclusions: We demonstrate that quantitative three-dimensional
analysis of the enhancer trap mp204a:GFP transgenic zebrafish line
can facilitate the identification and screening of candidate genes that
impact optic fissure closure.
Commercial Relationships: Pamela R. Pretorius, None; Yusuf
Agamawi, None; Julia M. Hatler, None; Stephanie L. Lerach,
None; Fei Qi, None; Bo Zhang, None; Brent R. Bill, None; Shuo
Lin, None; Lisa A. Schimmenti, None
Support: NIH Grant 5T32DE007288-16
Congenital eye defects due to failure of embryonic eyelid closure
Qinghang Meng, Maureen Mongan, Jinling Zhang, Ying Xia.
Universiity of Cincinnati, Cincinnati, OH.
Purpose: Mammalian eye development involves a transient closure
and re-opening of the eyelid. In humans, the upper and lower eyelids
fuse to each other at week 9-12 and they separate at 4-6 months post
fertilization. In mice, the eyelid development undergoes a similar
process. The mouse eyelid closes at embryonic day (E) E15-E16.5,
but different from the humans, the mouse eyelids remain closed at
birth and its re-openning takes place at postnatal day 12-14. Failure
of eyelid closure in mice leads to an eye-open at birth (EOB)
phenotype. We used genetic mutant mice with the EOB phenotype as
models to evaluate whether failure of embryonic eyelid closure is
associated with congenital eye diseases.
Methods: Three mouse strains that displayed the EOB phenotypes
were used for this study. One is the Map3k1ΔKD/ΔKD mice, which
expressed a kinase-dead mitogen-activated protein kinase kinase
kinase 1 (MAP3K1). Two is the Dkk2 knockout mice, which lack the
Wnt inhibitor DKK2. Three is the c-JunΔOSE/ΔOSE mice, in which the
transcription factor c-Jun is ablated only in ocular surface epithelium
by crossing c-Jun flox and lens epithelium (Le)-cre mice. The eyes of
wild type and knockout mice were collected at the prenatal pre-eyelid
closure (E15.5) and post-eyelid closure (E16.5-E18.5) stages, and the
postnatal pre-eye open (p1-p12) and post-eyelid open (after p14)
stages. These eyes were subjected to histology and
immunohistochemistry analyses.
Results: Besides the eyelid closure defect, all the knockout mice had
aberrant extraocular muscles (EOM). In addition, the Map3k1ΔKD/ΔKD
and c-JunΔOSE/ΔOSE mice displayed smaller lens, while the Dkk2-null
mice did not.
Conclusions: Studies in the mouse models suggest that failure of
eyelid closure may be responsible for congenital defects of
extraocular muscle, and MAP3K1 and c-Jun may have additional
roles in lens development.
Commercial Relationships: Qinghang Meng, None; Maureen
Mongan, None; Jinling Zhang, None; Ying Xia, None
Support: NIH Grant EY15227
The Developmental Roles of c-Jun in Ocular Surface Epithelium
Maureen Mongan1, Qinghang Meng1, Winston Kao2, Ying Xia1, 2.
1
Environmental Health, University of Cincinnati, Cincinnati, OH;
2
Ophthalmology, University of Cincinnati, Cincinnati, OH.
Purpose: At embryonic day (E) 15.5, the mouse eyelid closes as the
result of eyelid epithelial cell migration. The MAP3K1-JNK
signaling cascade is required for embryonic eyelid closure, but its
downstream effectors have not been identified. c-Jun is a
transcription factor and potential substrate of JNK. Here we used
genetic tools to investigate the roles of c-Jun in ocular surface
epithelium and its crosstalk with MAP3K1 in embryonic eyelid
closure
Methods: The c-Junflox mice were used to cross with the lens
epithelium (le)-cre mice to generate the c-JunΔOSE/ΔOSE mice, in which
c-Jun was ablated specifically in the ocular surface epithelium. The
Map3k1ΔKD/ΔKD mice express a kinase-dead MAP3K1 protein fused to
a β-Galactosidase. Standard crossing techniques were used to
generate the Map3k1+/ΔKDc-Jun+/ΔOSE double hemizygous mice.
Fetuses were collected at E15.5-18.5 and postnatal day 1. The eyelid
closure status was documented, eye morphology was examined by
H&E, and the expression of c-Jun was detected by
immunohistochemistry.
Results: In the wild type E15.5 fetuses, strong c-Jun expression was
found in the equatorial zone of the lens and the leading edge eyelid
epithelium, weaker expression was detected in cornea and inner
eyelid epithelium, but almost no expression was seen in the outer
eyelid and skin epithelium. Besides the ocular surface epithelium, cJun was strongly expressed within the dermis of the eyelid, extra
ocular muscles, brain and retina. In the c-JunΔOSE/ΔOSE fetuses, though
c-Jun expression in other tissues remained strong, its expression in
ocular surface epithelium was markedly decreased. In contrast to the
wild type mice, which were born with their eyelids closed, the cJunΔOSE/ΔOSE mice were born with the eye-open at birth (EOB)
phenotype due to failure of embryonic eyelid closure. Both cJunΔOSE/ΔOSE and Map3k1ΔKD/ΔKD mice displayed the EOB phenotype;
however, the Map3k1+/ΔKDc-Jun+/ΔOSE double hemizygous mice were
born with closed eyes.
Conclusions: Our results suggest that c-Jun expression in the ocular
surface epithelium is essential for embryonic eyelid closure. Lacking
genetic complementation between MAP3K1 and c-Jun suggests that
these factors may control embryonic eyelid closure through
independent mechanisms.
Commercial Relationships: Maureen Mongan, None; Qinghang
Meng, None; Winston Kao, None; Ying Xia, None
Support: EY15227
The functional significance of zinc-finger protein Nlz2 in optic
fissure closure
Grace Shih1, 2, Ramakrishna Alur1, Felix I. Onojafe1, Brian P.
Brooks1. 1National Eye Institute, Bethesda, MD; 2Howard Hughes
Medical Institute, Bethesda, MD.
Purpose: Currently, the genetic networks underlying the closure of
the optic fissure during vertebrate eye development are poorly
understood. Failure of optic fissure closure leads to a potentially
blinding congenital ocular malformation called uveal coloboma.
Profiling of global gene expression during optic fissure closure has
suggested a role for the C2H2 zinc finger proteins Nlz1 and Nlz2 in
early eye development. Knockdown of either gene in zebrafish using
a morpholino strategy results in optic fissure closure defects, perhaps
via dysregulation of the critical transcription factor, Pax2. The aim of
this project was to determine the role of the Nlz2 gene in optic fissure
closure in the mammalian eye.
Methods: Long range PCR and southern blotting confirmed the
homologous recombination of the knockout mouse construct. Betagalactosidase staining was used to study Nlz2 gene expression in
knockout mice. Histopathological and immunohistochemical stains of
Nlz2 mouse embryo sections at E11.5 and E13.5 days were evaluated
for ocular and systemic morphological differences. Fundus and slit
lamp photography were employed in adult mice. The two exons of
NLZ2 (ZNF503) were amplified and sequenced (Sanger
dideoxynucleotide sequencing) from genomic DNA of 174 patients
with clinically observed coloboma.
Results: Nlz2 demonstrates homozygous lethality in knockout mice
by E17.5 days. Beta-galactosidase staining of Nlz2 mice
demonstrated widespread expression, particularly in the eyes,
hindbrain, facial prominences, and limbs. Gross and histopathological
sections of the developing eye revealed a failure of the optic fissure
to close in homozygous knockout mice by E13.5-E14.5 days, but not
in heterozygous or wild-type mice. Fundus and anterior segment
evaluation of adult mice revealed no overt phenotypic differences
between heterozygotes and wild-type mice. No NLZ2 sequence
changes or SNPs were found in our coloboma patient population.
Conclusions: Knockout of Nlz2 in mice leads to a failure of the optic
fissure to close, a phenotype which closely resembles that seen in
human uveal coloboma. Additionally, the gene product appears to
function in the fusion of the closely apposed edges. As no direct
NLZ2 sequence mutations were found in a cohort of patients with
coloboma, further genetic studies will be conducted to elucidate the
mechanism by which Nlz2 interacts with other genes or players
causative of uveal coloboma.
Commercial Relationships: Grace Shih, None; Ramakrishna
Alur, None; Felix I. Onojafe, None; Brian P. Brooks, None
330 From Cytology to Proteomics: New Insights into the Vitreous
and Its Role in Ocular Disease - Minisymposium
Tuesday, May 07, 2013 11:00 AM-12:45 PM
608 Minisymposium
Hyalocytes in Ocular Development and Disease
Paul G. McMenamin. Dept of Anatomy & Dev Biology, Monash
University, Melbourne, VIC, Australia.
Commercial Relationships: Paul G. McMenamin, None
Proteomics of Embryonic Human Vitreous Development
Lloyd P. Aiello. Ophthalmology-Eye Res, Joslin Diabetes Center,
Boston, MA.
Commercial Relationships: Lloyd P. Aiello, Genentech (C),
Genzyme (C), Thrombogenetics (C), Ophthotech (C), Kalvista (C),
Pfizer (C), Proteostasis (C), Abbott (C), Vantia (C), Optos, plc (F)
Role of Exosomes in Aqueous Humor Dynamics
W Daniel Stamer. Ophthalmology, Duke University, Durham, NC.
Commercial Relationships: W Daniel Stamer, Allergan (F), Alcon
(F), Acucela (C), Aerie (C), Cytokinetics (C)
Identification of Protein Biomarkers in Vitreous Humor
Following Laser Exposure
Rachida Bouhenni. Ophthalmology, Summa-Health System, Akron,
OH.
Commercial Relationships: Rachida Bouhenni, None
Advances in Understanding and Treatment of Diabetic
Retinopathy from Vitreous Studies
Elia J. Duh. Ophthalmology, Johns Hopkins Wilmer Eye Inst,
Baltimore, MD.
Commercial Relationships: Elia J. Duh, None
Presentation Time: 12:12 PM - 12:26 PM
Anomalous PVD and Pharmacologic Vitreolysis
J Sebag. VMR Institute, Univ of Southern California, Huntington
Beach, CA.
Commercial Relationships: J Sebag, ThromboGenics (C),
ThromboGenics (I)
Proliferative Vitreoretinopathy: Od Problem, New Treatments
Andrius Kazlauskas. Ophthalmology, Schepens Eye Res Inst/
Harvard, Boston, MA.
Commercial Relationships: Andrius Kazlauskas, None
365 Myopia: Molecular/Genetic Mechanisms
Tuesday, May 07, 2013 2:45 PM-4:30 PM
608 Paper Session
Association studies of EGR-1, PCDHB9, NARF, OGDH and
SELENBP1 with myopia and myopia sub phenotypes reveals a
novel association of OGDH with corneal curvature
Paul N. Baird, Maria Schache. Ctr for Eye Res-Australia, University
of Melbourne, East Melbourne, VIC, Australia.
Purpose: The exact nature of the genetic mechanisms underlying
ocular growth in myopia has not been fully established. A number of
factors have been implicated through animal studies, in particular, the
early growth response protein Egr-1 in mice (Zenk, the avian
homolog of Egr-1 in chicks). In Egr-1 knockout mice, several other
differentially expressed genes; pcdhb9, narf, ogdh and selenbp1 have
also been identified. We wished to assess the association of these
genes in common myopia, refraction, axial length, anterior chamber
depth and corneal curvature in an Australian cohort.
Methods: Individuals of European background were recruited as part
of the Genes in Myopia (GEM) Study. Myopia was defined as -0.5D
or less in the right eye. A total of 543 individuals from the GEM
study were used for analysis including 314 myopic and 229 non
myopic individuals. Tag single nucleotide polymorphisms (SNPs)
were chosen to encompass 2kb upstream of the start codon through to
2kb downstream of the stop codon of the chosen genes. Statistical
analysis used logistic and linear regression methods including age
and sex as covariates. Bonferroni corrections were applied to account
for multiple testing.
Results: 28 SNPs were genotyped encompassing the EGR-1,
PCDHB9, NARF, OGDH AND SELENBP1 genes. Following
statistical analysis and correction for multiple testing (corrected
p=0.05/28=0.0018), association at SNP rs17133935 in the OGDH
gene remained statistically significant for corneal curvature (p
uncorrected=0.0008).
Conclusions: A variant in the OGDH gene was shown to be
associated with corneal curvature in this cohort. The ERG-1,
PCDHB9, NARF, OGDH AND SELENBP1 genes did not appear to
be associated with myopia, refractive error, anterior chamber depth
and axial length in this cohort. This is the first report of OGDH, a
gene involved in the tricarboxylic acid cycle, being associated with
corneal curvature suggesting there may be a potential mitochondrial
involvement in this phenotype.
Commercial Relationships: Paul N. Baird, None; Maria Schache,
None
Support: National Health and Medical Research Council
(NH&MRC) Canberra, Australia through the Centre for Clinical
Research Excellence in Translational Clinical Research in Eye
Diseases (grant no. 529923) and an NH&MRC research fellowship to
P.N.B (No. 1028444).
Evaluation of MicroRNA Expression Profiles for FormDeprivation Myopia in Mouse
Xiaoyan Luo1, 2, Tatiana V. Tkatchenko3, Andrei V. Tkatchenko3, 4,
Ravikanth Metlapally5, Pedro Gonzalez1, Terri L. Young1, 2.
1
Department of Ophthalmology, Duke University, Durham, NC;
2
Duke Center for Human Genetics, Duke University Medical Center,
Durham, NC; 3Department of Anatomy & Cell Biology, Wayne State
University, Detroit, MI; 4Department of Ophthalmology, Wayne
State University, Detroit, MI; 5School of Optometry, University of
California at Berkeley, Berkeley, CA.
Purpose: The study aim was to determine microRNA (miRNA)
expression profiles of ocular tissues in form-deprivation induced
myopic mice.
Methods: Fifteen C57BL/6J mice (Jackson Laboratory, Bar Harbor,
ME) were used in this study. Form-deprivation myopia was induced
in postnatal day 24 (P24) C57BL/6J mice by applying a frosted
hemispherical plastic diffuser over the right eye for 10 days. Total
RNA was isolated from the whole eye, retina, and sclera and used for
miRNA expression profiling with the Agilent mouse miRNA
microarray(Agilent Technologies, Inc., Santa Clara, CA). The
microarray raw data were processed by subtracting background noise
and performing log2 transformation. Sample outliers were removed.
The normalized microarray data were analyzed using ANOVA to
identify differences in miRNA expression level between myopic and
contralateral control eyes.
Results: ANOVA revealed 75 miRNAs with differential expressions
[fold change (FC) >= 1.5] from the whole eye, retina, and sclera.
Multiple members of the miRNA family -302 (b and c), -466 (c, g, h,
and j), and -669 (a, b, e, and o), and three members of the 290-295
cluster (290, 291 and 294) showed increased or decreased differential
expressions. Member examples included mmu-miR-302c [FC= 3.2;
p=2.1E-2], mmu-miR-466c [FC=2.3; p=2.0E-3], mmu-miR-466j
[FC=3.1; p=8.5E-4], and mmu-miR-669b [FC=2.1; p=9.0E-4] from
the whole eye; mmu-miR-294 [FC=1.5; p= 2.4E-2] and [FC=2.3;
p=8.0E-5] from retina and sclera separately. MiR-302 and the 290295 member cluster are involved in cell pluripotency maintenance.
Other differentially expressed miRNA examples included mmu-miR15a [FC=2.2; p=1.1E-3] and mmu-miR-16-1[FC=2.1; p=4.6E-4]
from the whole eye, and mmu-miR-21[FC=2.2; p=1.7E-3] from
retina. MiR-15, 16, and 21 are involved in growth and development
regulation.
Conclusions: Differential expression of miRNAs in several ocular
tissues upon induction of experimental myopia in mice suggests a
developmental and regulatory role in eye growth, and can potentially
serve as therapeutic targets for treating myopia.
Commercial Relationships: Xiaoyan Luo, None; Tatiana V.
Tkatchenko, None; Andrei V. Tkatchenko, None; Ravikanth
Metlapally, None; Pedro Gonzalez, None; Terri L. Young,
National Institutes of Health (F)
Support: R01 EY014685
Gene Expression Studies implicate Physiological Stress in Form
Deprivation Myopia
Sheila G. Crewther, Loretta Giummarra, Melanie J. Murphy.
Psychological Science, La Trobe University, Melbourne, VIC,
Australia.
Purpose: Myopia is the commonest visual disorder and a risk factor
for most disorders leading to visual compromise, yet the aetiology
remains elusive. Thus we aimed to examine the gene expression
changes underlying the morphological, ultrastructural and
physiological changes previously described, that suggest severe
physiological stress in the form deprived chick model.
Methods: Methods: Ten male hatchling chicks of which 6 were
occluded to induce form-deprivation (FD) myopia by attaching a
translucent polystyrene occluder to the periocular feathers of their
right eye from day 3 - 10. Choroid/retina/RPE preparations were
taken from the posterior eye cup, placed in RNA stabilizing buffer
and transferred to -20°C freezer. For Affymetrix microarrays,
samples were pooled and analysis was conducted using the one-cycle
process (The Australian Genome Research Facility LTD; Walter and
Eliza Hall Institute, Victoria, Australia). Data was exported as .cel
files containing probe level intensities and analysed using Pathway
Studio 8.0 (Ariadne Genomics Inc, Rockville, MD, USA). Gene Set
Enrichment Analysis (GSEA) using the Kolmogrov-Smirnov test
algorithm to identify statistically enriched (p<0.05) Gene Ontologies
(GO) as well as Ariadne Ontologies (AO), Ariadne Signalling
Pathways (ASP) and Ariadne Metabolic Pathways (AMP).
Results: At a FC>2.5, 309 genes were up- and 352 down-regulated
with Sorcin, a calcium binding protein showing a FC of +31.3 change
while acetylcholinesterase showed a FC of -10.5. Unlike previous
studies in this area we have extended our analyses to include
ontological (functional) pathways that may be involved in these
observed changes. The GSEA algorithm identified downregulation of
genes associated with cell structure and integrity and genomic strain.
An increase in cell metabolism was noted, specifically, fatty acid
oxidation as the main source of energy in these tissues.
Conclusions: The pathways associated with the myopic
pathophysiology include diminished glucose metabolism, cellular
stress responses and cell volume control. All pathways are indicative
of physiological stress and diminished ability to maintain retinal
homeostasis after a week of form deprivation. The pathways include
most genes described in previous studies. Human SNP research is
also inconsistent but mostly identifies extracellular matrix genes that
offer no indication of the mechanism underlying myopia progression.
Commercial Relationships: Sheila G. Crewther, None; Loretta
Giummarra, None; Melanie J. Murphy, None
Bidirectional Gene Expression in Tree Shrew Choroid during
Lens-Induced Myopia and Recovery
Li He, Michael R. Frost, John T. Siegwart, Thomas T. Norton. Vision
Sciences, Univ of Alabama at Birmingham, Birmingham, AL.
Purpose: To examine gene expression in the choroid that may
communicate retinally-generated GO and STOP signals to the sclera
to control axial elongation and refractive error development.
Methods: Two groups of tree shrews were used (n=7 per group).
GO: lens-induced myopia (LIM): 2 days of monocular −5 D lens
wear starting at 24 days of visual experience (DVE); STOP: 2 days of
recovery (REC) starting at 35 DVE, after compensation to a −5 D
lens. The untreated contralateral eyes served as controls. A total of 77
genes were examined by quantitative real-time PCR (qPCR),
including 14 genes that showed bidirectional expression in a wholetranscriptome analysis (RNA-Seq) of 3 animals from each group.
Results: Small refractive changes were observed in the LIM (−1.0 ±
0.2 D; mean ± SEM) and REC groups (+1.3 ± 0.3 D) indicating eyes
were early in the process of developing LIM and recovering from
LIM. As shown in the figure, 35 of 77 genes showed significant
regulation (treated eye - control eye). 13 genes (11 of 14 suggested
by RNA-Seq) showed bidirectional regulation. 2 were up-regulated in
both. 20 additional genes showed significant differences in either
LIM or REC, but not both. Of the 35 genes that showed significant
regulation, 18 are involved in signaling pathways including
TGFβ/BMP and retinoic acid signaling. The remainder are primarily
involved in extracellular matrix remodeling and some may have a
role in vascular regulation or angiogenesis. 10 additional genes that
showed consistent bidirectional changes in the RNA-Seq analysis,
but were not examined with qPCR, would likely also show
bidirectional gene expression.
Conclusions: Numerous genes in choroid show bidirectional
expression in LIM and REC as assessed with qPCR and even more
are suggested on the basis of RNA-Seq. In addition, many genes
show altered expression in either LIM or REC but not both. From
these results in choroid, it appears that many genes are involved in
conveying GO and STOP signals to the sclera. It is highly unlikely
that any single gene is primarily responsible for the control of axial
elongation and refractive error.
Commercial Relationships: Li He, None; Michael R. Frost, None;
John T. Siegwart, None; Thomas T. Norton, None
Support: NIH grants EY005922 and EY003039 (P30)
Regional variations in corneal and scleral mRNA expressions of
MMP2, TIMP2, TGFβ2 in highly myopic-astigmatic chicks
Chea-Su Kee1, Yan-yan Xi1, Chin-Hung Geoffrey Chu1, Shea Ping
Yip2, Jody A. Summers Rada3. 1School of Optometry, The Hong
Kong Polytechnic University, Kowloon, Hong Kong; 2Department of
Health Technology and Informatics, The Hong Kong Polytechnic
University, Kowloon, Hong Kong; 3Department of Cell Biology, The
University of Oklahoma, Oklahoma City, OK.
Purpose: To determine MMP2, TIMP2, TGFβ2 mRNA expressions
at different corneal and scleral regions in highly myopic-astigmatic
chicks.
Methods: High magnitudes of myopia and astigmatism were induced
by using diffuser to monocularly form-deprive chicks from P5 to P12
(n=8). Age-matched chicks without any treatment served as controls
(n=8). Refractive errors and corneal biometric parameters were
measured by, respectively, a modified Hartinger refractometer and a
custom-made corneal videokeratography system. The refractive and
corneal biometric components were expressed as interocular
difference between the treated/right and fellow/left eyes. Corneal
(1.5mm diameter) and posterior scleral tissues punches (5mm
diameter) were obtained using a disposable trephine from five
regions: central, superior, inferior, temporal and nasal. Real-time RT
PCR was used to quantify the mRNA expressions of MMP2, TIMP2,
TGFβ2, and 18S rRNA. Fold of change at each region was calculated
by 2-ΔΔCT method, where ΔΔCT=[(CT target gene - CT 18S gene)
treated eye - (CT target gene - CT 18S gene) fellow eye]. MannWhitney test was used to compare the changes between treated and
control groups.
Results: Compared to the control group, significantly higher mRNA
expressions in MMP2, TIMP2 and TGFβ2 were found at the superior
region of posterior sclera (all p≤0.014). In addition, there was higher
than normal expression of MMP2 at the nasal quadrant of posterior
sclera (p=0.046). When data from all birds were pooled for
correlation analyses, the correlations between refractive-error
components and mRNA expressions were generally higher in sclera
than those in cornea. All three target genes at the superior sclera
showed moderate-high correlations with refractive/corneal spherical
equivalent and J45 astigmatic components (r=0.51~0.88, all p≤0.01).
In addition, high correlations between TGFβ2 with MMP2 (r=0.88,
p<0.001) and TIMP2 (r=0.83, p<0.001) were found at the nasal
cornea, whereas high correlations between TGFβ2 with MMP2
(r=0.91, p<0.001) and TIMP2 (r=0.90, p<0.001) were found at the
superior sclera.
Conclusions: Regional variations in mRNA expressions of the three
target genes were found in corneal and scleral tissues. These results
suggest that the eye shape remodeling during myopia development
may be modulated by local molecular mechanism.
Commercial Relationships: Chea-Su Kee, None; Yan-yan Xi,
None; Chin-Hung Geoffrey Chu, None; Shea Ping Yip, None;
Jody A. Summers Rada, None
Support: RGC General Research Fund #561209
Atropine Prevents Myopia via a Nitric Oxide-Mediated Relay
Brittany Carr1, Neil M. Nathanson3, William K. Stell2. 1Neuroscience,
University of Calgary, Calgary, AB, Canada; 2Cell Biology &
Anatomy, Surgery, Neuroscience, and Hotchkiss Brain Institute,
University of Calgary, Calgary, AB, Canada; 3Pharmacology,
University of Washington, Seattle, WA.
Purpose: Myopia is an incurable refractive disorder that affects a
large minority of the world’s population. Atropine, a broad-spectrum
muscarinic receptor (mAChR) antagonist, is known to prevent
myopia progression, but unpleasant side-effects limit its wider use
and its mechanism is unknown. Although there is evidence for
coupling of muscarinic receptor activation with production of nitric
oxide (NO) by NO-synthase (NOS) in the retina (Cimini et al. JCN
‘08), this muscarinic-NO synthesis mechanism has not been applied
so far to myopia pathology. Here we tested the hypothesis that NO is
the primary mediator of myopia-inhibition by atropine, using a
nonselective NOS-inhibitor (L-NIO) to block NO-mediated
signaling.
Methods: The right eyes (T) of White Leghorn cockerels (P7-P8)
were goggled with diffusers to induce FDM (day 0); the left eyes
served as ungoggled controls (C). 20 µL of PBS, atropine (A; 45.3
nmol), L-NIO (300 nmol), or both (A+L-NIO) [all n=23-24] was
injected intravitreally on treatment days 1, 3, 5. On day 6, refractive
error, axial length, equatorial length, and eye weight were measured.
Control eyes were not affected by treatments to the goggled eyes so
the interocular difference (T-C; one-way ANOVA, Tukey post-hoc)
was taken as the measure of treatment effect.
Results: In PBS control, goggles induced myopic refractive error (14 ± 3D) and excessive axial length (0.5 ± 0.3mm) [T-C]. Atropine
significantly reduced [T-C] for refractive error (-9 ± 3D; p<0.001)
and axial length (0.3 ± 0.2mm; p=0.027). A+L-NIO (T) eyes were
not significantly larger or more myopic than PBS or L-NIO eyes.
Conclusions: These results show that atropine inhibits FDM in
chicks via a NO-mediated relay. M2/M4 mAChRs, the most likely
mediators of atropine’s effects, are Gi-coupled; they constitutively
inhibit cAMP synthesis, thus atropine acts as an inverse agonist at
these receptors (Tietje et al. JBC ‘90; Migeon et al. JBC ‘94). We
suggest that atropine stimulates cAMP synthesis in NO-ergic
neurons, accounting for the blockade of atropine’s anti-myopia effect
by L-NIO. Given that NO prevents FDM in chick, and that
prevention of FDM by dopamine requires activation of NOS via
D2/D4-like receptors, it is reasonable that atropine provides another
input to a common myopia-preventing network. Pathways such as
these (i.e. downstream effectors of atropine-activated receptors) may
be preferable to atropine as targets for anti-myopia drug therapy.
Commercial Relationships: Brittany Carr, None; Neil M.
Nathanson, None; William K. Stell, None
Support: NSERC Discovery Grant and Foundation Fighting
Blindness (Canada) - EYEGEYE Research Training Fund to W.K.
Stell
Reciprocal activities of dopamine D1 and D2 receptors on the
form deprivation myopia in pigmented guinea pigs
Xiangtian Zhou, Sen Zhang, Huangfang Ying, Jia Qu. School of
Ophthalmology and Optometry, Wenzhou Medical College,
Wenzhou, Zhejiang, China.
Purpose: Dopamine is involved in the development of myopia and
the exact mechanism has not been fully clarified. One of our previous
study showed that dopamine was involved in the progression of
spontaneous myopia in albino guinea pigs under natural visual
environments, probably due to a reciprocal action of the two
dopamine receptors: activation of the D1 receptor and inhibition of
the D2 receptor appear to prevent myopia. This study investigated
whether this reciprocal action also plays a role in the development of
form deprivation myopia (FDM) in pigmented guinea pigs (Cavia
porcellus).
Methods: Form deprivation was applied to pigmented guinea pigs
(age: 2 weeks) that received daily peribulbar injection of SKF38393
(selective dopamine D1 receptor agonist) at doses of 10ng, 100ng,
and 1000ng or quinpirole (selective dopamine D2 receptor agonist) at
doses of 10ng, 33ng and 100ng for a period of 2 weeks. The
refraction, corneal radius of curvature and axial components of the
eye were measured in all animals prior to, at 1week and at 2 weeks of
the experiments.
Results: The D1 receptor agonist SKF38393 inhibited the
development of FDM with the largest inhibition at a dose of 1000ng
(-4.54±1.92D vs. -7.44±2.21D, p<0.05) followed by the dose of
100ng (-5.18±2.24D vs. -7.44±2.21D, p<0.05) at 2 weeks of
experiments. The dose of 10ng of SKF38393 had no effect on FDM
in wide type guinea pigs. In contrast, the D2 receptor agonist
quinpirole promoted the development of FDM with dose dependent.
The dose of 100ng had largest promotion of FDM (-10.05±3.02D vs.6.86±2.39D, p<0.05) followed by the dose of 33ng (-8.87±2.62 D
vs.-6.86±2.39D, p<0.05) at 2 weeks of experiments. The dose of
10ng of quinpirole had no effect on FDM.
Conclusions: Our findings suggest that dopaminergic system
regulates FDM development through the reciprocal activities of two
type dopamine receptors in pigmented guinea pigs: D1 receptor
activation inhibits, while D2 receptor activation promotes myopia.
Commercial Relationships: Xiangtian Zhou, None; Sen Zhang,
None; Huangfang Ying, None; Jia Qu, None
Support: 973 project: 2011CB504602; National Natural Science
Foundation of China:30973278, 81000386
380 Retinoblastoma: Experimental and Clinical
Tuesday, May 07, 2013 2:45 PM-4:30 PM
Program #/Board # Range: 3967-3982/D0368-D0383
Program Number: 3967 Poster Board Number: D0368
Optical coherence tomography for screening of orthotopic
retinoblastoma xenografts
Andrea Wenzel1, Brian C. Samuels1, Timothy W. Corson1, 2.
1
Ophthalmology, Indiana University School of Medicine,
Indianapolis, IN; 2Biochemistry and Molecular Biology, Indiana
University School of Medicine, Indianapolis, IN.
Purpose: Retinoblastoma is the most common intraocular tumor in
children, often causing blindness. Orthotopic xenograft models are a
critical tool for studying new therapeutic methods. A successful
xenograft model has been generated by intravitreal injection of
newborn rats with a bioluminescent derivative of the Y79 human
retinoblastoma cell line. Although this model is powerful, screening
panels of xenografts from multiple cell lines would be valuable for
assessing inter-individual responses to novel therapeutics. We
evaluated whether optical coherence tomography (OCT) could be
used to identify successful xenografts of other, non-engineered
retinoblastoma cell lines and characterize xenograft growth patterns.
Methods: Sprague-Dawley rats (P0) received an intravitreal injection
of 1,000 to 250,000 retinoblastoma cells from either the standard
bioluminescent/EGFP+ Y79 cell line or other, non-engineered
retinoblastoma cell lines. The Micron III rodent imaging system was
used to obtain fundus photographs and OCT images regularly over
the course of 4 weeks to document xenograft development.
Results: Using in vivo, intraocular, EGFP fluorescence imaging as a
guide, previously described Y79-EGFP-luciferase xenografts were
characterized by OCT. The xenografts produced both small and large
tumors that were typically dense, highly vascularized, and had well-
defined edges. While direct retinal involvement was rare, xenografts
appeared to preferentially grow just posterior to the lens suggesting
that the regressing tunica vasculosa lentis and/or the regressing
hyaloid artery may be the preferred vascular source. Successful nonfluorescent xenografts had similar morphologic characteristics and
growth patterns to those from the Y79 line on OCT imaging.
Conclusions: OCT imaging of retinoblastoma orthotopic xenografts
is a novel way to spatially analyze and follow tumor growth in vivo.
When tumors were not always readily evident on brightfield imaging,
OCT proved to be a valuable tool to help identify hard-to-see, nonfluorescent tumors generated with these non-engineered
retinoblastoma cell lines. This approach will enable rapid screening
of additional cell lines in the future as well as quantitative spatial
analysis of response to therapeutics.
Commercial Relationships: Andrea Wenzel, None; Brian C.
Samuels, Merck & Co., Inc (F), Merck & Co., Inc (C), ICHE (C);
Timothy W. Corson, None
Support: Indiana CTSI, NCATS TR000006
Gene expression signature of putative Cancer Stem Cells in
Retinoblastoma Y79 cell line
Rohini M. Nair1, Murali Mohan Sagar Balla2, Imran Khan3, Ravi
Kiran Reddy Kalathur3, Paturu Kondaiah3, Santosh Honavar4,
Mohammad J. Ali4, Geeta K. Vemuganti1. 1School of Medical
Sciences, University of Hyderabad, Hyderabad, India; 2Ophthalmic
Pathology Laboratory, L.V.Prasad Eye Institute, Hyderabad, India;
3
Molecular Reproduction, Development and Genetics, Indian
Institute of Science, Bangalore, India; 4Ophthalmic and Facial Plastic
Surgery, Orbit and Ocular Oncology, L.V.Prasad Eye Institute,
Hyderabad, India.
Purpose: Gene expression studies in Cancer provide an insight into
the global functioning of tumor and their pathways. In our previous
studies on Retinoblastoma tumors, using a two parameter analysis
(size and phenotype), we observed a FSClo/SSClo population that
was CD133-CD44+ CD90- and expressed primitive stem cell
markers, lacking the expression of differentiation markers (Balla et
al. 2009). Since, CD44 and CD90 expression was absent in Y79 cell
line, we used CD133 to sort the cells and analysed for various stem
cell assays. This study highlights gene expression signature specific
to putative cancer stem cells in Y79 cell line.
Methods: Cultured Y79 cells were analysed after doublet
discrimination and sorted based on the expression of CD133 marker
using BD FACS Aria. Total RNA was isolated and quantified.
Microarray was performed in duplicates using human whole genome
(4x44K) cDNA arrays (Agilent technologies, USA) as per the
manufacturer’s instructions. The data was analyzed and normalized
using GeneSpring and Lowess algorithm. Data validation was done
using semi quantitative PCR. Pathway and interaction studies were
analyzed using GeneMania and String database.
Results: In comparison to CD133+ cells, the CD133- cells of Y79
cell line showed 2945 upregulated genes (≥1.5 fold) and 4531
downregulated genes (≤ 1.5 fold). There was down regulation of
Purine metabolism pathway (p=0.009), TGF-beta pathway(p=0.009),
p53 signaling (p=0.017), and oxidative phosphorylation pathway
(p=0.012) in CD133- cells. Pathways upregulated in CD133- cells
were involved in cell migration (3.53e-1), and cytokine signaling
(3.69e-6). Stem Cell genes such as BMI1, ABCB1, CD69, HOXA11,
KLF17 were found to be upregulated.
Conclusions: The gene expression data supports our hypothesis that
the FSClo/SSClo cells lacking CD133 expression are more
undifferentiated and possibly putative cancer stem cells in Y79 cell
line. Further studies to validate these pathways are warranted to
understand the role of cancer stem cells in Retinoblastoma.
a-d) FACS profile of CD133 sorted Y79 cells e,f) Heat map of sorted
CD133 cells
Network analysis of upregulated Genes in CD133- Y79 cells
Commercial Relationships: Rohini M. Nair, None; Murali Mohan
Sagar Balla, None; Imran Khan, None; Ravi Kiran Reddy
Kalathur, None; Paturu Kondaiah, None; Santosh Honavar,
None; Mohammad J. Ali, None; Geeta K. Vemuganti, None
Support: Indian Council of Medical Research, Hyderabad Eye
Research Foundation
Role of miR 106 - 25 family in Retinoblastoma tumouriogenesis:
in-vitro analysis of their functions using antagomirs
Subramanian Krishnakumar1, Nalini Venkatesan1, Vikas Khetan2,
Maddy A. Reddy3. 1L&T Ocular Pathology, Vision Research
Foundation, Chennai, India; 2Shri Bhagwan Mahavir vitreoretinal
services, Medical Research Foundation, Chennai, India; 3Ocular
oncology, Moorfields Eye Hospital Trust, Barts and London NHS
Trust, London, United Kingdom.
Purpose: Retinoblastoma (RB), a primary pediatric intraocular
tumor. Several novel molecular strategies are being developed for the
clinical management of RB. Here, the role of selected miRNA
cluster: miR106b~25 family have been analyzed for potential
diagnostic and therapeutic targeting in RB.
Methods: The miRNA cluster (miR-106b, miR-93, miR-25) and
their direct regulatory gene , Miniature chromosome maintenance
7(MCM7) complex were confirmed by quantitative Reverse
Transcriptase PCR (qRT-PCR) in primary retinoblastoma tissues (N=
21).Functional role of these miRNA cluster have been studied by
using antagomirs in cultured RB (Y79,Weri Rb-1) cells in-vitro. The
expression of gene targets - p21 / BIM regulated by miR-106b
family, was confirmed by western blot. Cell proliferation and
apoptotic studies have been performed using Cell viability assay
(MTT), Colony forming assay (Anchorage dependent assay), and
Annexin V binding assay, in the antagomirs-treated RB cells (Y79
and Weri Rb-1).
Results: In the 21 tumor tissues analysed, miR-106b was detected in
90.47%, miR-93 in 85.71%, miR-25 in 95.23% respectively. In
association with these miRNA clusters, we observed higher
expression of MCM7 in the RB primary tumors. In-vitro silencing of
these miRNAs in RB cells resulted in the reduction of cell
proliferation and in colony formation, through the induction of
apoptosis (confirmed by Annexin V assay).
Conclusions: The aberrant expression of miR-106b-25family in
primary retinoblastoma implicates its role in RB tumorigenesis.
Diagnostic/ Prognostic potential for miR 106b ~25 family miRNAs
in RB is indicated. The reduction in cancer cell viability by specific
antagomir suggests a potential target for RB therapy.
Funding agency: Childhood Eye cancer trust (CHECT Foundation),
London, UK
Commercial Relationships: Subramanian Krishnakumar, None;
Nalini Venkatesan, None; Vikas Khetan, None; Maddy A. Reddy,
None
Support: Childhood Eye cancer trust (CHECT Foundation), London,
UK
Immunohistochemical Analysis of PDGFR-α, PDGFR-β & c-Abl
in Retinoblastoma: Potential therapeutic targets
Mali Worme3, Leticia Rielo de Moura3, 2, Debra Sanft3, Bruno F.
Fernandes1, 3, Emilia Antecka3, Miguel N. Burnier3, 1.
1
Ophthalmology and Pathology, The McGill University Health
Center, Montreal, QC, Canada; 2Instituto Brasileiro de Oftalmologia,
Rio de Janeiro, Brazil; 3Henry C. Witelson Ocular Pathology
Laboratory, Montreal, QC, Canada.
Purpose: The aim of this study was to:
1/ Determine the immunoexpression of PDGFR-α, PDGFR-β and cAbl, known targets of imatinib mesylate (Gleevec), in human
retinoblastoma specimens.
2/ Determine if the expression of PDGFR-α, PDGFR-β and c-Abl in
retinoblastoma correlate with histopathological prognostic factors
(optic nerve invasion and choroid invasion).
Methods: Sixty-one paraffin-embedded retinoblastomas were
collected from the archives of the Henry C Witelson Ocular
Pathology Laboratory. PDGFR-α, -β and c-Abl immunostaining was
performed according to the protocol provided by Ventana Medical
System Inc., Arizona. Immunoreactivity was correlated with the
presence or absence of invasion into the choroid and optic nerve.
Optic nerve (ON) invasion was considered positive when tumor cells
could be identified beyond the lamina cribrosa, and choroidal
invasion was considered positive when tumor cells were seen to
infiltrate through Bruch's membrane. The Fisher Exact Test was used
to assess whether the immunostainining expression differed with
optic nerve or choroid invasion. Statistical significance was assumed
if the p value was less than 0.05.
Results: Overall, c-Abl expression was identified in 50 of 61
specimens (81.97%), PDGFR-α was identified in 20 of 60 specimens
(33.33%) and PDGFR-β expression was identified in 57 of 61
specimens (93.44%). The only correlation of statistical significance
was that PDGFR-β expression was negatively correlated with optic
nerve involvement (P=0.034).
Conclusions: This paper provides convincing evidence that
retinoblastoma (Rb) is a cancer that expresses the receptor tyrosine
kinases (RTKs) PDGFR-α, PDGFR-β and c-Abl. The Henry C
Witelson laboratory has shown a pharmacological effect of imatinib
mesylate (Gleevec) on Rb cells in vitro (manuscript in review).
Additional in vitro studies should be done to decipher if the
expression of the RTKs in question are relevant to the
pharmacological action of Gleevec on Rb cells, as this drug is known
to act through other pathways, not solely RTKs.
(Additionally, PDGFR-β expression is negatively correlated with a
poor histopathological prognosis).
Commercial Relationships: Mali Worme, None; Leticia Rielo de
Moura, None; Debra Sanft, None; Bruno F. Fernandes, None;
Emilia Antecka, None; Miguel N. Burnier, None
The role of endothelial progenitor cell in the tumor angiogenesis
of retinoblastoma
Dong Hyun KIM1, 2, Sung Wook Park1, 2, Byung Joo Lee1, 2, Dong
Hyun Jo1, 2, Jin Hyoung Kim1, Jeong Hun Kim1, 2. 1Fight against
Angiogenesis-Related Blindness Laboratory, Clinical Research
Institute, Seoul National University Hospital, Seoul, Republic of
Korea; 2Department of Ophthalmology, Seoul National University
Hospital, Seoul, Republic of Korea.
Purpose: To investigate the role of cancer stem cell and endothelial
progenitor cell in angiogenesis of orthotopic transplantation mouse
model of retinoblastoma.
Methods: Regarding orthotopic transplantation mouse model of
retinoblastoma, SNUOT-Rb1 cells (1x107) were intravitreallyinoculated into the eyes of the BALB/c nude mice. Four weeks after
inoculation, the mice were sacrificed and the eyes were enucleated.
To identify tumor development, H&E staining was performed in 4
um section slides. Immunofluorescence staining was performed on
mouse retinoblastoma tissue to evaluate the spatial distribution of
endothelial progenitor cell and tumor vessel using CD133 and CD34
as endothelial progenitor cell markers and collagen type IV as
endothelial cell marker.
Results: The Wintersteiner rosettes were identified in mouse
retinoblastoma tissue with SNUHOT-Rb1 cells. CD34 and CD133
were co-expressed in the Wintersteiner rosettes. CD133 was
expressed in the cells with collagen type IV consisting tumor vessels.
Conclusions: Our data demonstrated that orthotopic transplantation
mouse model of retinoblastoma was established by intravitreous
injection of SNUHOT-Rb1 cells. Endothelial progenitor cells might
contribute tumor angiogenesis in retinoblastoma.
Commercial Relationships: Dong Hyun KIM, None; Sung Wook
Park, None; Byung Joo Lee, None; Dong Hyun Jo, None; Jin
Hyoung Kim, None; Jeong Hun Kim, None
Metformin affects the growth and redox state of human
retinobastoma cells
Katarzyna A. Brodowska, Evangelos S. Gragoudas, Demetrios
Vavvas. Mass Eye and Ear Infirmary, Boston, MA.
Purpose: Metformin is an anti-diabetic drug with antigluconeogenesis and insulin sensitizing properties used for the
treatment of type 2 diabetes. Recent studies suggest that metformin
may reduce the risk of cancer, but its mode of action remains
uncertain. In this study we examined the effect of metformin on
human retinoblastoma cell proliferation in vitro.
Methods: Two different human retinoblastoma cell lines (Y79,
WERI) were treated with metformin (0.6-10mM) for 1, 3 and 5 days.
Cell proliferation and growth inhibition were assessed by MTT assay,
trypan blue exclusion and viability/cytotoxicity calcein AM/ethidium
homodimer test. Mitochondrial function was tested with Mitotracker
and ATP generation assays.
Results: Metformin inhibited retinoblastoma growth and
proliferation in a dose dependent manner. Treatment with metformin
resulted in increased doubling time of retinoblastoma cells and
significant impairment of the redox state of the cells leading to an
overestimation of doubling time measured by the MTT assay.
Conclusions: Metformin is a potent inhibitor of growth and
proliferation of retinoblastoma cell lines. It has a potential to be used
as non-chemotherapeutic adjuvant drug for retinoblastoma.
Commercial Relationships: Katarzyna A. Brodowska, None;
Evangelos S. Gragoudas, QLT Phototherapeutics, Inc. (P);
Demetrios Vavvas, MEEI (P), Genentech (C), Roche (C), Kala
Pharmaceuticals (C)
Support: National Eye Institute Grant, EY014104 (MEEI Core
Grant)
Small-Gauge Needles and the Potential for Tumor Cell Seeding
of Retinoblastoma and Melanoma
Abby Y. Liu, Hans E. Grossniklaus. Emory Eye Center, Decatur, GA.
Purpose: Tumor cell seeding has been reported with the use of 25-31
gauge needles during fine-needle aspiration biopsy of intraocular
tumors. The purpose of this study was to evaluate the safety profile of
small, 34-gauge needles for the diagnosis of uveal melanoma and
retinoblastoma.
Methods: Polymer beads 1, 10, and 20 microns in diameter were
aspirated using 26, 30, and 34-gauge needles, and bead count was
recorded for each group (n=10). All needles were used to aspirate
WERI-Rb and Mel-270 cells in culture, and DNA was detected using
spectrophotometry (n=1). To evaluate differences in in vitro growth,
needles were used to aspirate and deposit WERI-Rb and Mel-270
cells onto culture plates (n=3). Cell counts were performed after
initial aspiration and every two days for a total of ten days. Linear
regression analyses were performed to compare growth among the
aspirates. Aspirates were embedded in paraffin and cellular
morphology was examined microscopically. The extent of
intercellular adhesion within cells was quantified by counting clusters
in high-power microscopic fields.
Results: All needles aspirated similar amounts of beads, except the
34-gauge needle of 4-mm length, which aspirated significantly fewer
20-micron beads. All aspirates contained DNA. For WERI-Rb, there
was a direct relationship between gauge size and cells aspirated. This
difference was amplified over 10 days, as 34-gauge aspirates
demonstrated significantly less growth [beta = 0.194, t(49)= p<
0.0001]. For Mel-270, there was no difference in cells initially
aspirated or in 10-day growth [beta = 0.145; t(49) = -1.194; p =
0.238]. Aspirates from each group demonstrated intact cellular
morphology, although fewer numbers of cells were observed in the
34-gauge groups. Compared to Mel-270 cells, WERI-Rb cells
occurred in clusters more often [29% vs. 17% of total cells per hpf]
and their weighted diameter was higher [mean ± SD = 24.6 ± 16.0 vs.
18.2 ± 9.1; two sample t-test, p<0.01].
Conclusions: 34-gauge needles can obtain DNA for analysis of
tumor aspirates. Their use may decrease the spread and metastatic
growth of retinoblastoma cells owing to cellular clustering, whose
effect may be further amplified in eye tissue, where cells are more
tightly bound than in cell culture.
Commercial Relationships: Abby Y. Liu, None; Hans E.
Grossniklaus, None
Support: NIH NEIP3006360
Spatiotemporal Patterns of Intraocular Tumor Occurrence in
Children with Retinoblastoma
Benjamin King1, 2, Carlos Parra2, 3, Matthew W. Wilson5, 4, Robert J.
Ogg2. 1College of Medicine, University of Tennessee Health Sciences
Center, Memphis, TN; 2Radiological Sciences, St. Jude Children's
Research Hospital, Memphis, TN; 3Biomedical Engineering,
University of Memphis, Memphis, TN; 4Surgery, St. Jude Children's
Research Hospital, Memphis, TN; 5Ophthalmology, University of
Tennessee Health Sciences Center, Memphis, TN.
Purpose: To map the spatial distribution and foci of origin of
intraocular retinoblastoma at the time of diagnosis in a prospectively
enrolled patient cohort.
Methods: Orbital MRI in 98 consecutive retinoblastoma patients(39
bilateral) was analyzed in axial, coronal and sagittal imaging
planes(Syngo Viewer, Siemens AG, Germany). The central visual
axis was approximated relative to the optic nerve and the polar angle
and visual angle of eccentricity were measured for a series of points
along each tumor margin. The tumor outline was interpolated on a
polar coordinate system centered at the fovea. Tumor maps were
digitized and the centroid of the mapped area was calculated to
approximate tumor focus of origin.
Results: Eyes were excluded from mapping(31) for globe filling
disease or inadequate tumor visualization on MRI. Mapping failed in
some eyes(21) secondary to extensive retinal detachment and vitreous
seeding. Mapping was successful for 178 tumors in 85 eyes from 63
patients(35 bilateral). Cumulative tumor burden was highest within
the macula and posterior pole and was asymmetrically higher within
the ventral and nasal hemiretinas(Fig. 1). Tumor location varied with
age at diagnosis(Fig. 2). Tumor occurrence in patients <6 months was
highest within the macula and superonasal periphery. Most tumors
diagnosed at 6-9 months were in the inferotemporal quadrant of the
posterior pole. Tumor occurrence shifted into the inferonasal
quadrant in patients 9-16 months and extended diffusely throughout
the nasal periphery in patients >16 months. Clinical information for
38 of the unmapped tumors showed that only 5 (13%) involved the
superotemporal quadrant, consistent with the asymmetry of mapped
tumors.
Conclusions: Precise mapping of tumor on the retina revealed a
complex spatial pattern of occurrence which evolves with age. The
spatiotemporal distribution of tumors may provide valuable clues
regarding the cell of origin, the developmental context that facilitates
tumorigenesis, and the impact of disease on vision in children with
retinoblastoma.
Cohort cumulative tumor burden over the mapped the retina. Left eye
nasal-temporal coordinates were inverted, color indicates number of
tumors.
Distribution of tumor centroids. A) Location by laterality
(triangle=bilat), area (size= quartiles), and age (young-to-old
quartile=pink, orange, blue, green) B) Estimated tumor density
relative to a uniform density over the mapped retina.
Commercial Relationships: Benjamin King, None; Carlos Parra,
None; Matthew W. Wilson, None; Robert J. Ogg, None
Support: NIH Grant HD049888
Shifting Treatment Paradigms in Retinoblastoma: Dilemmas for
the clinician in evaluating the use of novel therapies
Timothy G. Murray. 1Ophthalmology, Murray Ocular Oncology and
Retina, Miami, FL; 2Ophthalmology, Miami Childrens Hospital,
Miami, FL.
Purpose: To evaluate shifting treatment paradigms in the
management of children with retinoblastoma over the last three
decades. Focus on the integration of evolving pilot clinical data and
basic/translational research data on a clinicians choice of primary
therapy.
Methods: IRB approved, retrospective review of children treated
between 1990 and 2012 with retinoblastoma by a single surgical
ocular oncologist. Demographics included age at presentation,
staging of disease, unilaterality versus bilaterality, familial versus
non-familial and date of institution of primary therapy by the treating
surgeon. All children underwent pre-treatment ocular and CNS
imaging, examination under anesthesia and ongoing followup
including serial EUA and awake examinations. Survival status,
anatomic globe status and visual function were serially obtained.
Treatments were grouped in three time intervals to evaluate treatment
trends during the study window reported as 1990's, 2000's and 2010's.
Results: 272 children were primarily treated with retinoblastoma,
150 boys and 122 girls. Mean age at presentation was 14 months
(range birth to 18 years). 108 children presented with bilateral disease
(39%) with virtually all children presenting prior to three months
developing bilateral eye involvement. In the initial review, Treatment
evolution for primary therapy: 1990's, 85% of children treated with
external beam radiotherapy (EBRT) and 25% enucleated, 2000's 85%
of children treated with systemic chemotherapy and 10% enucleated,
and 2010's 90 of children treated with focal chemotherapy (intraarterial/peri-ocular) and less then 5% enucleated (p<.01). Primary
retinoblastoma mortality rates were less then 1% for the entire study
window (no deaths within the last 15 years (p<.005).
Conclusions: Retinoblastoma treatment has undergone significant
shifts related to our ongoing understanding of molecular genomics,
pathophysiology, treatment options, and treatment related morbidity.
Over three decades treatment has moved from enucleation to external
beam radiotherapy, to systemic chemotherapy to focal chemotherapy
(including intra-arterial chemotherapy, peri-ocular chemotherapy and
intra-vitreal chemotherapy). Ultimately, the evolution of treatment
for retinoblastoma has remarkably improved survival outcomes,
globe preservation, and visual function.
Commercial Relationships: Timothy G. Murray, None
Support: Ocular Oncology Research Foundation 2012/2013
Genetic Characterization among 232 Retinoblastoma Patients
Jacob Pe'er1, Ofira Zloto1, Michael Weintraub2, Michal Sagi3,
Israela Lerer3, Avishag Nadel3, Ido Rot2, Naomi Shoshani2, Shahar
Frenkel1. 1Ophthalmology, Hadassah-Hebrew University Medical
Center, Jerusalem, Israel; 2Pediatric Hematology-Oncology,
Hadassah-Hebrew University Medical Center, Jerusalem, Israel;
3
Genetics, Hadassah-Hebrew University Medical Center, Jerusalem,
Israel.
Purpose: To describe the association between the existence of a
germline mutation and disease characteristics in patients with
retinoblastoma.
Methods: The study included 232 patients with retinoblastoma who
were treated at a single center between 1988 and 2012. Genetic
testing for RB1 mutation was performed in 107 patients. Patients
with a RB1 mutation were compared to patients without a mutation,
in terms of epidemiological factors and clinical presentation. Several
parameters were compared among groups by distribution analysis and
Pearson correlation.
Results: Among 107 families whose genetic status was evaluated, 62
patients had a RB1 germline mutation and 45 did not have a mutation
(57.94% vs 42.06%). Mutations were found in 92.00% of the patients
with bilateral disease, 28.07% of the patients with unilateral disease
and in 3 of 4 patients with unilateral multifocal disease (Pearson
correlation, p<0.0001). Six patients with mutations showed
mosaicism (5 monocular and 1 binocular). The most common type of
mutation was a stop codon mutation (41.94%). 85.0% of the patients
with macular involvement had a mutation (Pearson correlation,
p=0.0106). No significant differences were found in gender, age or
reason for referral.
Conclusions: As expected, mutations were found in most of the
patients with bilateral disease. Surprisingly, our genetic tests also
revealed mutations in 28.07% of patients with unilateral
retinoblastoma. These patients have an increased risk for other
cancers throughout their life, and their first-degree relatives have an
increased risk for retinoblastoma. Therefore, genetic testing for RB1
mutation should be offered to all patients, including the unilateral
cases.
Commercial Relationships: Jacob Pe'er, None; Ofira Zloto, None;
Michael Weintraub, None; Michal Sagi, None; Israela Lerer,
None; Avishag Nadel, None; Ido Rot, None; Naomi Shoshani,
None; Shahar Frenkel, None
Topotecan and Cyclophosphamide as Salvage Therapy in the
Treatment of Retinoblastoma
Qi N. Cui1, Vasiliki Aivaliotis1, Joan M. O'Brien2, Paul Stewart1.
1
Department of Ophthalmology, University of California, San
Francisco, San Francisco, CA; 2Department of Ophthalmology,
University of Pennsylvania, Philadelphia, PA.
Purpose: Retinoblastoma is the leading primary ocular malignancy
of childhood. Currently, first-line therapy for intraocular
retinoblastoma consists of systemic chemoreduction in combination
with focal consolidative therapy. More recently, intra-arterial
chemotherapy has gained momentum as a new option for the
management of unilateral intraocular retinoblastoma. In this study,
we examined the effectiveness of a novel two-agent chemotherapy
regimen as salvage therapy for children who failed initial treatment.
Methods: We conducted a cross-sectional retrospective chart review
looking at cases of retinoblastoma that received treatment at the
University of California, San Francisco between 1994 and 2010.
Fifty-two patients were identified. Of these, eight patients and 14
globes failed primary chemotherapy and subsequently received
salvage chemotherapy consisting of Topotecan and
Cyclophosphamide. Outcome evaluated include the ICRB disease
stage, tumor recurrence, the need for enucleation, side effects, and
mortality.
Results: Three globes were stage B, five were stage C, four were
stage D, and two were stage E at the time of diagnosis. The duration
of follow-up was 19.6 ±10.2 (mean ± SD) months, and the age at last
follow up was 54.3 ± 12.7 months. Treatment with Topotecan and
Cyclophosphamide was associated with globe salvage in 10 out of 14
eyes (71%) overall, and 10 out of 12 eyes (83%) in bilateral cases. In
those with OU involvement, two globes were enucleated and one
patient experienced recurrence following treatment. Both subjects
with unilateral involvement were enucleated. Systemic side effects
included anemia (1 subject), neurosensory hearing loss (1 subject),
neutropenia (2 subjects), and an allergic reaction to carboplatin (1
subject).
Conclusions: Administration of novel two-agent systemic
chemotherapy as salvage treatment for retinoblastoma was associated
with 71% globe preservation overall. This is comparable to the effect
of intra-arterial (ophthalmic artery) chemotherapy using melphalan,
carboplatin, and/or topotecan as reported by Abramson et al. 2008 (1)
In addition, systemic chemotherapy is not limited by laterality and
demonstrated 83% globe preservation in bilateral cases.
1). Abramson DH, Dunke IJ, Brodie SE et al. A phase I/II study of
direct intraarterial (ophthalmic artery) chemotherapy with melphalan
for intraocular retinoblastoma initial results. Ophthalmoloy 2008;
115: 1398-404.
Commercial Relationships: Qi N. Cui, None; Vasiliki Aivaliotis,
None; Joan M. O'Brien, None; Paul Stewart, None
Non-selectivity of ERG reductions in eyes treated for
retinoblastoma
Catherine Y. Liu1, Gowtham Jonna1, Jasmine H. Francis1, Brian
Marr1, 3, David H. Abramson1, 3, Scott E. Brodie1, 2. 1Ophthalmic
Oncology, Memorial Sloan Kettering Cancer Center, New York, NY;
2
Ophthalmology, Mt. Sinai Hospital, New York, NY; 3Weill-Cornell
Medical College, New York, NY.
Purpose: We have monitored retinal function in patients treated for
retinoblastoma (primarily, but not exclusively by intra-arterial
chemotherapy infusion) by ERG recordings for the past six years. We
here present data from the subset of patients who underwent a
complete ERG protocol including both photopic and scotopic
recordings to justify our frequent practice of reporting primarily 30Hz photopic flicker amplitude data.
Methods: Patients referred for treatment of retinoblastoma
underwent ERG recordings during examination under anesthesia
whenever possible at baseline, and following most treatment sessions,
especially after intra-arterial chemotherapy. All recordings included
photopic single flash (Photopic 3.0, “Phot SF”) and 30-Hz flicker
(“Phot 30 Hz”) stimuli; when time permitted, many also underwent
dark adaptation for 5 minutes (shorter than the ISCEV standard
protocol to minimize anesthesia duration) followed by recordings of
responses to scotopic rod-isolating (Scotopic 0.01, “Rod”) and
scotopic maximal (Scotopic 3.0, “Scot Max”) flash stimuli. Response
amplitudes were measured from averages of 10 replicate records to
suppress the effects of unsteady baselines caused by the sevofluorane
anesthesia. Correlations were calculated for the complete datasets
between the four sets of responses amplitude data.
Results: Complete photopic and scotopic ERG data was available
from over 600 ERG studies of 108 patients. The correlation matrix
for these ERG responses are detailed in Table I.
Conclusions: Under our recording conditions, ERG responses of
eyes with untreated retinoblastoma or following intra-arterial
treatment for retinoblastoma show very high correlations between 30Hz flicker amplitude responses and the three other standard photopic
and scotopic ERG response amplitudes. The reductions in ERG
amplitudes seen in these eyes do not appear to be selective for rod or
cone systems. These observations support the use of photopic
response amplitudes (especially in response to 30-Hz flicker) as the
primary ERG outcome measure in studies of treated and untreated
eyes with retinoblastoma when more complete ERG protocols may
be impractical.
Commercial Relationships: Catherine Y. Liu, None; Gowtham
Jonna, None; Jasmine H. Francis, None; Brian Marr, None;
David H. Abramson, None; Scott E. Brodie, None
Support: This work was supported by the Fund for Ophthalmic
Knowledge and by an unrestricted grant to the Mount Sinai
Department of Ophthalmology from Research to Prevent Blindness,
Inc.
Electroretinogram monitoring of retinal toxicity of ophthalmic
artery chemosurgery for retinoblastoma: Six year experience
Jasmine H. Francis1, David H. Abramson1, 2, Pierre Y. Gobin3, Brian
Marr1, 2, Ira J. Dunkel4, Elyn R. Riedel5, Scott E. Brodie6, 1.
1
Ophthalmic Oncology, Memorial Sloan Kettering Cancer Center,
New York, NY; 2Department of Ophthalmology, Weill Cornell
Medical College of New York Presbyterian, New York, NY;
3
Department of Neurosurgery, Weill Cornell Medical College of New
York Presbyterian, New York, NY; 4Department of Pediatrics,
Memorial Sloan Kettering, New York, NY; 5Department of
Epidemiology and Biostatistics, Memorial Sloan Kettering, New
York, NY; 6Department of Ophthalmology, Mount Sinai School of
Medicine, New York, NY.
Purpose: The effectiveness of ophthalmic artery chemosurgery
(OAC) for retinoblastoma derives from the very high local tissue
concentrations achieved in the distribution of the ophthalmic artery,
before the drug is safely diluted in the systemic venous circulation.
We have recorded electroretinogram (ERG) responses before and
after OAC to monitor retinal function as a gauge of local toxicity, and
we herein report these results.
Methods: Prospective review of 6-year experience of retinoblastoma
patients receiving OAC. This study recorded the ERG 30 Hz flicker
amplitude response changes from baseline, at 3 and 12 months
following treatment completion. Inclusion criteria consisted of eyes
treated with OAC with ERG measurements a. before OAC, and b. 3
months after the last OAC treatment +/- 1 month, and if available, c.
12 months after last OAC treatment +/- 2 months. Both univariate
and multivariate linear regression models were performed, with
generalized estimating equations to correct for correlations within
patients. Independent numerical variables included maximum and
cumulative doses of melphalan, topotecan and carboplatin.
Results: ERG data were available for 103 eyes of 81 patients; a total
of 270 recordings were analyzed. Results from the univariate and
multivariate regressive analysis are shown in figures 1 and 2,
respectively.
Conclusions: Melphalan has the strongest, and carboplatin the
weakest association to change in ERG response. By univariate
analysis, both melphalan and topotecan appear to be associated with
changes in ERG amplitude at both 3 and 12 months; but for the most
part, these changes are minimal and likely clinically insignificant. By
multivariate analysis, maximum and cumulative melphalan have a
modest, temporary effect on the ERG amplitude change, which is
apparent at 3 months but no longer evident at 12 months after
completing treatment. By multivariate analysis, topotecan and
carboplatin do not appear to adversely effect the change in ERG
response.
Univariate regression analysis
clinical data with angiographic findings.
Results: A total of 43 eyes with advanced, untreated retinoblastoma
evaluated with Retcam FA were identified. For the Group D cases
(18 eyes), FA findings included: iris neovascularization (4/18),
abnormal retinal vascular dilatation (15/18), small retinal vessel
telangiectasia (13/18), late vascular leakage (16/18), retinal vein
periphlebitis (9/18), and terminal vascular bulbs (1/18). Among the
Group E cases (25 eyes), FA findings included: iris
neovascularization (23/25), retinal vascular dilatation (20/25), retinal
vessel telangiectasia (14/25), late vascular leakage (20/25), retinal
vein periphlebitis (7/25), and terminal vascular bulbs (1/25).
Conclusions: Advanced intraocular retinoblastoma is associated with
characteristic retino-vascular findings on Retcam FA. Iris
neovascularization, retinal vascular dilatation, and capillary
telangiectasia are common findings in these eyes, but terminal
vascular bulbs are rare. An important finding was the identification of
a distinctive retinal periphlebitis on FA, which correlated with the
presence of subretinal fluid produced by the tumor. Characteristic
angiographic findings in retinoblastoma may be helpful in correctly
classifying an eye with advanced disease and also distinguishing this
tumor from other pediatric ocular conditions.
FA findings in retinoblastoma: (A) retinal periphlebitis, Group D; (B)
iris neovascularization (NVI), Group E; and (C) NVI and
periphlebitis, Group E.
Commercial Relationships: Yohko Murakami, None; Lynn Ngai,
None; Jonathan W. Kim, None
Multivariate regression analysis
Commercial Relationships: Jasmine H. Francis, None; David H.
Abramson, None; Pierre Y. Gobin, None; Brian Marr, None; Ira
J. Dunkel, None; Elyn R. Riedel, None; Scott E. Brodie, None
Retcam Fluorescein angiography findings in eyes with advanced
retinoblastoma
Yohko Murakami1, Lynn Ngai1, Jonathan W. Kim2, 1.
1
Ophthalmology, Doheny Eye Institute, Los Angeles, CA; 2Children's
Hospital Los Angeles, Los Angeles, CA.
Purpose: The Retcam II is a wide-field, contact digital fundus
imaging system that also allows the performance of intraoperative
fluorescein angiography (FA). We propose that Retcam FA can be an
indispensible tool in the evaluation of retinoblastoma patients, both to
confirm the diagnosis and to define the extent of disease.
Methods: We performed an IRB-approved retrospective case series
to evaluate Retcam FA results in eyes with advanced retinoblastoma.
All new retinoblastoma patients who have undergone FA in the past
10 years on the Retcam II were identified through a database search.
Inclusion criteria included: 1) patients diagnosed with advanced
retinoblastoma (Group D or E), studied with early, mid-phase, and
late-phase FA photographs, 2) no previous treatment 3) follow-up for
at least one year. For the cases meeting inclusion criteria, the FA
images were analyzed and a chart review was performed to correlate
Radiation induced cataract surgery in patients treated for
retinoblastoma
Carlos Y. Chen1, Guadalupe Márquez2, Humberto C. Matiz2,
Federico Graue1, Norma C. Lara3, Marco Ramírez3. 1Retina and
Vitreous, Inst de Oftalmol Conde de Valenciana, Mexico City,
Mexico; 2Anterior Segment, Inst de Oftalmol Conde de Valenciana,
Mexico City, Mexico; 3Ophthalmology, Hospital Infantil de México
Federico Gómez, Mexico City, Mexico.
Purpose: To report the visual outcome and complications after
phacoemulsification and intraocular lens implantation in radiation
induced cataract.
Methods: Five eyes of five patients we retrospectively analyzed, 3
boys and 2 girls, the mean age was 6.2 years (4-8), all patients were
treated for bilateral retinoblastoma, all received chemotherapy and 4
patients were enucleated 1 eye. Five eyes received radiation therapy 4
with external beam and 1 brachytherapy.
Results: The mean time to cataract development was 1 year. The
follow up was 2.10 years (0.44-4.17). The best corrected visual
acuity(BCVA) pretreatment was 1.87 log mar (0.47-3) and the posttreatment BCVA was 0.54 log mar (0.30-1). The mean spheric
equivalent was -1.60dp (+1.25-7.50). To the last visit all eyes were
complety free of tumor activity.
Conclusions: We can concluded that phacoemulsification and
intraocular lens implantation is safe and improves the visual acuity
and the clinical examination of the tumor.
Commercial Relationships: Carlos Y. Chen, None; Guadalupe
Márquez, None; Humberto C. Matiz, None; Federico Graue,
None; Norma C. Lara, None; Marco Ramírez, None
Intraocular Calcification as a Diagnostic Marker for
Retinoblastoma
Wendy Ma1, Michele M. Bloomer2, Tina Rutar2. 1UCSF School of
Medicine, San Francisco, CA; 2Ophthalmology, University of
California, San Francisco, San Francisco, CA.
Purpose: The aim of this study was to determine pediatric conditions
other than retinoblastoma (Rb) that may be associated with posterior
segment intraocular calcification.
Methods: We reviewed 405 pathology reports from enucleation
specimens of children ages 0 to 5 years collected between January
1960 and May 2011 at one U.S. ocular pathology laboratory. An
ophthalmic pathologist re-reviewed a minimum of 5 representative
H&E stained slides for each of the 154 specimens that had no
mention of intraocular calcification in their reports. Specimens were
classified according to pathological diagnosis and the
presence/absence of calcification within the anterior segment of the
eye (cornea, ciliary body, lens) or posterior segment (vitreous,
choroid, retina, sclera, optic nerve). Optic disc drusen were not
studied. We then performed a PubMed literature search for
“intraocular calcification,” “osseous metaplasia eye,” and “leukocoria
calcification” to find English-language reports of posterior segment
intraocular calcification among children ages 0 to 5 years without Rb.
We also searched the reference lists of the resulting articles.
Results: Posterior segment intraocular calcification was present in
280 of 330 (84.8%) Rb specimens and 13 of 75 (17.3%) non-Rb
specimens. The diagnoses of these 13 non-Rb specimens included
phthisis (4; due to sympathetic ophthalmia; complications of
intraocular surgery; congenital rubella; endophthalmitis), congenital
microphthalmos with cyst (2), persistent fetal vasculature (1), Coats’
disease (1), astrocytic hamartoma (1), retinal dysplasia (1),
panophthalmitis with retained intraocular foreign body (1), retinal
detachment of unknown etiology (1), and congenital uveitis of
unknown etiology leading to autolytic degeneration of the globe (1).
The literature review identified the following pediatric conditions
with posterior segment intraocular calcification: astrocytic
hamartoma, ocular choristoma in linear nevus sebaceous syndrome,
medulloepithelioma, and retinal detachment in incontinentia
pigmenti.
Conclusions: Diagnoses other than Rb should be considered in a
young child with intraocular calcification, including phthisis from
myriad causes, congenital anomalies, persistent fetal vasculature,
Coats’ disease, astrocytic hamartoma, retinal dysplasia, and
endophthalmitis.
(H&E stain) Intraocular psammomatous calcification in child with
postoperative phthisis secondary to complications of congenital
rubella.
Commercial Relationships: Wendy Ma, None; Michele M.
Bloomer, None; Tina Rutar, None
Support: This study was supported by an unrestricted grant from
Research to Prevent Blindness, New York, NY, by the Quarterly
Research Fellowship from UCSF Dean’s Office Medical Student
Research Program, and by an institutional P30 core grant from the
National Institutes of Health NEI EY002162-31.
406 Myopia and Emmetropization
Wednesday, May 08, 2013 8:30 AM-10:15 AM
608 Paper Session
Distribution and Determinants of Eye Size and Shape in
Newborn Children: a Magnetic Resonance Imaging Analysis
Laurence S. Lim1, 2, Pei Ting Tan2, Gim Hong Chong2, Yap-Seng
Chong2, Marielle V. Fortier3, Peter Gluckman4, 5, Seang-Mei Saw2, 1,
Anqi Qiu2. 1Ophthalmology, Singapore National Eye Center,
Singapore, Singapore; 2National University of Singapore, Singapore,
Singapore; 3KK Women’s and Children’s Hospital, Singapore,
Singapore; 4Singapore Institute for Clinical Sciences, the Agency for
Science, Technology and Research, Singapore, Singapore; 5Liggins
Institute, University of Auckland, Auckland, New Zealand.
Purpose: To determine the range and distribution of measures of eye
size and shape obtained by Magnetic Resonance Imaging (MRI), and
to determine the associations of race and parental myopia with eye
dimensions and shape in newborn children.
Methods: This study was conducted on a subset of the Growing Up
in Singapore Towards healthy Outcomes (GUSTO) birth cohort
study. 173 newborn children underwent MRI. Eye volume and
surface area were measured. Longitudinal axial length (LAL, the
length from the posterior corneal surface to the retinal surface),
horizontal width, and vertical height along the cardinal axes were
measured. Eye shape was assessed qualitatively from threedimensional models, and quantitatively by calculation of an index of
eye shape, the oblateness (oblateness = 1-AL/width or 1-AL/height).
A spherical shape was oblateness=0, and prolate and oblate shapes
were oblateness <0 and >0 respectively.
Results: In total, 346 eyes of 173 full-term newborn children were
included. Eyes with longer AL had greater widths, heights, volumes
and surface areas (p<0.001 for all). The mean oblateness value
calculated in relation to width was -0.06±0.05(95% confidence
interval -0.23 - 0.08), and the mean oblateness calculated in relation
to height was -0.05±0.04 (-0.19 - -0.001). Using width to calculate
oblateness, most eyes were prolate (312 eyes(90.2%)), and, using
height, most eyes were also prolate(194 eyes(56.1%)). With
increasing AL, eyes assumed increasingly prolate profiles using both
eye width and height to calculate oblateness. However, even amongst
the eyes with the longest AL, the globe shape appeared spherical
from inspection, without evidence of a marked prolate or oblate
bias.(Figure) Race but not parental myopia was associated with the
size of the eye at birth, with children of Malay mothers having larger
eye volumes and surface areas.
Conclusions: Most newborn Singaporean Asian children are born
with spherical or slightly prolate eyes. Race but not parental myopia
is associated with the size of the eye at birth.
Figure: Three dimensional models of eyes with the longest and
shortest axial lengths.
Commercial Relationships: Laurence S. Lim, None; Pei Ting
Tan, None; Gim Hong Chong, None; Yap-Seng Chong, None;
Marielle V. Fortier, None; Peter Gluckman, Nestle (R), Danone
(R), Abbott (R); Seang-Mei Saw, None; Anqi Qiu, None
Support: This study is supported by National Medical Research
Council (NRMC) grant NMRC/1009/2005 and NMRC/TCR/004NUS/2008, Agency for Science, Technology and Research
(A*STAR) grant SICS-09/1/1/001, the Young Investigator Award at
the National University of Singapore (NUSYIA FY10 P07), and the
National University of Singapore MOE AcRF Tier 1.
Central retinal thickness and axial length in the Copenhagen
Child Cohort 2000 study: data from 432 11-year-old children
Xiao Q. Li1, Michael Larsen1, Inger C. Munch2. 1Department of
Ophthalmology, Glostrup Hospital, Glostrup, Denmark; 2Department
of Ophthalmology, Roskilde Hospital, Roskilde, Denmark.
Purpose: To investigate central retinal thickness in relation to axial
length.
Methods: The Copenhagen Child Cohort 2000 (CCC2000) is a
population-based study where 1417 children have been examined
with best corrected visual acuity, non-cycloplegic refraction, noncontact ocular biometry (IOL-master) and enhanced depth imaging
spectral-domain optical coherence tomography (EDI-SD-OCT).
Central retinal thickness (average thickness in the central 1000-μm
diameter area) was calculated in 432 randomly selected children from
the CCC2000 study. Scaling factors were as found in the instrument
manufacturer's proprietary software.
Results: Data from 432 children (199 boys; 233 girls) were analyzed.
The mean age was 11.67 ± 0.33 years. All had a best corrected visual
acuity of 50 letters on the ETDRS-chart or better and the mean axial
length was 23.18 ± 0.76 mm. The mean spherical equivalent
refraction was +0.12 ± 0.90 D. Mean central retinal thickness was
275.87 ± 19.03 μm. Boys had significantly longer eyes (0.42 mm,
CI95 0.28 - 0.55 mm ; p < 0.001) and thicker central retinae (6.16
mm, CI95 2.59 - 9.72 μm ; p = 0.008) compared with girls (Student’s
t-test analysis). Central retinal thickness increased 3.84 μm (CI95 0.88
- 8.23 μm ; p = 0.0019) per mm increase in axial length and was 4.55
μm (CI95 1.43 - 6.25 μm ; p < 0.0152) thicker in boys compared with
girls in a multiple linear regression analysis including both sex and
axial length. There was no significant interaction between sex and
axial length.
Conclusions: Central retinal thickness increased with increasing
axial length and was thicker in boys than in girls in this subset of 432
children from a Danish population-based cohort of 1417 Danish
children. The results are sensitive to errors of axial magnification
scaling factors, especially those that vary with axial length.
Commercial Relationships: Xiao Q. Li, None; Michael Larsen,
None; Inger C. Munch, None
Neurochemical Ablation of Peripheral Glucagonergic Amacrine
Cells in the Chick Retina and its Effects on Axial Eye Growth
and Refractive Error
Diane Nava1, 2, Alice Chang2, Lisa A. Ostrin2, Zhi Chen2, 3, Christine
F. Wildsoet1, 2. 1Vision Science Group, UC Berkeley, Berkeley, CA;
2
Center for Eye Disease and Development, UC Berkeley School of
Optometry, Berkeley, CA; 3Department of Opthalmology and Vision
Science, Fudan University Eye and ENT Hospital, Shanghai, China.
Purpose: Several recent myopia studies have pointed to important
influences of the peripheral retina on emmetropization. The purpose
of this study was to investigate whether mini-bullwhip glucagonergic
amacrine cells (MBW), which reside mostly in the dorsal peripheral
retina of chicks, contribute to eye growth regulation.
Methods: To ablate MBW cells, an intravitreal injection of 250 ng of
colchicine was given at p7 (Fischer et al 2008); control birds received
saline injections. The retinal effects of the latter treatment were
assessed using in vivo SD-OCT imaging and immunohistochemistry
(anti-glucagon labeling) on retinal wholemounts and vertical sections.
Retinal function was assessed with flash and pattern
electroretinography (ERG). Monocular defocusing lenses (+5D single
vision (SV), or multifocal (MF) with a +5 D peripheral power and
4.5, 5.5 or 6.5 mm central zone diameters (CZD), were fitted 5 days
after injections, and worn for 5 days. Refractive errors and ocular
axial dimensions were tracked using retinoscopy and high-resolution
A-scan ultrasonography respectively. Finally, equatorial diameters
were obtained from photographs of enucleated eyes.
Results: Colchicine induced significant retinal thinning in both
peripheral and central retina (p<0.01; p<0.05), and significantly
reduces the number of MBW in the peripheral retina (p=0.004)
without affecting glucagonergic cells in central retina (p=0.449). It
also decreased the amplitudes of b and c-waves as well as the
Oscillatory Potential 1 in flash ERG recordings; in pattern ERG
recordings, the p50 amplitude was reduced and delayed. All lens
treatments inhibited axial ocular elongation and induced hyperopia.
There was no difference between the responses to SV and MF lenses
in colchicine-treated eyes, contrasting with the previous finding that
MF lenses inhibited axial elongation more than SV lenses in normal
eyes (Liu and Wildsoet 2011). There was no difference in equatorial
diameters between control and colchicine groups.
Conclusions: MBW cells appear to be part of the pathway involved
in spatial pattern processing by the retina and also appear to
contribute to the response to peripheral myopic defocus. The changes
in the c-wave component of the ERG suggest changes in RPE
function, which may also contribute to the observed changes in
responses to MF lenses.
Mini-bullwhip cells in the chick retina
Commercial Relationships: Diane Nava, None; Alice Chang,
None; Lisa A. Ostrin, None; Zhi Chen, None; Christine F.
Wildsoet, None
Support: NEI T32-EY007043 and NEI R01-EY12392
Ocular growth guided by dual focal fresnel lenses requires an
intact optic nerve
Sally A. McFadden1, Dennis Y. Tse2, Chi-ho To2, Christine F.
Wildsoet3. 1School of Psychology, University of Newcastle,
Callaghan, NSW, Australia; 2School of Optometry, The Hong Kong
Polytechnic University, Kowloon, Hong Kong; 3School of
Optometry, University of California, Berkeley, CA.
Purpose: Hyperopic defocus imposed with a negative lens induces
myopia from excessive ocular growth while positive lenses cause
ocular growth to slow. When negative and positive powers are
combined 50:50 in a Fresnel lens, ocular growth is matched to the
average imposed defocus. We asked whether this apparent integration
occurs in eyes with sectioned optic nerves.
Methods: 47 guinea pigs underwent either optic nerve section (ONS)
or sham surgery at 3 days of age in one eye. The treated eye then
wore either a single vision lens (-5 D or 0 D) or a Fresnel lens
composed of two powers in equal ratios (-5/+5 D, -5/0 D, +5/0 D) for
11 days (from 6-16 days of age). Refractive error and axial ocular
dimensions were measured before and after treatments. Interocular
differences in refractive error and axial length after treatment are
reported.
Results: After ONS, eyes with -5 D lenses became excessively
myopic (-6.4 ± 1.6 D, p = 0.006) and elongated (150 ± 22 µm, p =
0.02) while there was little effect of ONS with no defocus (0 D: -1.4
± 1.7 D, p = 0.08 and 87 ± 103 µm, p = 0.02). After sham surgery,
eyes wearing -5/+5 D Fresnel lenses responded to the average
imposed defocus (Refractive Error: -0.8 ± 0.7 D, p = 0.4; Axial
Length: 65 ± 13 µm, p = 0.2), while after ONS, the same -5/+5 D
lenses induced myopia (-5.2 ± 1.5 D) and excessive elongation (151
± 36 µm). Similarly, ONS eyes wearing -5/0 D or +5/0 D lenses
failed to integrate the dual defocus, and the degree of myopia and
ocular elongation was similar to that in animals wearing -5/+5 D or 5 D lenses (-5/0 D: -5.8 ± 1.2 D; +5/0 D: -7.2 ± 1.3 D; F(3,38) = 0.3,
p = 0.8).
Conclusions: An intact optic nerve is required for the integration of
imposed optical defocus. Therefore, either ONS rapidly alters retinal
processing and causes this failure, or neural processes beyond the eye
contribute to the inhibition of mammalian eye growth and are
required for the ocular growth response to dual focal lenses.
Commercial Relationships: Sally A. McFadden, None; Dennis Y.
Tse, None; Chi-ho To, None; Christine F. Wildsoet, None
Support: Australian DIIRSE International Science Linkage
CG120160 (SAM), NIH R01 EY12392 (CFW, SAM), Niche Area
Fund Hong Kong JBB7P (CHT, DYT)
Effects of Long-Wavelength-Pass Filters on Refractive
Development in Rhesus Monkeys
Earl L. Smith1, 2, Li-Fang Hung1, 2, Baskar Arumugam1, 2, Juan
Huang1, 2, Maureen Neitz3, Jay Neitz3. 1College of Optometry,
University of Houston, Houston, TX; 2Vision Cooperative Research
Centre, Sydney, NSW, Australia; 3Department of Ophthalmology,
University of Washington Medical School, Seattle, WA.
Purpose: Because the refracting power of the eye varies with
wavelength, altering the spectral distribution of environmental
lighting can potentially influence refractive development. We
investigated whether restricting the vision of infant monkeys to
relatively long wavelength light would predictably alter refractive
development.
Methods: Beginning at 25 ± 2 days of age, infant monkeys were
fitted with helmets that secured long-wavelength-pass (red) filters in
front of one (n=7) or both eyes (n=7). For the monocularly treated
animals, neutral density filters were fitted in front of the fellow eyes
to balance the luminance levels in the two eyes. The housing area
was illuminated primarily with fluorescent tubes; tungsten lamps
were added to provide broad-spectrum lighting. The animals wore the
helmets continuously until 132 ± 15 days of age. Refractive
development and the eye’s axial dimensions were assessed
periodically throughout the observation period by retinoscopy and Ascan ultrasonography, respectively. Control data were obtained from
32 normal monkeys.
Results: At the end of the lens-rearing period, 5 of 7 monocularly
treated monkeys exhibited anisometropias that were larger than the
anisometropias found in 95% of the normal monkeys. In each case,
the treated eye was more hyperopic than the fellow eye. Similarly, by
120 days of age, the median refractive error for the binocularly
treated monkeys was significantly more hyperopic than that for
normal monkeys (right eye ametropia: +4.41 D vs +2.53 D; p =
0.0002). The relative hyperopia observed in the red-filter-treated eyes
was associated with slower vitreous chamber elongations rates.
Conclusions: Although the nature of the mechanism that mediated
the observed filter-induced refractive-error changes is not known,
contrary to predictions based on longitudinal chromatic aberration,
the red lenses employed in this study produced hyperopic shifts in
refraction rather than myopic shifts.
Commercial Relationships: Earl L. Smith, Ziess (P); Li-Fang
Hung, None; Baskar Arumugam, None; Juan Huang, None;
Maureen Neitz, Genzyme (F), Alcon (F), Alcon (P); Jay Neitz,
Alcon (F), Alcon (P)
Support: NIH Grants EY03611 (ELS), EY07551 (ELS), EY01730
(MN & JN), EY09393 (MN). EY09620 (JN) and Vision CRC,
Sydney
The role of central retina in chicks during form deprivation
myopia
Jianchao wang1, 2, Rachel K. M Chun1, K. k Li1, Quan Liu1, 2, Chiho
To1, 2. 1Laboratory of Experimental Optometry, Centre for Myopia
Research, School of Optometry, The Hong Kong Polytechnic
University, HongKong, China; 2State key Laboratory of
Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-sen
University, Guangzhou, China.
Purpose: This study aimed to examine if the central retina plays any
role in the development of form deprivation myopia in chicks.
Methods: Chicks (n=6-7) wore either central diffuser lenses (plano
lens with either 4 or 6 mm diameter diffuser zone at the centre;
C4/6mm FD) or full field diffuser lenses (full FD) in front of their
right eyes for 7 days . Plano lenses were mounted to their left eyes as
control. Refractive errors were measured both on-axis (centrally) as
well as 45 degrees off-axis nasally and temporally by retinoscopy.
The axial ocular dimensions were also measured by a high-resolution
A-scan ultrasound system respectively before and after 7 days of lens
wear.
Results: All treated eyes had significant changes of both central
refractive errors (C4mm FD vs control: -3.21± 0.57 vs -0.71 ± 0.23;
C6mm FD vs control: -5.50 ± 0.83 vs -2.16 ± 0.42; Full FD vs
control: -17.50 ± 0.99 vs -1.14 ± 0.34; mean changes [D] ± SEM,
independent-samples t-test, p<0.01 for all groups) and vitreous
chamber depth (C4mm FD vs control: 0.48 ± 0.02 vs 0.38 ± 0.02;
C6mm FD vs control: 0.53 ± 0.04 vs 0.35 ± 0.04; Full FD vs control:
1.02 ± 0.09 vs 0.31 ± 0.04; mean changes [mm] ± SEM; independentsamples t-test, p<0.05 for all groups) after 7 days of lens wear. The
refractive errors in temporal retina and nasal retina also became
myopic when compared with the control eyes. The myopic shift in
the central refractive error of the treated eyes was due primarily to the
increase in vitreous chamber depth. The interocular differences of
both central refractive error and the vitreous chamber depth increased
with the size of central form-deprived retina.
Conclusions: Form depriving the central retina with different sizes of
diffuser produced graded responses in terms of myopic growth even
in the presence of normal peripheral vision. The magnitude of
myopia induced in both central and peripheral retina increased with
the size of central diffuser. Apparently, central retina also plays a
significant role in form deprivation myopia in chicks. The graded
response of myopia may be due to integration between retinal regions
that received opposing optical inputs.
Commercial Relationships: Jianchao wang, None; Rachel K. M
Chun, None; K. k Li, None; Quan Liu, None; Chiho To, None
Support: PolyU research grants (GUA32 & GYK89); RGC General
Research Funds (GRF: BQ15Q & BQ29N); PolyU Myopia Niche
Funding JBB7P; Specialized Research Fund for the Doctoral
Program of Ministry of Education of China (3030902103012)
Photoreceptor Outer Retinal Function as the Primary Controller
of Ocular Growth
David P. Crewther1, 2, Sarah N. Kiely2, Melanie J. Murphy2, Nina
Riddell2, Sheila G. Crewther2. 1Centre for Human
Psychopharmacology, Swinburne Univ of Technology, Melbourne,
VIC, Australia; 2Psychological Science, La Trobe University,
Melbourne, VIC, Australia.
Purpose: Although balanced modulation of retinal responses to light
onset and offset have been implicated in refractive compensation
(RC) to defocus, the locus of ocular growth control has not been
established. Thus, we compared the effects on RC, of
pharmacological inhibition of the ON bipolar MGluR6 receptors by
2-amino-4-phosophonobutyrate (APB), and OFF bipolar ionotrophic
receptors by cis-2,3-piperidicarboxylic acid (PDA), in combination
with either normal diurnal light (ND) or low frequency flicker (LFF)
Methods: Hatchling chicks were reared from days 5 -9 in one of
three lens conditions (+ or -10D or No Lens) after receiving a single
intravitreal injection of APB, PDA or Phosphate Buffered Saline
(PBS) in one of three lens conditions under either ND or 1 Hz LFF
light. Biometric responses were measured on day 9.
Electroretinograms (ERGs) were measured at 0 and 96 hours to
determine acute and long term effects of drugs in No Lens chicks
(n=22).
Results: Neither APB nor PDA significantly perturbed RC in ND
compared with PBS. Under LFF, APB did not affect expected RC cf
PBS. However, PDA_LFF chicks were relatively more hyperopic
under both -10D and +10D lens conditions than the PBS chicks and
affected both -10D and +10D lens ultrasound biometrics. ERGs
demonstrated that APB suppressed b-wave amplitude and greatly
reduced c-wave amplitude over the rearing period. PDA initially
induced acute suppression of photoreceptor a- and d-waves and
enhanced the b-wave, and increased the amplitude of the c-wave,
with a-, c- and d-waves showing some recovery at 96 hours.
Conclusions: Results in ND light conditions indicate that neither
mGLuR6 nor inner retinal ON and OFF pathways play a dominant
role in RC to optical defocus. By comparison, the dramatic effect of
LFF modulation of the photoreceptors and the surrounding ionic
environment on RC to positive lens defocus was unaffected by APB.
However LFF + PDA affected RC of both ± lens groups, and after
consideration of the ERG results, this suggests a primarily outer
retinal driver of abnormal ocular growth mechanisms.
Commercial Relationships: David P. Crewther, None; Sarah N.
Kiely, None; Melanie J. Murphy, None; Nina Riddell, None;
Sheila G. Crewther, None
416 Uveal Melanoma
Wednesday, May 08, 2013 8:30 AM-10:15 AM
Contributing Section(s): Physiology/Pharmacology
Comparison of Two Melanoma Cell Lines as Mouse-Models of
Uveal Melanoma
Marta M. Kilian1, Karin U. Loeffler1, Hans E. Grossniklaus2, Frank
G. Holz1, Christiane Pfarrer3, Christian Kurts4, Martina C. Herwig1.
1
Department of Ophthalmology, University of Bonn, Bonn,
Germany; 2Department of Ophthalmology, Emory University,
Atlanta, GA; 3Department of Anatomy, University of Veterinary
Medicine Hannover, Hannover, Germany; 4Institutes of Molecular
Medicine and Experimental Immunology, University of Bonn, Bonn,
Germany.
Purpose: The purpose of this study was to compare the
immunologic, growth and metastatic characteristics of HCmel12, a
new murine macrophage-attractive skin melanoma cell line, and
B16LS9 melanoma cells in a murine ocular melanoma model.
Methods: Eight CX3CR1 GFP knock-in mice and eight C57Bl/6
mice were injected intravitreally with HCmel12 or B16LS9
melanoma cells. The latter represent a previously established mousemodel for uveal melanoma when injected intravitreally. Local tumor
growth and distribution of macrophages in CX 3CR1 GFP knock-in
mice were verified in vivo by scanning laser ophthalmoscopy. Tumor
characteristics such as inflammation, tumor invasion into the uvea,
extrascleral growth and sites of metastases were monitored
histologically. Draining lymph nodes were investigated by
immunohistochemistry with HMB45/MART-1 markers for
melanoma cells. Immunohistochemical stains were performed for
F4/80 and CD163 to identify macrophages and their subtypes.
Results: HCmel12 and B16LS9 cells grew intravitreally resulting in
solid tumor growth in all mice within two days after injection. Both
melanoma cell lines invaded the uvea and proceeded to extraocular
extension if enucleation was not performed after 5-9 days. Distant
metastases were found in the lung for HCmel12 cells and in the liver
and spleen for B16LS9 cells. For HCmel12 cells, they occurred only
after extrascleral tumor growth and under participation of the
lymphatic system. Both tumors exhibited inflammatory cells
including macrophages.
Conclusions: Both melanoma cell lines showed similar
characteristics in terms of local and extraocular tumor growth and
they both exhibited involvement of draining lymph nodes. Metastatic
behaviour was not influenced by the ocular microenvironment since
both cell lines showed the same metastatic behaviour when injected
subcutaneously. However, due to the fact that distant metastases of
HCmel12 cells established exclusively in the lung they do not qualify
as a model of uveal melanoma.
Commercial Relationships: Marta M. Kilian, None; Karin U.
Loeffler, None; Hans E. Grossniklaus, None; Frank G. Holz,
Acucela (C), Allergan (C), Genentech (F), Heidelberg Engineering
(F), Zeiss (F), Novartis (F), Novartis (C), Optos (F), Merz (C), Bayer
(F), Bayer (C), Boehringer Ingelheim (C); Christiane Pfarrer,
None; Christian Kurts, None; Martina C. Herwig, None
Support: Bonfor (in-house grant, University of Bonn)
Calcium regulation by transient receptor potential channels in
human uveal melanoma cells
Stefan Mergler1, Arina Boehm1, Raissa Derckx1, Lisa Schmelzer1,
Fabian Garreis2, Aline Riechardt1. 1Department of Ophthalmology,
University Medicine Charite Berlin, Berlin, Germany; 2Department
of Anatomy II, University Erlangen-Nuremberg, Erlangen, Germany.
Purpose: Uveal melanoma (UM) is not only the most common
primary intraocular cancer, but also the leading primary intraocular
disease being fatal in adults. Differences in transient receptor
potential channels (TRPs) and cannabinoid receptor type 1 (CB1)
expression levels can serve as prognostic factors for uveal melanoma
(UM) tumor progression. Here, we postulated that such differences
indicate characteristics of UM tissue or healthy human uvea tissue.
Therefore, we investigated in malignant human UM and healthy uvea
putative TRP channel and CB1 gene expression and their functional
activity.
Methods: Gene and protein expression was investigated by RT-PCR
and immunohistochemistry in various human UM cell lines and
human uvea. Intracellular free Ca2+ ([Ca2+]i) was measured in
fura2-loaded UM cells, which were kindly provided by M. Jager et
al., Leiden University (Netherlands). Whole-cell currents were
measured by the planar patch-clamp technique.
Results: RT-PCR analysis revealed TRPV1, TRPM8 and CB1
expression in UM cells. TRPA1 was highly expressed in human uvea
but not in all UM cell lines. Capsaicin (CAP) increased whole-cell
currents (Fig. 1). The cooling agents menthol (500 µM)/icilin (20-50
µM) or moderate cooling (18 °C) increased [Ca2+]i and whole-cell
currents, which could be blocked by the TRPM8 channel blocker
BCTC (10 µM). Notably, CAP-induced Ca2+ influxes could be
suppressed by capsacepine (CPZ) (20 µM) or by activation of CB1
(10 µM WIN55,212-2) (WIN). Furthermore, 10 µM WIN induced
outwardly rectifying whole-cell currents (Fig. 2).
Conclusions: This study demonstrates for the first time functional
expression of TRPV1, TRPM8 and TRPA1 subtype and CB1 in UM
cells as well as a CB1-TRPV1 communication. This may promise to
improve and extend of the arsenal of diagnostic and therapeutic tools
available to date in this aggressive neoplastic disease by utilizing
TRPs.
Fig.1: CAP induced increase of whole-cell currents in UM cells could
be suppressed by CPZ. A: Experimental design. B: voltage
stimulation protocol. C: Mean whole-cell currents (n = 6-9).
Fig. 2: Application of the CB1 agonist WIN led to outwardly
rectifying whole-cell currents. Left: Time course of currents at -60
mV (lower trace) and 130 mV (upper trace) showing the current
activation by WIN in UM cells following washout of the TRP
channel blocker La3+. Right: Traces of WIN-activated current
responses to voltage ramps (500 ms).
Commercial Relationships: Stefan Mergler, None; Arina Boehm,
None; Raissa Derckx, None; Lisa Schmelzer, None; Fabian
Garreis, None; Aline Riechardt, None
Support: DFG ME 1706/13-1, DFG ME 1706/14-1
Unusual sodium-potassium dynamics and Na,K-ATPase
expression in Mel-290 uveal melanoma cells
Elena B. Rodriguez de Turco, Nicholas A. Delamere, Mohammad
Shahidullah. Physiology, University of Arizona, Tucson, AZ.
Purpose: Uveal melanoma is a common malignant intraocular tumor
and metastasis is frequent. The mortality for metastatic uveal
melanoma approaches 90 %. Here we report on abnormal sodiumpotassium dynamics in a particular uveal melanoma cell line, Mel290.
Methods: Melanoma cells were cultured in RPMI 1640 medium
supplemented with 10% FBS, 3mM glutamine and 2% penicillin +
streptomycin. The ratio of cell sodium concentration to potassium
concentration was determined by atomic absorption spectrometry.
Abundance of Na,K-ATPase and other proteins was examined by
western blot analysis.
Results: The Na:K concentration ratio in Mel-290 cells was 0.57
compared to 0.1 in a different melanoma cell, OCM-3, and 0.15 in
pig ciliary epithelium. Assuming cell Na+K = 140 mM, the resting
sodium concentration in Mel-290 cells is approximately 50 mM
which is several fold higher than the concentration in normal cells.
Western blot studies showed lower abundance of Na,K-ATPase alpha
1 subunit protein in Mel-290 compared to OCM-3. Differences in
Na,K-ATPase alpha subunit phosphorylation also were observed. In
contrast to Na,K-ATPase, the abundance of H/K-ATPase beta subunit
and H-ATPase (v-ATPase) beta subunit was higher in Mel-290
compared to OCM-3. A high Na:K ratio was detected in two other
melanoma cell types, suggesting the possible existence of a high
sodium sub-group of melanomas.
Conclusions: Certain uveal melanoma cells display a high-sodium
phenotype. It is interesting that high cytoplasmic sodium has been
reported in certain liver, brain and breast tumors by MRI and other
techniques. It remains to be determined whether the high-sodium
phenotype is related to a particular pattern of ATPase expression or
regulation.
Commercial Relationships: Elena B. Rodriguez de Turco, None;
Nicholas A. Delamere, None; Mohammad Shahidullah, None
Host Pigment Epithelium-Derived Factor (PEDF) Prevents
Progression of Uveal Melanoma Metastasis in the Liver and
Inhibits Formation of Blood Vessels in the Metastatic
Microenvironment
John M. Lattier1, Hua Yang1, Susan Crawford2, Hans E.
Grossniklaus1. 1Dept. of Ophthalmology, Emory University, Atlanta,
GA; 2Dept. of Pathology, University of St. Louis, St. Louis, MO.
Purpose: The most common form of eye cancer in adults, uveal
melanoma has a 5-year mortality rate of 30% primarily due to liver
metastasis. Angiogenesis is required for the progression of liver
metastasis, thus our lab is focusing on factors that may prevent
angiogenesis in the metastasis microenvironment. Pigment
epithelium-derived factor (PEDF), a potent anti-angiogenic protein, is
produced by liver hepatocytes. Our aim is to analyze the effect of
host PEDF on the size, frequency, and mean vascular density of liver
metastases in a mouse model of uveal melanoma. We hypothesize
that PEDF inhibits the progression of liver metastasis through a
mechanism involving angiogenesis.
Methods: Transgenic mice lacking one or both copies of PEDF were
injected in the posterior compartment of the eye with B16LS9 mouse
melanoma cells and monitored for one month while melanoma
metastasized to their livers, alongside control C57BL/6 mice.
Afterwards, livers were sectioned, stained, and analyzed for
metastasis size and frequency, and mean vascular density. All data
are reported as n=5.
Results: PEDF-/- mice exhibited a 34.6x increase in metastasis area
over PEDF+/+ controls, and PEDF+/- mice showed a 7.1x increase,
both significantly greater than wild-type. However, there was no
difference in the frequency of metastasis. The number of
macrometastases (diameter>200μm) was significantly greater in
PEDF-/- mice, with 6 per liver section, while PEDF+/- averaged 2
macrometastases per liver section, and PEDF+/+ averaged 0. The
mean vascular density (MVD) was 20.8x greater in PEDF-/metastases versus controls, while the MVD in PEDF+/- metastases
was 7.3x greater than controls. In conclusion, PEDF inhibits the
growth, but not frequency, of metastases in a mouse model of uveal
melanoma while suppressing the formation of blood vessels, thus
preventing the progression to macrometasases.
Conclusions: PEDF is an anti-angiogenic protein secreted by
hepatocytes. In our mouse model, the host lacks the ability to
synthesize PEDF. Blood vessels rapidly form within the metastases,
and the metastases grow much larger. These experiments suggest that
host PEDF may be a candidate for therapeutic development for
patients with aggressive uveal melanoma due to its ability to inhibit
metastasis growth.
Commercial Relationships: John M. Lattier, None; Hua Yang,
None; Susan Crawford, None; Hans E. Grossniklaus, None
Support: Biology of the Eye T32 EY007092-24
Pre-clinical analysis of Crizotinib in MAPK independent uveal
melanoma metastases
Pieter van der Velden, Mark de Lange, Mieke Versluis, Gregorius P.
Luyten, Martine M. Jager. Ophthalmology, LUMC, Leiden,
Netherlands.
Purpose: Uveal melanoma (UM) is an intraocular neoplasm with an
annual incidence of 7 per million. UM originates from melanocytes
just like cutaneous melanoma (CM) and similar to CM, the MAPK
pathway is involved in the development of UM. However, not all UM
seem to depend on the activation of this pathway. Loss of ERK
signalling may be correlated with progression as the ERK negative
cells are derived from UM metastasis. Metastases apparently use
different mechanisms to proliferate and survive. In this study, we
aimed to determine the pathway that is up-regulated in UM
metastases and to inhibit its function with targeted therapy.
Methods: We used antibody arrays to identify the pathways that are
activated in UM metastases and based on this analysis, kinase
inhibitors were selected to treat UM. Treated UM cells were in vitro
characterized for metastatic potential with anchorage independent
colony formation and migration assays using soft agar and 3D sphere
assays.
Results: In MAPK independent UM metastases constitutive c-Met
activation was observed. Malignant UM displayed MAPK activation
as well as c-Met activation. Treatment with Crizotinib reduced c-Met
activation and this resulted in reduced growth that was aggravated in
anchorage independent cell cultures. UM cells with activated c-Met
that were captured in 3D spheres revealed a reduced capacity to
migrate upon Crizotinib treatment compared to cells that lack
activated c-Met.
Conclusions: In malignant UM and UM metastases c-Met activation
was observed and Crizotinib was selected as a potential treatment
option. Growth inhibition by Crizotinib occurred by MAPK
dependent and MAPK independent mechanisms while inhibition of
migration was correlated with c-Met status. Combined our data
revealed c-Met signalling in progression of UM and support the use
of Crizotinib for malignant and metastatic UM treatment.
Commercial Relationships: Pieter van der Velden, None; Mark
de Lange, None; Mieke Versluis, None; Gregorius P. Luyten,
None; Martine M. Jager, None
Support: KWF UL2011-4991
Vemurafenib as a therapeutic option for treatment of melanoma
Simon F. Leicht, Christian M. Wertheimer, Raffael Liegl, Anselm
Kampik, Kirsten Eibl-Lindner. Department of Ophthalmology,
Ludwig-Maximilians-University, Munich, Germany.
Purpose: Uveal melanoma is the most common intraocular tumor.
To date, effective pharmacological therapies are lacking. In a set of
proof-of-concept experiments, we evaluated the effect of
Vemurafenib, a selective BRAF-inhibitor, on A375 melanoma cell
line cells.
Methods: The human melanoma cell line A375 was incubated with
Vemurafenib for 24 hours in different concentrations in the presence
of Dulbecco’s MEM medium under standard cell-culture conditions.
A Live-dead assay as well as a MTT toxicity assay on confluent cells
were performed to exclude toxic concentrations. To determine cell
proliferation, 30k cells per well were placed in a 12-well-plate and
incubated with Vemurafenib for 24 hours before the tetrazolium dyereduction assay (MTT test) was performed. After cells were seeded
on coated 12-well plates, incubated with Vemurafenib, and rinsed
with phosphate-buffered saline, cell proliferation was assessed by the
MTT test.
Results: Vemurafenib was an effective inhibitor of human melanoma
cell proliferation at nontoxic concentrations in vitro. The 50%
inhibitory concentration was close to 500 nM. A Vemurafenib
concentration of 5.0 µM accounted for an inhibition of cell
proliferation of more than 60%. This effect was dose dependent. No
toxic effect was detected compared with controls.
Conclusions: Vemurafenib might have a potential for the treatment
of melanoma cells. However, further studies are required to
determine if these findings also apply to uveal melanoma cells.
Commercial Relationships: Simon F. Leicht, None; Christian M.
Wertheimer, None; Raffael Liegl, None; Anselm Kampik, None;
Kirsten Eibl-Lindner, PCT/EP2010/051490 (P)
additionally, treatment with 5-Aza decreases metastasis formation in
vivo. These results provide proof of concept for an exciting potential
therapy to reduce mortality from this disease. Future work will focus
on identifying pathways that mediate these changes.
5-Azacytidine Reduces Growth, Invasiveness, and Clonogenicity
of Uveal Melanoma Cells and Decreases the Rate of Metastasis
Fatemeh Rajaii, Laura Asnaghi, Shannath L. Merbs, James T.
Handa, Charles Eberhart. Department of Ophthalmology, Wilmer
Eye Institute, Johns Hopkins University, Baltimore, MD.
Purpose: Uveal Melanoma is the most common primary intraocular
malignancy in adults. About 50% of cases are complicated by lethal
metastases. Epigenetic silencing of tumor suppressor genes is an
important factor in tumorigenesis and metastasis of many cancers.
Promoter methylation is an epigenetic mechanism that can alter target
gene transcription. A number of tumor suppressors have been shown
to be methylated in uveal melanoma; such epigenetic modifications
may contribute to pathogenicity. We hypothesized that demethylation
of uveal melanoma cells may cause a decrease in growth,
invasiveness, clonogenicity, and reduce metastatic transformation.
Methods: We used 5-azacytidine (5-Aza), a drug approved for
therapy in myelodysplastic syndrome to demethlyate DNA in uveal
melanoma cells. Invasiveness was characterized using transwell
migration assays. MTS assays were used to analyze cell growth.
Clonogenicity was examined using soft colony agar assays.
Metastasis formation was assayed using a murine melanoma
intraocular xenograft model which metastasizes hematogenously.
Results: Treatment of OCM3, 92.1, OCM1, OMM1, Mel285, and
Mel290 cells with 1 uM or 2 uM of 5-Aza caused decreased growth
compared to DMSO controls at day 7 (p<0.05). Transwell migration
assays of OCM3 cells treated with 0.5 uM or 1 uM of 5-Aza
demonstrated a decrease in invasion compared to controls (p<0.001).
Clonogenicity assays revealed that OCM3, 92.1, and OCM1 cells
pretreated with 0.5 uM or 1 uM 5-Aza formed fewer and smaller
colonies than controls (p<0.001). In vivo assays of 15 C57Bl6 mice
injected with vehicle-treated B16F10 cells and 14 mice injected with
5-Aza-treated cells revealed significantly fewer lung metastases in
the latter group.
Conclusions: Using 5-Aza to demethylate DNA in uveal melanoma
cells reduces growth, invasiveness, and clonogenicity in vitro;
A. 5-Aza decreases cell growth in 6 cell lines. B. Transwell migration
assays. C. Treatment of OCM3 cells with 5-Aza decreases invasion.
A. Clonogenicity assays in OCM3, 92.1, and OCM3 lines. B. 5-Aza
decreases the number of colonies formed. C. 5-Aza decreases the size
of colonies formed.
Commercial Relationships: Fatemeh Rajaii, None; Laura
Asnaghi, None; Shannath L. Merbs, None; James T. Handa,
Genentech, Inc. (C); Charles Eberhart, None
Support: Research to Prevent Blindness
Cytotoxic Effects of the Combination of Resveratrol and Nacetylcysteine on Cultured Human Uveal Melanoma Cells
Tommaso Vagaggini1, 2, Dan-Ning Hu2, Anthony Sclafani2, Steven A.
McCormick2, Joan E. Roberts1. 1Natural Sciences, Fordham
University, New York, NY; 2Pathology, New York Eye and Ear
Infirmary, New York, NY.
Purpose: Resveratrol (3,5,4′-trihydroxytrans-stillbene) is a
stillbenoid non-flavonoid contained in various natural compounds,
particularly in the skin of grapes. It is a powerful antioxidant and has
been proven in vitro to inhibit proliferation and induce cell death in a
number of cancer cell lines. Zahid et al. (2011) had previously shown
that resveratrol combined with N-acetylcysteine (NAC) had increased
potency in the inhibition of cancer emergence and proliferation. The
purpose of this study was to assess whether the cytotoxic effect of
resveratrol could be enhanced by the addition of NAC on uveal
melanoma cells as well.
Methods: A metastatic (C918) and a non-metastatic (SP6.5) human
uveal melanoma cell lines were treated with resveratrol (10, 30, 100
and 300 μM), NAC (0.3, 1, 3, 10 and 30 mM) and a combination of
the two compounds (resveratrol at 30 μM with NAC at 1, 3, 10 and
30 mM). The effects of these compounds on cell viability were
assessed by using the MTT (3-[4,5-dimethylthiazol-2-yl]2,5diphenyltetrazolium bromide) assay.
Results: Resveratrol reduced cell viability of cultured human uveal
melanoma cells in a dose-dependent manner (10, 30, 100 and 300 μM
) and time-dependent manner (3-48 hr), with IC50 at 91.8 ±16.2 μM
and 62.7 ±32.0 μM in SP6.5 and C918 cell lines, respectively. NAC,
on the other hand, reduced cell viability in a dose dependent manner
only in the non-metastatic cell line (SP6.5) with IC50 at 18.7 ± 2.6
mM, but did not affect cell viability of the metastatic cell line up to
30 mM. The combination of resveratrol and NAC was not effective
on both SP6.5 and C918 cell lines, e.g., cell viability in SP6.5 treated
with resveratrol (30 μM ) and resveratrol (30 μM ) with NAC (10
mM) was 66.7 ± 7.4 % and 80.0 ± 10.8%, respectively (P = 0.162),
indicating that the addition of NAC did not increase the cytotoxic
effect of resveratrol.
Conclusions: Resveratrol has a powerful cytotoxic effect on both cell
lines of uveal melanoma, whereas NAC is effective only in the nonmetastatic cell line (SP6.5). Addition of NAC to resveratrol did not
enhance cytotoxic effect in uveal melanoma cells.
Commercial Relationships: Tommaso Vagaggini, None; DanNing Hu, Zeovision (F); Anthony Sclafani, None; Steven A.
McCormick, None; Joan E. Roberts, None
Support: Fordham University Dean's Research Grant
Simultaneous Inhibition of the HGF/MET and Erk1/2 Pathways
Affect Uveal Melanoma Cell Growth and Migration
Chandrani Chattopadhyay, Elizabeth A. Grimm, Scott E. Woodman.
Melanoma Medical Oncology, UT MD Anderson Cancer Ctr,
Houston, TX.
Purpose: About 50% of primary uveal melanoma metastasize, and
almost all metastatic uveal melanomas involve the liver. Based on the
biological evidence of metastatic uveal melanoma tropism to the
liver, hepatocyte growth factor (HGF) has been proposed to be an
important micro-environmental element in attracting and supporting
uveal melanoma metastasis through activation of MET, the HGF
receptor. It is also known that unlike cutaneous melanoma, the
majority (>85%) of uveal melanoma express mutations in the Gproteins (GNAQ or GNA11) that drive the MEK/ERK1/2 pathway.
Therefore, we propose that the combination of MET and MEK
inhibition, using clinically relevant small molecule inhibitors, would
have selective inhibitory effects on the growth and migration of wellcharacterized mutant versus non-mutant uveal melanoma cell lines.
Methods: Western-blot analysis was done to determine the relative
protein levels of ERK1/2 and MET in uveal melanoma cells. Drug
treatment studies were performed to determine the singular and
combinatorial effect of AZD6244 (MEKi) and MK-8033 (METi) on
downstream markers. Further studies were performed to evaluate the
effect of combination MEKi and METi treatment on cell growth,
apoptosis and cell migration. Cell growth was tested by MTT assays,
cell migration by in vitro Boyden chamber assays and apoptosis by
monitoring PARP processing.
Results: All uveal melanoma cell lines with a GNAQ mutation
express MET mRNA and protein. The level of mRNA expression
correlated well with protein expression. MEKi treatment, but not
METi treatment, resulted in markedly reduced ERK1/2
phosphorylation. Either MEKi or METi treatment alone resulted in
reduced cell proliferation, but only modest induction of apoptosis.
The combination MEKi + METi resulted in significant reduction of
proliferation only in GNAQ mutant cells. Uveal melanoma cell
migration was blocked by the METi, but not the MEKi treatment.
Conclusions: MET protein expression showed no correlation with
GNAQ mutation status. Combining MEKi with METi treatment may
have added benefit to either treatment alone in reducing cell growth
in GNAQ mutant cells, but not wild-type cells. In addition,
combining METi treatment with a MEKi regimen adds the effect to
limiting uveal melanoma cell migration.
Commercial Relationships: Chandrani Chattopadhyay, EMD
Sorono (F); Elizabeth A. Grimm, Merck (F), EMD-Serono (F);
Scott E. Woodman, GlaxoSmithKline (F), Life Technologies (C)
Cyclooxygenase-2 (COX-2) expression in primary uveal
melanoma and the potential role for adjuvant treatment with
COX-2 inhibitors
Lindsay E. Adam, James B. Massengill, Colleen M. Cebulla,
Mohamed H. Abdel-Rahman. Ophthalmology, The Ohio State
University, Columbus, OH.
Purpose: Cyclooxygenase-2 (COX-2) has previously been shown to
have an influential role in tumorigenesis in a variety of malignancies.
The objective of this study was to examine COX-2 expression and
function in primary uveal melanoma and uveal melanoma cell lines
and to evaluate correlation with histological, genetic, and clinical
markers.
Methods: The expression of COX-2 was assessed by
immunohistochemistry. The cytotoxicity of COX-2 inhibitor
Celecoxib was assessed on five uveal melanoma cell lines (C918,
OCM3, MEL270, MEL202, 92.1) using in-vitro cell proliferation
assay.
Results: Using immunohistochemistry, 4 of the 15 specimens
(26.7%) showed intense staining for COX-2. No statistically
significant associations (p <0.05) were identified between COX-2
expression and survival, cell type, tumor size, tumor genetics, pattern
distribution, or tumor invasion. The IC50 ranged from 11.4 to 29.3
microMolar in the tested cell lines.
Conclusions: Increased COX-2 expression does not appear to be
strongly correlated with poor prognostic indicators in primary uveal
melanoma. COX-2 could be a potential adjuvant therapy in a small
subset of patients.
Commercial Relationships: Lindsay E. Adam, None; James B.
Massengill, None; Colleen M. Cebulla, None; Mohamed H. AbdelRahman, None
Support: The Patti Blow Research Fund in Ophthalmology; The
American Cancer Society Grant #IRG-67-003-47
Antimicrobial Peptide Expression and Potential Roles in Uveal
Melanoma Pathogenesis
Joseph C. Manarang, Deborah C. Otteson, Adrian Glasser, Alan R.
Burns, Alison M. McDermott. College of Optometry, University of
Houston, Houston, TX.
Purpose: The purpose of this project was to elucidate the potential
roles of AMPs in UM pathogenesis by determining expression of
human neutrophil peptide-1 (HNP-1), cathelicidin LL-37, and human
beta defensins (hBD) -1, -2, -3 and -4 in UM cells, and their effects
on apoptosis induction, cell migration and vasculogenic mimicry
(VM).
Methods: AMP mRNA expression by UM cell lines OCM8,
OMM2.5, SP6.5 and MUM2b and primary normal and UM
melanocytes was determined by RT-PCR. The presence of DNA
laddering was sought in UM cells treated with HNP-1 (75μg/ml), LL37 (100μg/ml), Magainin II (250μg/ml) or Cecropin B (800μg/ml) for
24 hrs. To test migration, UM cell monolayers were scratched then
HNP-1 (5 and 17.5μg/ml) or LL-37 (5 and 20μg/ml) added. Digital
images were collected at 0, 6, 12, 24 and 48 hrs and analyzed by a
Matlab program. AMP effects on VM formation were investigated by
exposing MUM2b cells growing in a 3D gel matrix to HNP-1 (12.5
and 17.5μg/ml) and LL-37 (5 and 10μg/ml) for upto 21 days. Gels
were stained with DAPI and Phalloidin or Calcein AM, then imaged
and reconstructed in 3D using a Deltavision microscope running
SoftWorx.
Results: LL-37 was expressed by all UM cell lines (n=3). None
expressed hBD-1, hBD-2, nor HNP-1 while some expressed hBD-3
and hBD-4. Primary cultured normal and UM melanocytes expressed
hBD-4, but variably expressed the other AMPs (n=2-3). DNA
laddering characteristic of apoptosis was found in control
staurosporine treated cells, but not in AMP treated cells (n=3). In the
presence of 10% FBS, UM cells migrated significantly faster than
cells in serum free media. However AMPs did not significantly
modulate cell migration rates (n=3). Light and fluorescence
microscopy revealed MUM2b cells on gels grew in multiple levels
forming 3D looping networks with characteristic acellular channels
(n=3). These VM patterns were not affected by treatment with AMPs.
Conclusions: AMP mRNA was variably expressed by UM, and there
was no marked difference in the expression profiles between UM
melanocytes and normal melanocytes. Likewise, our findings suggest
that AMPs do not modulate UM pathogenesis with respect to
apoptosis induction, cell migration or VM formation. While this
indicates that AMPs play no role in UM tumor pathogenesis, their
ability to exert a cytotoxic effect on UM cells (Manarang et al.
ARVO 2009) may be capitalized upon for development of novel
treatments.
Commercial Relationships: Joseph C. Manarang, None; Deborah
C. Otteson, None; Adrian Glasser, None; Alan R. Burns, None;
Alison M. McDermott, None
Support: NIH EY13175, NIH EY007551, Texas ARP, EY021792,
SVGR 2011
Improved Specimen Handling for Optimizing Diagnostic Yield
from Fine Needle Aspiration Biopsy of Uveal Tumors
Nieraj Jain, Victor M. Elner, Hakan Demirci. University of
Michigan, Ann Arbor, MI.
Purpose: To describe and evaluate a novel strategy of tissue handling
of transscleral fine needle aspiration biopsy (FNAB) specimens in
unknown uveal tumors.
Methods: This is a retrospective series of consecutive patients
undergoing FNAB of uveal tumors from September 2010 through
November 2012. The diagnostic yield and adverse outcomes are
documented, including large (in excess of 3 disc diameters in greatest
linear dimension) or fovea-involving subretinal hemorrhage,
rhegmatogenous retinal detachment, and orbital metastasis.
Surgical technique involves FNAB via a pars plana approach under
indirect ophthalmoscopy. Upon collection, the specimen is placed in
a microcentifuge tube and immediate low speed centrifugation is
performed.
Results: FNAB samples of 10 patients were analyzed.
Cytopathological diagnosis was established in 8 of 10 patients (80%).
Of these, 6 were choroidal melanoma, 1 was metastatic
adenocarcinoma from the lung, and 1 was peripheral exudative
hemorrhagic chorioretinopathy. The two subjects with non-diagnostic
FNAB were later found by incisional biopsy to have melanoma of the
ciliary body.
Conclusions: This technique compares favorably with those in the
literature, despite a high number of small tumors. Improved specimen
handling involving the use of a microcentrifuge tube for specimen
collection, immediate low speed centrifugation, and formation of a
cellular pellet may improve the diagnostic yield of this procedure.
Commercial Relationships: Nieraj Jain, None; Victor M. Elner,
OcuSciences, Inc. (I), OcuSciences, Inc. (P); Hakan Demirci, None
Support: The Heed Ophthalmic Foundation
Heterogeneity of Monosomy 3 in Fine Needle Aspiration Biopsy
of Choroidal Melanoma
Melinda Chang, Nagesh Rao, Lariza Johnson, Tara McCannel. Jules
Stein Eye Institute, Los Angeles, CA.
Purpose: To report on the heterogeneity of monosomy 3 within a
fine needle aspiration biopsy obtained transsclerally from choroidal
melanoma for prognostication.
Methods: All clinical records of patients who were diagnosed with
choroidal melanoma and underwent iodine-125 plaque brachytherapy
or enucleation with intraoperative transscleral fine needle aspiration
biopsy from January 2005 to August 20, 2011 and who had a positive
result for monosomy 3 by fluorescence in situ hybridization as
reported by clinical cytogenetics testing were collected. Patient age
and gender, total number of cells evaluated and number of cells
positive for monosomy 3, tumor size, and metastatic outcome were
recorded for each patient.
Results: A positive result for monosomy 3 was reported in 98
patients who underwent transscleral fine needle aspiration biopsy.
Two patients were lost to follow-up immediately post-operatively,
and the remaining 96 patients were included in this study. The mean
number of cells evaluated in the biopsy was 275 (range 28 to 520).
The mean percentage of cells positive for monosomy 3 was 62.0%
(range 4.7-100%). Five patients (5%) were treated by enucleation,
and 91 patients (95%) underwent brachytherapy. The mean tumor
height was 6.20 mm (range 1.99 to 19.42 mm). Tumor size did not
correlate with percentage of monosomy 3 in the biopsy. In an average
follow-up interval of 28.7 months (range 3-76 months), choroidal
melanoma metastasis developed in 21 (22%) patients. There was no
significant difference in the percentage of monosomy 3 in the biopsy
of patients who developed metastasis (68.6%) compared to those who
did not develop metastasis (60.2%) (p=0.31) during the follow-up
period.
Conclusions: Cytogenetic heterogeneity of fluorescent in-situ
hybridization for monosomy 3 exists within a biopsy sample. Tumor
size did not correlate with the percentage of monosomy 3 in the
biopsy. Furthermore, the percentage of monosomy 3 within the
biopsy did not correlate with the development of choroidal melanoma
metastasis during the follow-up interval. Therefore, a positive finding
of monosomy 3 within a tumor biopsy, regardless of percentage of
cells positive, may portend a poor prognosis for metastasis.
Commercial Relationships: Melinda Chang, None; Nagesh Rao,
None; Lariza Johnson, None; Tara McCannel, None
Support: Research to Prevent Blindness and the George E. and Ruth
Moss Trust
Prognostic FNAB of Uveal Melanoma
Arun D. Singh1, 5, Mary E. Aronow1, Charles V. Biscotti2, Raymond
Tubbs3, Lynn Schoenfield2, Pierre Triozzi4, 5. 1Ophthalmic Oncology,
Cole Eye Institute, Cleveland, OH; 2Anatomic Pathology, Cleveland
Clinic, Cleveland, OH; 3Molecular Pathology, Cleveland Clinic,
Cleveland, OH; 4Solid tumor Oncology, Cleveland Clinic, Cleveland,
OH; 5Taussig Cancer Center, Cleveland Clinic, Cleveland, OH.
Purpose: To report experience with prognostic fine needle aspiration
biopsy of uveal melanoma.
Methods: Prospective interventional series of 150 consecutive
patients with uveal melanoma treated at the Cleveland Clinic Cole
Eye Institute between May 2009 and August 2012. FNAB was
performed with 25-gauge needle. Chromosome 3 status was assessed
by FISH using both directly labeled Spectrum Green® and Spectrum
Orange® enumeration probes for the alphacentromeric locus of
chromosome 3 (CEP3). Cut off value of 20% was used to define
monosomy fro chromosome 3.
Results: 41 eyes were enucleated, 102 treated with plaque radiation
therapy and 7 treated by local resection. Enucleated eyes were
subjected to transcleral FNAB in all cases. Patients undergoing
plaque radiation therapy underwent trans corneal (6) transcleral (32)
or trans vitreal biopsy (64) based upon tumor location. Overall,
sample was adequate for FISH analysis in 135 eyes (90%). Eyes that
underwent enucleation revealed greater proportion of monosomy 3
cells (67% vs 40%) respectively. 32 (53%) patients with monosomy 3
entered into an adjuvant treatment trial.
Conclusions: Prognostic biopsy FNAB for uveal melanoma with 25gauge needle can yield sufficient samples in 90% of cases. High-risk
cases identified by FISH FNAB can be recruited into an adjuvant
treatment trial.
Commercial Relationships: Arun D. Singh, None; Mary E.
Aronow, None; Charles V. Biscotti, None; Raymond Tubbs, None;
Lynn Schoenfield, None; Pierre Triozzi, None
Support: Falk Trust
Extraocular Extension of Uveal Melanoma After Fine Needle
Aspiration, Vitrectomy, or Open Biopsy
Amy C. Schefler1, Daniel Gologorsky2, Brian Marr3, Carol L.
Shields4, Ignacio Zeolite5, David H. Abramson3. 1Retina Consultants
of Houston, Houston, TX; 2Geisel School of Medicine at Dartmouth,
Hanover, NH; 3Ophthalmic Oncology, Memorial Sloan-Kettering
Cancer Center, New York, NY; 4Oncology Service, Wills Eye
Institute, Philadelphia, PA; 5Ophthalmology, Universidad de
Mendoza, Mendoza, Argentina.
Purpose: The use of fine needle aspiration biopsy (FNAB) for uveal
melanoma has increased dramatically in recent years. Some clinicians
have expressed concern for orbital recurrence of melanoma in part
because of published experimental evidence indicating that tumor
cells can remain in a needle track after experimental biopsy
procedures in animal and human eyes. The incidence of tumor cells
seeding the FNAB site and causing orbital extension of uveal
melanoma in the context of routine clinical care is unknown.
Methods: This was a retrospective, multicenter cases series
examining cases of orbital recurrence of uveal melanoma after fine
needle biopsy, excisional biopsy, or surgery. An international survey
was initiated among ocular oncologists who had attended the
International Conference of Ocular Oncology in 2011. All reported
cases were pooled and clinical and pathologic details were gathered
from each contributing institution. Risk factors for recurrence were
identified. Four cases were identified, bringing the total number of
cases ever reported in the world literature to five.
Results: Four cases worldwide were identified with sufficient
information to explore. Two patients underwent excisional biopsy
and vitrectomy , one underwent excisional biopsy only, and one
underwent FNAB only. All patients had histopathologic confirmation
of uveal melanoma and all developed histopathologically confirmed
orbital extension. Three developed systemic metastases and one
patient died of metastases.
Conclusions: Extraocular extension of uveal melanoma following
FNAB, incisional biopsy, and vitrectomy is extremely rare but not
impossible. Details related to technique appear to be critical such as
needle gauge, number of passes of needle into tumor, manipulation of
needle while in the tumor, removal of needle from the eye with
compression of the globe at the entry site, number of cells extracted,
leakage of vitreous outside the globe, lack of subsequent
brachytherapy, and use of cryotherapy at the biopsy site. Continued
multicenter collaboration and honest reporting of these rare cases is
needed to definitively identify risk factors for this devastating
complication.
Commercial Relationships: Amy C. Schefler, None; Daniel
Gologorsky, None; Brian Marr, None; Carol L. Shields, None;
Ignacio Zeolite, None; David H. Abramson, None
A comparison of gene expression profiling versus multiplex
ligation-dependent probe amplification in metastatic risk
prediction in choroidal melanoma
Sarah E. Coupland1, Bertil E. Damato1, Helen Kalirai1, Marcela
Baudo1, Chris Bergstrom2, Jill R. Wells2, Tero Kivela3, Hans E.
Grossniklaus2. 1Pathology, Univ of Liverpool/Sydney Jones Library,
Liverpool, United Kingdom; 2Department of Ophthalmology, Emory
University School of Medicine, Atlanta, GA; 3Department of
Ophthalmology, Helsinki University Central Hospital, Helsinki,
Finland.
Purpose: To compare gene expression profiling (GEP) using
DecisionDX-UM® with multiplex ligation-dependent probe
amplification (MLPA) in molecular typing and predicting metastatic
risk of choroidal melanomas (CM).
Methods: 20 enucleated formalin-fixed, paraffin-embedded (FFPE)
CM, previously examined by DecisionDX-UM®, were tested with
MLPA, and then categorized with both methods independently in a
masked manner as having “minimal”, “low” or “high” metastatic
potential. Results were correlated with clinical and histologic
metastasis predictors, and the 10-year risk of metastatic death was
estimated by multivariate analysis using Liverpool Uveal Melanoma
Prognosticator Online (LUMPO).
Results: The patients’ median age was 60 years, and the CM a
median largest basal diameter and height of 18.4 mm and 10.2 mm.
The TNM stages of the CM were: IIA (n=2), IIB (3), IIIA (3) IIIB (9)
and IIIC (3). DecisionDX-UM® classified the CM as Class 1A (n=4),
Class 1B (n=7) and Class 2 (n=9). MLPA indicated “minimal”, “low”
and “high” risk in 4, 5 and 11 patients, respectively. Discordant
results occurred in 5 CM (25%; 95% confidence interval, 9-49), with
MLPA indicating a higher risk of metastasis in 3 and DecisionDXUM® in 2. Associations with clinical and histomorphologic
metastasis predictors were similar. Multivariate LUMPO predictions
of the 10-year metastatic mortality for all patients had medians of
37% and 41% according to DecisionDX-UM® and MLPA results,
respectively. Four patients with "high" risk had succumbed to their
disease; all of the UM patients with "minimal" or "low" risk were still
alive at time of abstract submission.
Conclusions: DecisionDX-UM® and MLPA in FFPE agreed well
with each other, with metastasis predictors and with survival
estimates. For most CM patients both methods are likely to provide
similar results in clinical use.
Commercial Relationships: Sarah E. Coupland, None; Bertil E.
Damato, None; Helen Kalirai, None; Marcela Baudo, None; Chris
Bergstrom, None; Jill R. Wells, None; Tero Kivela, None; Hans E.
Grossniklaus, None
Prognostic implications of GEP class 2 in clinical practice: a
single center experience with 273 cases
James J. Augsburger, Zelia M. Correa. Ophthalmology, University
of Cincinnati, Cincinnati, OH.
Purpose: Several reports from the developers of the gene expression
profile (GEP) test of uveal melanomas (Washington University, St.
Louis [WUSTL] group) currently licensed for commercial use in the
United States (DecisionDX UM test, Castle Biosciences, Inc.,
Pheonix, AZ) have suggested that patients with a GEP Class 2 tumor
have a median survival time until death from melanoma metastasis of
approximately 3 years and almost no chance of long-term survival.
Our group has submitted FNAB specimens for GEP testing of
patients with a clinically diagnosed posterior uveal melanoma prior to
treatment since 9/2007. We analyzed our series of patients (9/2007 5/2012) to determine if our experience mirrored or differed from the
published and presented survival results.
Methods: Retrospective analysis of tumor size and GEP class in 273
primary posterior uveal melanomas evaluated by FNAB prior to or at
the time of intraocular tumor treatment and actuarial assessment of
cumulative probability of death from metastatic uveal melanoma in
this group of patients.
Results: Tumor size in our series ranged from 2.5 - 22.0 mm in
largest basal diameter (mean 11.8 mm) and from 1.0 to 18.0 mm in
thickness (mean 5.6 mm). 103 patients (37.3%) had a tumor ≤ 10 mm
in largest linear tumor dimension (LTD) while 123 (45.1%) had a
tumor > 10 but ≤ 15 mm in LTD and only 47 (17.2%) had a tumor >
15 mm in LTD. In contrast, 50.0% of tumors in the WUSTL group's
validation series had LTD > 15 mm and only 10.4% had LTD ≤ 10
mm). 193 of the tumors in our series (70.7%) were categorized as
GEP class 1 while only 77 (28.2%) were categorized as GEP class 2.
In contrast, 51.2% of the tumors in the WUSTL validation series had
a class 2 tumor. Three-year melanoma-specific cumulative actuarial
mortality in patients with a GEP class 2 tumor in our series was
32.8%; in contrast, 3-year mortality in the class 2 cases in the
WUSTL series was 44.3%.
Conclusions: In our series, patients with a GEP class 2 tumor did not
experience a short-term mortality rate from uveal melanoma
metastasis as unfavorable as that reported by the WUSTL in their
validation study. This observation suggests that (1) tumor size
smaller than medium to large is associated with a longer metastasisfree latent interval, (2) GEP class 2 is not as predictably linked to
short-term development of metastasis as indicated by the developers
of this test, or (3) both.
Commercial Relationships: James J. Augsburger, None; Zelia M.
Correa, None
Support: Unrestricted Departmental Grant from Research to Prevent
Blindness, Inc., New York, NY, to University of Cincinnati
Department of Ophthalmology (James J. Augsburger, Chairman)
Choroidal Melanoma pronostication: a robust and costless
classifying system
Nathalie Cassoux1, Laurence Desjardins1, Corine Plancher2,
Bernard Asselain2, Christine Levy-Gabrielle1, Livia Lumbroso-Le
Rouic1, Manuel J. Rodrigues4, Xavier Sastre5, Sophie PipernoNeumann4, Jerome Couturier3. 1Ophthalmology, Institut Curie, Paris,
France; 2biostatistics, Institut Curie, Paris, France; 3Genetics, Institut
Curie, Paris, France; 4Oncology, Institut Curie, Paris, France;
5
Pathology, Institut Curie, Paris, France.
Purpose: This paper investigate the capability to predict high risk
patients using genetic analysis of choroidal melanoma on enucleated
eyes, local resection or FNAB material.
Methods: Patients with choroidal melanoma were prospectively
enrolled. Demographic data were collected including patient age,
gender, tumor diameter, and tumor thickness, ciliary body invasion or
extrascleral extension. Tumor samples were taken on enucleated
globe, at the time of clips or plaque positioning with FNAB, or after
endoresection. Array-CGH techniques were performed on two
different platforms. Home-made BAC-arrays (CIT / INSERM U830,
Institut Curie, Paris) and NimbleGen 4x72K arrays (Roche
NimbleGen, Madison, WI). Genomic DNA was extracted with Phase
Lock Gel Light and analyzed versus normal reference DNA. Gains
are defined as a log2ratio ≥0.58 and deletions ≤-1. Patients were
managed for their primary tumor then monitored for metastasis.
Results: 3 groups were classified by Array-CGH. “Low risk” patients
were defined by disomy3, “intermediate risk” was defined by
monosomy 3 alone, and “high risk” patients were defined by
monosomy 3 and 8q gain. The media follow-up was 25 months
[range; 22-29 months]. At the end of the study, 128 patients
developed metastasis (33.7%) and 96 patients died. Univariate cox
analysis showed that metastatic free interval at 2 years was 92% for
low risk patients, 67% for intermediate risk and 33% for high risk
patients. There is a statistical association between genomic high risk
results and other known prognostic factors especially for
mixt/epitheliod cell type. Univariate Cox proportional hazard analysis
factors associated in our study with metastasis included basal tumor
diameter p=0.001, tumor thickness p=0.03, mixt/epithelioid cell type
p=0.006, genetic data p<0.0001. High risk profile was more strongly
associated with metastasis than the other prognostic factor p<0.001.
Multivariate cox modeling analysis showed that genetic result was
the only variable that contributed independently to prognosis with
p=0.001 IC 95%[1.6-7.8] for chromosome 3 monosomy alone and
p<0.0001 IC 95% [5.2-20.4] for chromosome 3 monosomy and 8q
gain.
Conclusions: Array-CGH is a robust and not too expensive method
for choroidal melanoma prognostication. This method is to date
currently used in France. High risk patients are included in an
adjuvant therapy trial.
Commercial Relationships: Nathalie Cassoux, None; Laurence
Desjardins, None; Corine Plancher, None; Bernard Asselain,
None; Christine Levy-Gabrielle, None; Livia Lumbroso-Le Rouic,
None; Manuel J. Rodrigues, Janssen-Cilag (R), Chugai Pharma (R);
Xavier Sastre, None; Sophie Piperno-Neumann, None; Jerome
Couturier, None
Relationship between rate of choroidal melanoma flattening
following plaque radiotherapy and GEP class of tumor cells
Zelia M. Correa, James J. Augsburger. Ophthalmology, University
of Cincinnati, Cincinnati, OH.
Purpose: Several retrospective clinical studies prior to the advent of
gene expression profile (GEP) testing of posterior uveal melanomas
showed faster rate of flattening of the tumor over the first 6 months
following focal radiotherapy to be predictive of significantly higher
actuarial probabilities of death from metastasis. To date, there have
been no reports describing the relationship between GEP class of
tumor cells obtained by FNAB prior to primary tumor treatment and
post-treatment tumor regression following plaque radiotherapy. We
hypothesized that GEP class would be strongly associated with faster
tumor shrinkage post-plaque.
Methods: Retrospective analysis of the relationship between GEP
class of posterior uveal melanoma cells and rate of post-irradiation
tumor flattening during the first 6 post-treatment months in patients
managed by I-125 plaque radiotherapy and evaluated by FNAB at or
shortly prior to the start of treatment. To minimize the impact of
initial tumor thickness on rate of tumor flattening, we employed a
paired case approach in which tumor thickness at baseline in class 1
and class 2 cases was matched to within ± 0.5 mm. Paired t-testing
was used to compare the mean thickness of the tumors in the GEP
subgroups at the 3 and 6 month post-treatment assessments.
Results: Our base study group (9/7/07 - 4/27/12) consisted of 269
patients. 77 of these tumors (28.6%) were GEP class 2. Thirty-seven
of these 77 patients were treated by I-125 plaque radiotherapy postFNAB and returned for ultrasonographic measurement of tumor
thickness at both 3 and 6 months post-treatment. We identified a
matching patient from the GEP class 1 tumor cases for each of the
class 2 cases. The tumors in the 37 pairs of patients ranged in
thickness from 2.0 mm to 11.0 mm at baseline. The mean tumor
thickness was 5.6 mm in both GEP subgroups at baseline. At 3
months post-plaque, mean tumor thickness was 4.1 mm in the class 1
cases and 4.5 mm in the class 2 cases (paired t = 0.88, P = 0.061). At
6 months post-plaque, mean tumor thickness was 3.5 mm in the class
1 cases and 3.9 mm in the class 2 cases (paired t = 1.66, P = 0.11).
Conclusions: Association with GEP class of tumor cells does not
appear on the basis of this study to explain the previously reported
unfavorable prognostic impact of faster tumor flattening following
focal irradiation of posterior uveal melanomas.
Commercial Relationships: Zelia M. Correa, None; James J.
Augsburger, None
Support: Unrestricted Departmental Grant from Research to Prevent
Blindness, Inc., New York, NY, to University of Cincinnati
Department of Ophthalmology (James J. Augsburger, Chairman)
The role of Osteopontin expression in the mechanism of invasion
and metastasis potential in Uveal Melanoma
Bin Li. Beijing Institute of Ophthalmology, Beijing TongRen
Hospital, Beijing, China.
Purpose: To investigate the relationship between Osteopontin(OPN)
expression with invasion and metastasis potential in UM.
Methods: (1)50 UM samples were collected in Beijing Tongren
Hospital. The OPN expression of the samples was detected by using
immunohistochemistry staining. (2)The expression of OPN mRNA in
different metastasis potential uveal melanoma cell lines (MUM2B,C918,OCM-1A) was analyzed by using Q-PCR. (3)To bulid up
OPN siRNA lentiviral vector and transfect it into UM cell lines C918
and MUM-2B to detect the proliferation and cell invasion capacity of
cells by using MTT and Transwell method.
Results: (1)OPN was positively expressed in 10/13 patients with
metastasis and 14/37 patients without metastasis (P=0.000). There
were 11 samples and 13 samples exhibiting the positive expression in
15 epithelial cell type and 35 non-epithelial cell type of UM
respectively (P=0.000). The OPN positive expression were 20/30
affected tumors and 4/20 unaffected ciliary tumors(P=0.000). No
significant differences in OPN expression were found between age,
gender, eyes, largest basal diameters and (P=0.536, 0.256, 0.802,
0.848, 0.555).(2)The relative expression value of mRNA of OPN was
in turn 1±0.04, 0.91±0.03, 0.08±0.01 in MUM-2B,C918,OCM-1A
(P<0.05). The OPN mRNA expression was significant enhanced in
high metastatic potential cell lines(MUM-2B,C918) compared with
low metastatic potential cell lines OCM-1A.(P<0.05), but no
significant difference was observed between two high metastatic
potential cell lines (P=0.804). (3)OPN siRNA lentiviral vector
transfected UM cell lines MUM-2B,C918, the transfection efficiency
is over 80%, and the expression level of the OPN is lower than that
before transfection ( P<0.05).(4)After transfection of OPN siRNA
lentiviral vector, the cell proliferation capacity of C918 cells in
Konck Down group is lower than cells in Negative Control group and
Control group, while the cell proliferation capacity of MUM-2B cells
in Konck Down group is a little lower than cells in the other two
groups. (5) After transfection of OPN siRNA lentiviral vector, the
cell invasion capacity of C918 cells in Konck Down group is lower
than cells in other two groups.
Conclusions: OPN expression is associated with invasion and
metastasis potential in uveal melanoma cell line. The results imply
that OPN expression maybe as an indicator of uveal melanoma
invasion and metastasis ability.
Commercial Relationships: Bin Li, None
BAP1 mutations in uveal melanoma
Anna E. Koopmans1, 2, Robert M. Verdijk3, Thierry van den Bosch3,
Mike M. van den Berg1, 2, Jolanda Vaarwater1, Dion Paridaens4,
Emine Kilic1, Annelies de Klein2. 1Ophthalmology, Erasmus Medical
Centre, Rotterdam, Netherlands; 2Clinical Genetics, Erasmus Medical
Centre, Rotterdam, Netherlands; 3Pathology, Erasmus Medical
Centre, Rotterdam, Netherlands; 4The Rotterdam Eye Hospital,
Rotterdam, Netherlands.
Purpose: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. It has a strong tendency to metastasize to
the liver. Monosomy of chromosome 3 is the most frequent found
chromosomal aberration in UM and predominantly found in
metastasizing tumours. Inactivating somatic mutations have been
described in the BRCA-associated protein 1 (BAP1) gene, located on
chromosome 3q21.1, in class II UMs (Harbour et al, 2010). In this
study we determine the prevalence of BAP1 mutations in specific
subgroups of UM patients and examine BAP1 expression in UM
tissue.
Methods: Ciliary body and choroidal melanomas were subjected to
BAP1 mutation analysis using several techniques such as targeted
Next Generation Sequencing, deep sequencing with a custom
designed HaloPlex Target Enrichment kit, and exome sequencing.
Variations were validated by Sanger sequencing. Protein expression
of BAP1 in UM samples was analyzed by immunohistochemical
staining in which expression in the RPE functioned as our internal
positive control. BAP1 mutation status was correlated with diseasefree survival, clinical and histopathological factors and copy number
variations detected by fluorescence in situ hybridization (FISH) and
SNP-array analysis.
Results: Mutations in BAP1 have hitherto been identified in 31.4%
(16/51) of the tumors. More specific, the analyzed set of UM samples
contained 3 missense mutations, 2 nonsense mutations, 5 frameshift
deletions, 2 non-frameshift deletions, and 4 splice site mutations.
Positive BAP1 immunohistochemical staining was observed in 52.9%
(27/45) of UMs and no expression was seen in 35.3% (18/45) of the
cases. Two out of the 15 tumors with BAP1 mutations and 5 out of
30 wild type BAP1 tumors showed no staining. Correlations were
found between BAP1 mutation status and BAP1 expression at tissue
level, and also between BAP1 mutations and monosomy 3 (P=0.000
and P=0.000, respectively). Univariate analyses of BAP1 mutated
cases compared to wild type showed a significantly decrease in
disease-free survival (43.2 versus 94.0 months, respectively,
P=0.002).
Conclusions: Deep sequencing revealed somatic mutations in the
BAP1 gene in nearly a third of the UMs. BAP1 mutations and gene
expression seems to play a role in the UM pathogenesis towards
metastatic disease.
Commercial Relationships: Anna E. Koopmans, None; Robert M.
Verdijk, None; Thierry van den Bosch, None; Mike M. van den
Berg, None; Jolanda Vaarwater, None; Dion Paridaens, None;
Emine Kilic, None; Annelies de Klein, None
Support: SNOO, CORR
Gender differences and estrogen and progesterone receptor
expression in uveal melanoma
Lynn Schoenfield1, Thomas Plesec2, Erinn Downs-Kelly2, Mary Beth
Aronow3, Paula Carver2, Raymond Tubbs2, Arun D. Singh3.
1
Pathology, Ohio State University Wexner Medical Center,
Columbus, OH; 2Pathology, Cleveland Clinic, Cleveland, OH; 3Eye
Institute, Cleveland Clinic, Cleveland, OH.
Purpose: Older reports have not demonstrated estrogen receptor
(ER) expression in uveal melanoma (UVM). However, recent
publications and SEER data prompted a re-examination of gender
differences and hormome receptor status with comparison to clinical
outcome and chromosome 3 status.
Methods: Incidence and outcome data of 57 cases of UVM from
2004-10 were analyzed. The study was approved by the IRB, and this
research adhered to the tenets of the Declaration of Helsinki. Of the
57 cases, 34 were enucleations and were stained with ER/PR by IHC
using a red chromagen. Cases were considered positive if at least 1%
of the tumor nuceli stained and were scored by 3 surgical pathologists
who routinely do either ocular or breast pathology or both.
Chromosome 3 status was determined by FISH and SNP array
analysis.
Results: There were 31 men (54%) and 26 women (46%). 17 patients
were DOD: 13 men (76%) and 4 women (24%); 3 patients were alive
with metastasis: 2 men (67%) and 1 woman (33%). Of the 34 stained
cases, ER was positive in 20 (59%): 8 (40%) men and 12 (60%)
women. ER was negative in 14 cases, 86% of which were from men.
PR was positive in 18 cases (53%), 13 of which also expressed ER.
Comparing IHC results to outcome showed: 10/20 ER positive cases
(50%) were DOD or were alive with metastases. Of the 14 negative
cases, only 3 (21%) were DOD. If male and ER positive, 5/8 (63%)
were DOD or had metastases compared to if male and ER negative,
3/12 (25%) were DOD. If female and ER positive, 5/12 (42%) were
DOD or with metastases and if female and ER negative, 0/2 (0%)
were DOD or with metastases. When compared to monosomy 3
status, 18/20 ER positive cases (90%) showed monosomy 3
compared to 6/14 ER negative cases (43%) with monosomy 3.
Agreement in scoring the IHC's for hormone receptors was excellent
for ER (29/31 cases, 94%) but less for PR (19/31, 61%).
Conclusions: Gender may play a role in the incidence and behavior
of UVM. ER expression was present in 59% of cases, with or without
PR expression, and was only slightly more likely in women (60%)
than men (40%). These findings are different than previous reports,
and male gender and ER positive tumors may suggest a worse
prognosis. These findings raise the possibility of treatment options
with tamoxifen or other SERMs in the management of UVM. We
propose studying more cases along with quantitative evaluation of
receptor positivity using image analysis
Commercial Relationships: Lynn Schoenfield, None; Thomas
Plesec, None; Erinn Downs-Kelly, None; Mary Beth Aronow,
None; Paula Carver, None; Raymond Tubbs, None; Arun D.
Singh, None
Detection of circulating tumor cells in uveal melanoma using the
CellSearch® system
Martina Angi1, Leila Khoja3, 4, Bertil E. Damato2, Paul Lorigan3,
Caroline Dive4, Sarah E. Coupland1, Helen Kalirai1. 1Department of
Molecular & Clinical Cancer Medicine, University of Liverpool,
Liverpool, United Kingdom; 2Royal Liverpool University Hospital,
Liverpool, United Kingdom; 3The Christie NHS Foundation Trust,
Manchester, United Kingdom; 4Paterson Institute for Cancer
Research, Manchester, United Kingdom.
Purpose: Circulating tumor cells (CTC) have been identified
previously in the peripheral blood of uveal melanoma (UM) patients
using either PCR analysis of tumor-associated RNA or
immunomagnetic cell-sorting based on the expression of the
melanoma associated chondroitin sulphate proteoglycan (MCSP)
antigen. Published data, however, vary significantly between groups.
The CellSearch® system (Veridex, New Jersey, USA) is the only
FDA approved technology for the enumeration of CTC in the clinical
setting.
The aim of our study was to apply the CellSearch® system for the
detection of CTC in UM patients at different stages of disease.
Methods: Blood samples from UM patients with and without
metastases were prospectively collected prior to treatment and
processed according to the CellTracks® Circulating Melanoma Cell
(CMC) kit standard protocol. Patients with primary UM were
classified either as high (HR) or low (LR) risk of developing
metastases on the basis of clinical, histopathological and genetic
features of the ocular tumor, and correlations were drawn with CTC
number.
Additional validation of the expression of the CMC enrichment
(CD146) and detection (MSCP) antigens was performed by flow
cytometry using directly conjugated immunofluorescence antibodies
in 5 UM cell lines (Mel270, 92.1, Omm1, Omm2.3, Omm2.5) grown
as adherent and non-adherent cultures.
Results: CTC were detected in 7/13 metastatic (range 1-525), in 5/14
HR (range 1-2) and in 0/6 LR UM patients. The presence of CTC
correlated significantly with monosomy 3 (Chi-square, p<0.01) but
not with age, sex, tumor dimensions, ciliary body involvement,
epithelioid cellularity or mitotic count (all p>0.05).
CD146 was detected in >98% of all cell lines and culture conditions,
although the fluorescence intensity (FI) varied between cell lines
(range18.1-299.1). Expression of MCSP showed a high degree of
heterogeneity within and between cell lines. In general, the FI for
MCSP was low across all cell lines (range 10.5-37.2).
Conclusions: The CellSearch® system can detect CTC in the
peripheral blood of UM patients at different stages of the disease;
however, further studies are necessary to establish its clinical value.
CD146 appears suitable for UM cell enrichment, while MCSP may
not be an optimal antigen for detection. Additional markers, which
can be added to the customizable channel present in the CMC kit, are
being investigated to further characterize CTC in UM.
Commercial Relationships: Martina Angi, None; Leila Khoja,
None; Bertil E. Damato, None; Paul Lorigan, None; Caroline
Dive, None; Sarah E. Coupland, None; Helen Kalirai, None
Quantification of circulating tumour cells in patients with
choroidal melanocytic tumours: correlation with clinical risk
factors
Manuel F. Bande1, Maria Santiago1, Purificacion Mera1, Maria Jose
Blanco1, Laura Muinelo-Romay3, Carmela Capeans1, Maria Pardo2,
Antonio Piñeiro1. 1Ocular Oncology Unit. Servizo de Oftalmoloxía.
Complexo Hospitalario Universitario de Santiago. Universidade de
Santiago de Compostela. Santiago de Compostela. Spain, Santiago de
Compostela, Spain; 2Grupo Obesidómica, Laboratorio de
Endocrinología Molecular y Celular, Hospital Clínico Universitario
de Santiago (CHUS/SERGAS), Spain, Santiago de Compostela,
Spain; 3Unit of circulating tumor cells analysis. Traslational
laboratory/ Medical Oncology Department. Complexo Hospitalario
Universitario de Santiago de Compostela Santiago de Compostela,
Spain., Santiago de Compostela, Spain.
Purpose: Detect and quantify circulating tumour cells (CTCs) in
peripheral blood of patients with choroidal melanocytic tumours and
study the relationship of the CTCs with clinical risk factors.
Methods: Prospective study with three clinical groups: 4 patients
diagnosed with choridal nevus, 8 patients with choroidal melanoma
prior to treatment and 2 enucleated patients with metastases (liver)
detected at 65 and 37 months after treatment. A single sample of 7.5
mL of peripheral blood was taken and the CTCs were isolated using a
Celltrack system that captures positive cells for the CD146 antigen
(MUC18).
Results: No patients with choroidal nevus had CTCs in peripheral
blood. Of the 8 patients with choroidal melanoma prior to treatment,
50% had more than one CTC/7.5 mL. The higher level of CTCs cells
in peripheral blood (3/7.5 mL) was detected in the patient with the
larger choroidal melanoma which also presented extrascleral
extension and epithelioid pathology. Furthermore, we were unable to
detect any CTCs in those patients (n=2) with postenucleation liver
metastases.
Conclusions: Performing an analysis with the Celltrack system
allows to quantify the choroidal melanoma CTCs in peripheral blood.
This finding highlights the potential usefulness of this technique to
achieve the correct stratification and monitoring of the treatment.
Commercial Relationships: Manuel F. Bande, None; Maria
Santiago, None; Purificacion Mera, None; Maria Jose Blanco,
None; Laura Muinelo-Romay, None; Carmela Capeans, None;
Maria Pardo, None; Antonio Piñeiro, None
Support: Instituto de Salud Carlos III (FIS PI11/00972)
2
Department of Ophthalmology, Cleveland Clinic Foundation,
Cleveland, OH; 3Quantitative Health Sciences, Cleveland Clinic
Foundation, Cleveland, OH.
Purpose: Uveal melanoma metastasizes preferentially to liver that
manifests several years after treatment of the primary tumor.
Although, there is lack of consensus regarding surveillance protocols,
periodic liver imaging studies is increasingly utilized. We analyzed
the effectiveness of hepatic ultrasonography in the diagnosis of
asymptomatic hepatic metastases in patients with uveal melanoma.
Methods: We conducted a retrospective chart review of patients
diagnosed and treated for uveal melanoma at the Cole Eye Institute
from the year 2003 to 2011. Inclusion criteria included patients with a
diagnosis of primary uveal melanoma that were assessed using a
standardized protocol; initial staging with contrast enhanced CT scan
of the chest, abdomen and pelvis immediately prior to the treatment
of the primary and subsequent surveillance with hepatic function
panel and liver ultrasound (USG) every six months. Abnormal USG
findings that were not present on baseline CT were categorized as
cyst and hemangioma, indeterminate lesion, suspicious for
metastases, and consistent with metastases. Lesions that were
indeterminate, suspicious or consistent for metastases were further
evaluated by a CT/MRI or PET scan and the diagnosis of metastatic
lesion was confirmed with a tissue biopsy. The rest were serially
imaged by USG.
Results: A total of 265 patients were included in the study. 2 patients
(0.8%) were found to have metastases at initial staging. The liver
USG scan data (1390 scans) was analyzed for 263 patients. The
number of scans per patient ranged from 1 to 17 (mean: 5.3),
performed over a median period of 34 months (range: 0.2-125).
Overall 86 scans of 71 patients [27%] were reported as abnormal
(Table 1). 36 (13.6%) had biopsy confirmed hepatic metastases
(Table 2). None of the patients with indeterminate lesions developed
metastases where as only one patient initially diagnosed with a cyst
was later found to have metastasis. 30 of the 45 patients with
suspicious lesions (66.7%) and 100% of patients with consistent
lesions proved to have a positive biopsy (positive predictive value of
53%).
Conclusions: Periodic ultrasonography of the liver performed as part
of systemic surveillance protocol is able to identify asymptomatic
metastatic lesions. However, because of its low positive predictive
value other imaging techniques need to be evaluated.
Systemic Surveillance for Metastases in patients with Uveal
Melanoma
Maria M. Choudhary1, Akshay Gupta2, James Bena3, Arun D. Singh2.
1
Internal Medicine, Cleveland Clinic Foundation, Cleveland, OH;
Commercial Relationships: Maria M. Choudhary, None; Akshay
Gupta, None; James Bena, None; Arun D. Singh, None
Detecting hepatic metastases from ocular melanoma using MRI
with a novel designed contrast agent
Hua Yang1, Hans E. Grossniklaus1, Qing Zhang1, Shenghui Xue2,
Fan Pu2, Jingjuan Qiao2, Zhi-Ren Liu2, Robert C. Long3, Jenny
Yang2. 1Ophthalmology, Emory University Eye Center, Atlanta, GA;
2
Chemistry, Georgia State University, Atlanta, GA; 3Radiology,
Emory University, Atlanta, GA.
Purpose: Despite successful local tumor control, one half of patients
with ocular melanoma will die from hepatic metastases. The 10-year
mortality rate in patients with undetected metastatic tumors is
approximately 30%. Metastases-related deaths occur as long as 35
years after diagnosis of primary tumor. The mean survival time is
around 12 to 14 months once the hepatic metastasis is diagnosed. The
purpose of this study is to develop a novel contrast agent to detect
early stage hepatic metastases from ocular melanoma.
Methods: The novel protein-based MRI contrast agent (ProCA) was
designed and generated. ProCAs has more than 10 times high
relaxivity than that of clinical MRI contrast agents. ProCAs also
enhance liver with high dose efficiency and specificity. One eye of
PEDF deficient homozygote mice (n=6) was inoculated with mouse
melanoma cell B16LS9. At the 10th day after inoculation, the mice
were injected with the novel contrast agent ProCA or a control
protein through the tail vein, and imaged using a 4.7 T small animal
MRI at 30 minutes post injection of the contrast agent. After imaging,
the mice were sacrificed with their eyes and livers processed for
histology and IHC staining for S100A and Ki67.
Results: Hepatic metastases could be detected with the novel proteinbased contrast agent ProCA, but not with the control protein or
without a contrast agent using MRI. Histology confirmed that the
sizes of hepatic metastases which had been detected with MRI were
less than 1 mm diameter. IHC identified that the melanoma marker
S100A was expressed in the hepatic metastases. Hepatic metastases
showed hypo-intensity with the T1 weighted spin echo sequence in
vivo. The hypo-intense tumors also had been verified the lack of
ProCA in tumors and strong fluorescence of fluorescently labeled
ProCA in hepatic tissues using the Leicamz16FA microsystem in
vitro.
Conclusions: The novel contrast agent, ProCA, is able to detect the
<1 mm diameter metastatic ocular melanomas in the mouse liver with
MRI. This provides the possibility and a noninvasive method to
detect small, early stage hepatic metastases.
Pu, None; Jingjuan Qiao, None; Zhi-Ren Liu, None; Robert C.
Long, None; Jenny Yang, None
Support: NIH EB007268
In vivo contrast-enhanced high-frequency ultrasonography of
experimental uveal melanoma: imaging features and
histopathologic correlations
Hans E. Grossniklaus, Shin J. Kang, Qing Zhang. Dept of Ophthal,
School of Med, Emory University, Atlanta, GA.
Purpose: To evaluate the utility of in vivo imaging of a rabbit model
of uveal melanoma utilizing high-frequency contrast-enhanced
ultrasound (HF-CE-US) with 2- or 3- dimensional modes, and to
correlate the sonographic findings with histopathologic
characteristics.
Methods: Five New Zealand white rabbits were inoculated into their
right eyes with aliquots of 92.1 human uveal melanoma cells. The
rabbits were immune suppressed with subcutaneous cyclosporine
15mg/kg qd from three days prior to inoculation to four weeks after
inoculation. Tumor-bearing eyes were imaged using HF-CE-US to
determine the 2- and 3-dimensional tumor size and relative blood
volume. The rabbits were sacrificed and their eyes with melanoma
were histologically examined to determine melanoma size and mean
vascular density (MVD).
Results: Melanoma grew in the choroid in all eyes. Utilizing HF-CEUS, melanomas were visualized as relatively hyperechoic regions in
the images. The correlation coefficient of sonographic size or volume
compared with histologic area were r square =0.72 and 0.70,
respectively. The sonographic tumor relative blood volume correlated
with histologic tumor vascularity (MVD) (r=0.92).
Conclusions: There is a positive correlation between in vivo
sonographic tumor volume/size and histologic tumor size in our
rabbit ocular melanoma model. HF-CE-US corresponds to
microvascular density and blood volume. HF-CE-US is a real-time,
non-invasive method for evaluation of intraocular melanoma tumor
area and relative blood volume.
Rabbit model of uveal melanoma demonstrating fundus view of
melanoma, histology of melanoma, ultrasound and HF-CE-US
Commercial Relationships: Hua Yang, None; Hans E.
Grossniklaus, None; Qing Zhang, None; Shenghui Xue, None; Fan
Correlation between HF-CE-US tumor area and histologic tumor size
(r squared=0.70)
Commercial Relationships: Hans E. Grossniklaus, None; Shin J.
Kang, None; Qing Zhang, None
Support: NIH P30EY06360 Research to Prevent Blindness Inc.
Enhanced Depth Imaging Optical Coherence Tomography of
Choroidal Melanoma Before and After Plaque Radiation
Therapy
Denise S. Kim, Hakan Demirci. Ophthalmology & Visual Sciences,
University of Michigan Kellogg Eye Center, Ann Arbor, MI.
Purpose: To evaluate characteristics of choroidal melanoma using
enhanced depth imaging optical coherence tomography (OCT-EDI)
before and after treatment with plaque radiation therapy.
Methods: A retrospective case series was designed to evaluate OCTEDI characteristics of choroidal melanoma before and after treatment
with plaque radiation therapy. All patients who underwent plaque
radiation therapy at the Kellogg Eye Center for choroidal melanoma
during a 2-year period between October 1, 2010, and October 1,
2012, and who were imaged with OCT-EDI in the pre-operative and
post-operative period were evaluated. Kellogg Eye Center Review
Board approval was obtained.
Evaluated OCT-EDI features included optical qualities of the
melanoma and retinal layer abnormalities. The presence of subretinal
fluid, subretinal lipofuscin deposition, and intraretinal edema was
noted. The tumor thickness was measured by ultrasonography
(millimeters) and OCT-EDI (millimeters) when possible. In OCTEDI, the tumor thickness was measured in the scan showing greatest
tumor thickness by placing the calipers between the base of retina
pigment epithelium and the tumor base judged to be at the junction of
hyperreflective inner sclera.
Results: A total of 31 choroidal melanomas underwent OCT-EDI
imaging before and after radioactive plaque therapy. Mean age at
surgery was 63.1 years (range 33-83 years). Initial post-operative
OCT-EDI imaging was obtained at a mean of 141 days (range 79-289
days). Concurrent resolution of subretinal fluid and shaggy
photoreceptors was seen in 5 (16.1%) of patients, and resolution of
intraretinal edema was seen in 4 (12.9%) of patients, in first postoperative OCT-EDI. Average length of follow-up was 260 days
(range 86-597 days). Average time for resolution of subretinal fluid
and shaggy photoreceptors was 221 days. Average time for resolution
of intraretinal fluid was 176 days.
Conclusions: Following plaque radiotherapy, OCT-EDI allows a
more precise assessment of the intraretinal, subretinal, and choroidal
layers. OCT-EDI can be useful in assessing the post-operative
response to radioactive plaque therapy of choroidal melanoma.
OCT-EDI of choroidal melanoma with subretinal fluid and shaggy
photoreceptors
OCT-EDI of same tumor 100 days after plaque radiation therapy,
with resolution of subretinal fluid and shaggy photoreceptors
Commercial Relationships: Denise S. Kim, None; Hakan Demirci,
None
Enhanced Depth Imaging Optical Coherence Tomography of
Tumors of the Retina and Choroid: Comparison to B-scan
ultrasonography
Euiyong Kweon1, 2, Jaysun G. Frisch3, Peter G. Hovland1. 1Colorado
Retinal Associates, Denver, CO; 2Department of Ophthalmology,
Chonbuk National University Hospital, Jeonju, Republic of Korea;
3
Roocky Vista University Medical School, Denver, CO.
Purpose: To evaluate the characteristics of choroidal nevus,
choroidal melanoma, metastatic lesions, and congenital hypertrophy
of the retinal pigment epithelium (CHRPE) using the enhanced depth
imaging spectral-domain optical coherence tomography (EDI-OCT),
with a comparison to the B-scan ultrasound (BUS) findings.
Methods: A retrospective observational study design was used to
evaluate characteristics of retinal and choroidal tumors using
spectral-domain OCT. 40 sequential patients who presented to an
ocular oncology practice were selected based on a posterior location
of the lesions in question; this allowed for EDI-OCT imaging and
BUS. EDI-OCT was performed with the Heidelberg Spectralis
HRA+OCT (Heidelberg Engineering, Heidelberg, Germany) using a
custom scan image acquisition protocol of up to 13 raster lines of 9
mm image length. Ultrasound was performed with an I-cubed
instrument in most cases.
Results: EDI-OCT appears to yield greater anatomic detail and better
thickness measurements in tumors of less than 2 mm thickness when
compared to BUS. EDI-OCT is not able to create full-thickness
imaging in lesions greater than 2 mm; yet in these lesions the
technique has utility in that it can identify the location of the lesion
(retina or choroid), as well as associated characteristic changes of the
surrounding tissues (subretinal fluid, retinal edema, photoreceptor
changes). EDI-OCT can detect tumor growth under subretinal fluid;
as the components of fluid v. tumor are better distinguished than by
BUS.
Conclusions: Imaging of choroidal and retinal tumors with EDI-OCT
show superior measurement of certain tumor characteristics
compared with ultrasonography. The clinical utility of this modality
is emerging; this presentation demonstrates the usefulness of EDIOCT in distinguishing suspicious nevi from small melanoma and
CHRPE, detecting tumor re-growth along the treatment margin, and
demonstrating retinal vs. choroid tumor location.
Commercial Relationships: Euiyong Kweon, None; Jaysun G.
Frisch, None; Peter G. Hovland, None
EDI-OCT Findings in Unilateral Choroidal Melanocytosis with
Macular Involvement
Sruthi Arepalli, Marco Pellegrini, Carol L. Shields. Ocular
Oncology, Wills Eye Institute, Philadelphia, PA.
Purpose: To assess retinal and choroidal features in eyes with
unilateral choroidal melanocytosis using enhanced depth imaging
optical coherence tomography (EDI-OCT).
Methods: Retrospective case series with review of chart, fundus
photography and EDI-OCT images. Included patients had unilateral
choroidal melanocytosis with macular involvement (sectoral or
complete) evaluated by EDI-OCT (Spectralis HRA + OCT,
Heidelberg Engineering, Heidelberg, Germany). For each patient
retinal and choroidal layers were assessed. Mean subfoveal choroidal
thickness was manually determined independently by 2 physicians
(SA and MP) using the caliper function on a cross-line scan passing
through the foveola (averaging equal 100 frames) from inner
choroidal limit to sclero-choroidal junction. Choroidal thickness of
study fovea was then compared with the unaffected opposite (control)
fovea.
Results: Of 13 patients enrolled in the study (9 males,4 females), the
mean age was 26 years (range 5-76 years). The choroidal
melanocytosis was diffuse in 4 and sectoral in 9. Associated features
included dermal (4), scleral (9) iris (1) and palate (2) melanocytosis.
Spectral domain EDI-OCT showed normal inner retina (n=13) and
normal outer retina (n=11). The affected choroid showed smooth
surface contour without nodularity (n=13), dense shadowing (n=13),
and compression of vascular tissue inward (n=12). The mean
choroidal thickness was 317.2 in the study eye and 263.1 in the
control eye. (p=0,23). The subfoveal choroidal thickness was 21%
greater in the eye with melanocytosis compared with control.
Conclusions: EDI-OCT revealed eyes with choroidal melanocytosis
show 21% thicker subfoveal region compared to opposite unaffected
eye. Overlying retinal features were generally preserved.
Commercial Relationships: Sruthi Arepalli, None; Marco
Pellegrini, None; Carol L. Shields, None
Macular Choroidal Thickness in Uveal Melanoma Patients
treated with Plaque Radiotherapy
Ira H. Schachar, Hakan Demirci. Ophthalmology, University of
Michigan, Ann Arbor, MI.
Purpose: To evaluate the macular choroidal thickness in uveal
melanoma patients following plaque radiotherapy
Methods: Macula of both eyes of eleven uveal melanoma patients
who were treated with plaque radiotherapy were scanned by using
enhanced depth imaging feature of Heidelberg Spectralis HRA+OCT
(Heidelberg Engineering, Heidelberg, Germany). A cross-section of
the retina and choroid was identified for each patient. The choroid
was identified as the area between Bruch’s membrane and the
anterior scleral border, which were manually identified using ImageJ
(v.145s, NIH). A choroidal area was measured for a 3000 micronwide section of the choroid and centered on the fovea for both eyes of
every patient. For each patient, choroidal areas were compared
between their plaque-treated and untreated control eye. All patients
underwent complete eye examination to assess radiation retinopathy
and maculopathy without knowing the result of OCT-EDI.
Results: The mean time interval following plaque radiotherapy was
50 months (range: 12mo-121mo). Seven of 11 patients exhibited
clinical findings of radiation maculopathy and retinopathy in their
plaque-treated eye. In all patients, the average difference in macular
choroidal area between plaque-treated and untreated eyes was
35000μm2 vs 41000μm2, respectively (p = 0.06). A priori subgroup
analysis of 7 patients with radiation retinopathy showed a similar
trend towards decreased choroidal thickness in plaque-treated eyes
(38000μm2 vs 33000μm2, p=0.17). The decrease choroidal area eyes
corresponds to, on average, a 15% reduction in choroidal thickness in
plaque-treated eyes regardless of whether clinical radiation
retinopathy was present.
Conclusions: A trend towards decreased choroidal thickness in eyes
with a history of plaque-radiotherapy was observed. Further
investigations are warranted into the effect of plaque-radiotherapy on
choroidal structure and function.
Commercial Relationships: Ira H. Schachar, None; Hakan
Demirci, None
Radiation Therapy for Small Choroidal Melanoma: 10-years
Experience with Palladium-103 Plaque Radiation Therapy
Kimberly J. Chin, Ekaterina Semenova, Paul T. Finger. The New
York Eye Cancer Center, New York, NY.
Purpose: To evaluate outcomes (vision, local control, complications)
after ophthalmic plaque radiation therapy for small choroidal
melanomas.
Methods: Ninety one patients with small choroidal melanomas (<2.5
mm apical height and <10 mm wide) were treated with palladium103 plaque brachytherapy between 2002-2012. Pre-operative tumor
thickness ranged from 1.5 to 2.5 mm (mean 2.2 mm, CI 2.1-2.3 mm).
Tumor base diameters ranged from 5.5 to 10.0 mm (mean 8.4 mm, CI
8.2-8.7). Thirty one patients (34.1%) in this study were observed for
change from 2 months to 16 years prior to radiation treatment.
Results: Plaque radiotherapy provided local tumor control in 98.9%
at a mean 55 months of observation (95% CI 48-62 months). One
patient failed treatment. Mean tumor apex dose was 82.3Gy (range
70.0-102 Gy). The most common long-term brachytherapy-related
complications were radiation retinopathy (47.3%) and radiation optic
neuropathy (19.8%) developing 9 to 72 months after brachytherapy
(mean 26 months). These complications were typically stabilized by
periodic anti-VEGF intravitreal injections. In this series 85 patients
(93.4%) maintained 20/200 or better vision. Metastasis developed in
one patient (1.1%).
Conclusions: These findings support palladium-103 plaque
radiotherapy as an effective method to treat small choroidal
melanomas. While informed consent plays a critical role in small
melanoma case selection, treated patients should expect excellent
local control rates and better visual acuity outcomes compared to
treatment of larger choroidal melanomas.
Commercial Relationships: Kimberly J. Chin, None; Ekaterina
Semenova, The Eye Cancer Foundation (F); Paul T. Finger, None
Support: Supported by The Eye Cancer Foundation, Inc.
http://eyecancerfoundation.net
Plaque Radiation Therapy for Large and Extra-large Choroidal
Melanoma
Ekaterina Semenova1, 2, Paul T. Finger1, 2. 1Ocular Oncology, The
New York Eye Cancer Center, New York, NY; 2Ocular Oncology,
The New York Eye and Ear Infirmary, New York, NY.
Purpose: To evaluate outcomes (vision, local control, complications)
after ophthalmic plaque radiation therapy for large uveal melanomas.
Methods: We evaluated the results of ophthalmic plaque
brachytherapy of 48 patients with American Joint Committee on
Cancer (AJCC) T3 and T4 uveal melanomas treated within the period
of 2002-2012 (with a minimum follow up of 6 months). Pretreatment
comparative dosimetry studies resulted in the use of palladium-103
(n=47) or iodine-125 seed (n=1) sources. The mean dose to the tumor
apex was 69 Gy (range 49-87Gy).
Results: Plaque radiotherapy provided local tumor control in 91.7%
and eye retention in 89.6% of patients at a mean 4 years observation
(range 6 to 125 months). The most common long-term
brachytherapy-related complications were radiation retinopathy
(66.7%) and radiation optic neuropathy (47.9%) developing 5 to 36
months after brachytherapy (mean 19 months). These complications
were typically stabilized by periodic bevacizumab or ranibizumab
intravitreal injections. Secondary cataract developed in 36.4% phakic
eyes within 2-45 months after irradiation (mean 26 months). Preexisting and persistent exudative retinal detachments were observed
in 29.2% and were the major cause of vision loss. Iris
neovascularization developed in 18.8% of the patients and secondary
glaucoma in 16.6% requiring enucleation in 3 patients (6.3%). In this
series 54.2% patients maintained 20/200 or better vision. Metastasis
developed in 31.3% of patients.
Conclusions: In this series, palladium-103 plaque radiotherapy
offered superior local control, visual acuity and eye retention rates as
compared to those reported in the literature.
Commercial Relationships: Ekaterina Semenova, The Eye Cancer
Foundation (F); Paul T. Finger, None
Support: The Eye Cancer Foundation, Inc
(http://eyecancerfoundation.net)
Proton beam radiation for the treatment of large choroidal
melanoma
Shideh Schoenfeld. charite Berlin, ophthalmology, Berlin, Germany.
Purpose: To evaluate long term outcomes of proton beam radiation
therapy in the treatment of large semiperipheral choroideal
melanoma.
Methods: A retrospective, nonrandomized long term follow-up of 62
patients with a tumor to disc and tumor to macular distance of more
than 2 mm without previous treatment. Average tumor thickness was
7.6 mm, base 12.8 mm, fovea tumor distance 4.6 mm, disc tumor
distance 5.2 mm.
Results: The mean follow-up including eye examination was 64.4
months with a mean acquisition time of a minimum of information as
to the patients’ overall survival of 67.0 months. The 5-year KaplanMeier estimate of average survival rate was 96.1%. Rate of
enucleation was 1.7% and metastasis 13.4%.
71% of the PBRT group received endoresection of the tumor. Only
18 patients did not require additional surgery and were considered as
a separate group. The most common adverse treatment effects were
radiation retinopathy in both groups and radiation papillopathy in the
endoresection group. Vitrectomy and cataract surgery were the most
frequent secondary treatments.
Conclusions: In large size consecutively treated tumors we
demonstrate the effectiveness of proton beam irradiation to control
tumor growth and preservation of the globe. This study demonstrates
no higher rate of metastasis in larger tumors after treatment of proton
therapy and an excellent local tumor control.
Commercial Relationships: Shideh Schoenfeld, None
Outcomes of Choroidal Melanomas Treated with Eye Physics®
plaques: a 20-year Review
Jesse L. Berry1, Savita V. Dandapani2, Marta Stevanovic3, Melvin
Astrahan2, A. L. Murphree4, Jonathan W. Kim1, 4. 1Doheny Eye
Institute and Department of Ophthalmology, Keck School of
Medicine of the University of Southern California, Los Angeles, CA;
2
Department of Radiation Oncology, Keck School of Medicine of the
University of Southern California, Los Angeles, CA; 3Harvard
College, Cambridge, MA; 4Ophthalmology, The Vision Center at
Children’s Hospital Los Angeles, Los Angeles, CA.
Purpose: To review the University of Southern California
(USC)/Doheny Eye Institute experience using Eye Physics® (EP)
plaques and Plaque Simulator™ (PS) software to treat medium-sized
choroidal melanomas and to compare the outcomes with results from
the Collaborative Ocular Melanoma Study (COMS).
Methods: A retrospective case series of 82 patients treated for
choroidal melanoma from 1990 to 2010 using plaques and treatment
simulation software developed at USC. The dosimetric goal was to
deliver 85 Gy to a conformal volume enclosing the tumor apex and a
2 mm margin surrounding the base. Plaque localization was guided
by PS computer modeling system using preoperative imaging studies.
Primary outcome measures were local tumor control, globe
preservation, and metastases. Secondary outcome measures were late
radiation side effects, including vision changes, optic neuropathy,
radiation retinopathy, and cataract.
Results: Median follow-up for 82 patients was 46.8 months (range,
1-171 months). Globe preservation was achieved in 80 (97.5%)
patients; two patients underwent enucleation for local recurrence.
Metastatic disease developed in 9 (10.9%) patients during the followup period. Retinopathy was seen in 31 (37.8%), optic neuropathy in
12 (14.6%) and cataracts in 26 (31.7%) patients. Postoperatively, 21
(25.6%) patients lost >6 lines of Snellen visual acuity.
Conclusions: When considering rates of local recurrence, metastases,
and late radiation side effects, the results of treating medium-sized
choroidal melanomas with EP plaques compare favorably with the
COMS data. The PS three-dimensional tumor modeling program
developed at USC is a reliable method for determining plaque
positioning preoperatively and for treating this cohort of patients.
Commercial Relationships: Jesse L. Berry, None; Savita V.
Dandapani, None; Marta Stevanovic, None; Melvin Astrahan, Eye
Physics, LLC (I); A. L. Murphree, Clarity Medical Systems (P),
3TOphthalmics, inc. (P), iCheck Health Connections (P); Jonathan
W. Kim, None
103
Pd versus 125I Plaque Radiation Dose to Normal Ocular
Structures in the Treatment of 319 Uveal Melanomas
Di Zhou1, 4, Ekaterina Semenova1, 3, Ping Wong2, Nina Kalach2,
Walter Choi2, 3, Paul T. Finger1, 4. 1The New York Eye Cancer
Center, New York, NY; 2Beth Israel Comprehensive Cancer Center,
New York, NY; 3The New York Eye and Ear Infirmary, New York,
NY; 4New York University School of Medicine, New York, NY.
Purpose: To calculate and compare the pre-operative radiation doses
to normal intraocular structures in a large series of patients scheduled
for plaque brachytherapy using Pallidum-103 (103Pd) versus Iodine125 (125I).
Methods: We performed a retrospective chart review of 319 patients
treated for uveal melanoma in 2006-2012. IRB approval was obtained
and the study followed the guidelines of the Declaration of Helsinki.
The radiation dose to tumor and normal ocular structures (outside the
targeted zone) were calculated prior to surgery for both 103Pd and 125I
Specific intraocular locations included: a 5mm depth, opposite retina,
macula, optic disc, lens and the tumor apex (prescription point). Dose
measurements were then related to tumor characteristics such as
location (anterior versus posterior location), size, and T-stage
according to the American Joint Committee on Cancer (AJCC)
System.
Results: Three hundred nineteen patients were diagnosed with T1-T4
uveal melanomas ranging in maximum (apical) height from 1.8 to
14.2 mm (mean 4.0 mm,CI 2.4-2.8). The range of the maximum basal
diameter is 1.8 to 21 mm (mean 9.9 mm, CI 9.5-10.3). 21.6% of the
tumors are in the anterior location and 78.4% of the tumors are in the
posterior location. Table I shows the comparative dosimetry of 103Pd
versus that of 125I to intraocular locations for all tumors. By design,
the dose to the prescription point (tumor apex) for 103Pd and 125I
plaques was equivalent. However, 103Pd shows a reduced mean doses
to all the normal intraocular structures outside the targeted zone when
compared to that of 125I in all tumors (p < 0.05).
Conclusions: Radioactive 103Pd seeds emit lower energy photons (21
KeV) versus 125I (28 KeV). This shift in photon energy leads to shift
in radiation exposure within the eye. That is, for an equivalent tumor
apex dose the less energetic photons are more quickly absorbed
within the melanoma and continue to be more quickly absorbed
before they reach most normal intraocular structures. Thus, in a large
cohort of patients with uveal melanoma, this study demonstrates that
103
Pd seeds in standard gold eye plaques offer a method to deliver
relatively more radiation within the tumor (mean base to apex) and
less dose to most essential intraocular structures.
Table I: Pre-operative Comparative Dosimetry for All Tumors
Commercial Relationships: Di Zhou, None; Ekaterina Semenova,
The Eye Cancer Foundation (F); Ping Wong, None; Nina Kalach,
None; Walter Choi, None; Paul T. Finger, None
Support: The Eye Cancer Foundation, Inc.
High Dose Rate Interstitial Radiation Therapy for Orbital
Melanoma
Paul T. Finger1, 3, Lawrence B. Tena2, Paul Aridgides2, Ekaterina
Semenova1, 3, Walter Choi2, 3. 1Ophthalmic Oncology, The New York
Eye Cancer Center, New York, NY; 2Radiation Oncology, Beth Israel
Comprehensive Cancer Center, New York, NY; 3Ophthalmology,
The New York Eye and Ear Infirmary, New York, NY.
Purpose: To report out experience with a novel radiation
brachytherapy technique used in treatment of orbital melanoma
secondary to extrascleral extension of uveal melanoma. Further, to
compare the dose deposition versus external beam radiation therapy.
Methods: A clinical case series of patients received high dose rate
iridium-192 interstitial orbital radiation therapy. Tumors were staged
according to the American Joint Committee on Cancer system as
T1c, T2c, T2d, T3d, (three) T4c, (two) T4d and one R2. Typically 810 orbital catheters were surgically placed into the orbit. Then a
target dose of 32.85 Gy was delivered in 9-10 twice daily fractions
over 5 consecutive days.
Results: Ten patients were entered into the study. Nine successfully
underwent high dose rate therapy. With a mean follow up 18 months
(range 1 to 62) there has been no orbital recurrence. Cosmetic results
were invariably better than our experience with 6MV photon external
beam radiation therapy with almost no eyelash or eyebrow loss nor
retracted sockets. All were able to maintain ocular prostheses.
Conclusions: High dose rate, interstitialiridium-192 brachytherapy
can be considered as an alternative to external beam radiation
treatment for post-enucleation extrascleral extension. This series
reports complete local control, few side effects and excellent
cosmetic results.
Table: Patient, Treatment and Outcome Data
Commercial Relationships: Paul T. Finger, None; Lawrence B.
Tena, None; Paul Aridgides, None; Ekaterina Semenova, The Eye
Cancer Foundation (F); Walter Choi, None
Support: The Eye Cancer Foundation, Inc.
Intravitreal Bevacizumab Therapy In the Management of
Radiation Maculopathy
Andrew W. Stacey, Hakan Demirci. Ophthalmology, University of
Michigan - Kellogg Eye Center, Ann Arbor, MI.
Purpose: Radiation maculopathy is a common, vision threatening
complication of plaque radiotherapy in the management of uveal
melanoma. It is reported that intravitreal bevacizumab (IVB)
injections might be effective in the management of radiation
maculopathy. However, the treatment regimen and long term results
are not known. In this study, we evaluated long-term effects of IVB
in the management of radiation maculopathy.
Methods: A retrospective review of patients who received IVB for
radiation maculopathy from October 2010 through October 2012 was
performed. The patients received monthly injections for 3 months,
followed by injection intervals adjusted to every 2 or 3 months
depending on patient response. Patients were evaluated by visual
acuity (VA), status of radiation maculopathy, and macular thickness
on optical coherence tomography (OCT). Student's t-tests were used
to compare observations.
Results: A total of 17 patients were included into the study. An mean
of 4.25 injections were given per patient over a mean of 195 days. All
patients experienced vision loss documented on Snellen chart prior to
therapy. The mean VA on the day of first injection was 0.78
logMAR. Following injection, vision loss was stabilized with no
significant change in VA on first follow up (VA=0.77, 41 days after
1st injection), second follow up (VA=0.80, 86 days), or third follow
up (VA=0.78, 186 days) (all p-values < 0.05).
When injections were spaced longer than two months apart, VA was
negatively affected. Patients who received repeat injections within
eight weeks of the previous injection (n=44) demonstrated a mild
improvement in visual acuity when compared to vision at time of the
last injection (-0.02 change in logMAR) while patients who received
the repeat injections longer than eight weeks (n=40) had significantly
more vision loss (+0.12 logMAR, p=0.01).
The mean central macular thickness obtained from OCT at initial
IVB treatment was 377 μm. Follow up OCTs were obtained an
average of 71 days after first IVB injection and showed a
significantly decreased mean macular thickness of 310 μm (p=0.048).
Conclusions: IVB seems to be effective in the management of
radiation maculopathy by preventing further visual loss. Eight-week
intervals between injections seems to give the best visual acuity
result. Identifying factors that lead to treatment success will be
essential for future therapy.
Commercial Relationships: Andrew W. Stacey, None; Hakan
Demirci, None
Autocrine impact of VEGF ligands in uveal melanoma cells
Konrad R. Koch1, Deniz Hos1, Birgit Regenfuss1, Felix Bock1,
Jacobus J. Bosch2, Claus Cursiefen1, Ludwig M. Heindl1. 1Center of
Ophthalmology, University of Cologne, Cologne, Germany;
2
Department of Internal Medicine 5, Hematology and Oncology,
University of Erlangen-Nuremberg, Erlangen, Germany.
Purpose: Members of the VEGF growth factor family have been
shown to promote melanoma-associated angiogenesis crucial for
tumor growth as well as for hematogenous and lymphatic metastatic
spread. Additional to vascular processes, autocrine effects (migration,
survival, proliferation) of VEGF molecules on several malignoma
cells (e.g., ovarian carcinoma, pancreatic carcinoma, dermal
melanoma) have been described. The purpose of this study was to
investigate in vitro (I) whether uveal melanoma cells express
different VEGF molecules, and (II) whether melanoma cell
proliferation is influenced by VEGF or the anti-VEGF-antibody
bevacizumab.
Methods: Human primary uveal melanoma cell lines (OCM-1,
OCM-3, MEL-270) were cultured, RNA was isolated (RNeasy Mikro
Kit, Quiagen), and cDNA was synthesized (SuperScript 3,
Invitrogen). The cDNA was used to measure VEGF-A/-B/-C mRNA
expression by quantitative real time RT-PCR.
For the proliferation assays, melanoma cells seeded on a 96-wellplate (2000 cells/well) were incubated for 24 hours in medium
containing VEGF-A (100ng/ml), or bevacizumab (250μg/ml). BrdU
was added for 6 hours. Colorimetric analysis was performed after
fixation and staining.
Results: All melanoma cell lines expressed VEGF-A, -C and -D,
with VEGF-A showing the most prominent expression.
Melanoma cells incubated with VEGF-A showed increased
proliferation in comparison with VEGF-free-medium (p<0.05).
Incubation with bevacizumab resulted in decreased tumor cell
proliferation compared with VEGF- and bevacizumab-free medium
(p<0.05).
Conclusions: VEGF ligands, expressed by ocular melanoma cells
may serve as autocrine growth factors beyond their well-known
proangiogenic attributes. Accordingly, potential adjuvant therapeutic
strategies with anti-VEGF-drugs such as bevacizumab seem to be
particularly useful by addressing malignant melanoma progression in
a twofold approach.
Commercial Relationships: Konrad R. Koch, None; Deniz Hos,
None; Birgit Regenfuss, None; Felix Bock, None; Jacobus J.
Bosch, None; Claus Cursiefen, Gene Signal (C), Alcon (R),
Allergan (R), Bayer (R); Ludwig M. Heindl, None
Symptomatic Macular Melanocytic Lesions Treated with
Intravitreal Bevacizumab
Jella A. An1, Christine Corriveau2, Mikael Sebag1, 2, Sonia A.
Callejo1, 2. 1Ophthalmology, McGill University, Montreal, QC,
Canada; 2Ophthalmology, Centre Hospitalier de l'Université de
Montréal, Montreal, QC, Canada.
Purpose: To review 9 cases of symptomatic macular melanocytic
lesions with or without associated choroidal neovascularization
(CNV) managed with intravitreal bevacizumab.
Methods: All patients were examined by an ophthalmologist and
imaged with serial color fundus photography, optical coherence
tomography (OCT), fluorescein angiography, and B-scan
ultrasonography before and after treatment with intravitreal
bevacizumab (1.25mg/0.05cc) on a PRN basis. Treatment outcomes
were retrospectively analyzed.
Results: Nine patients with chronic macular choroidal melanocytic
lesions presented with acute decrease in vision due to tumorassociated intraretinal and subretinal fluid (SRF), pigment epithelial
detachment (PED) and/or subretinal heme (SRH). Tumor location
was subfoveal in 5 patients and within 1 disc-diameter of the fovea
with extension of fluid into the macula in 4 patients. Two patients
had no angiographic evidence of CNV associated with tumor. Initial
best-corrected visual acuity (VA) was 20/20 to 20/40 in 3, 20/50 to
20/150 in 5, and 20/200 or worse in 1 case. All patients were
followed for minimum of 1 year (range 1-12, mean 3.5 years). After a
mean of total 12 injections (range 2-25), final VA improved by mean
of 4 lines (range 1-8) in 6 patients and remained unchanged in 3
patients. All 9 cases responded with decreased fluctuation in VA and
stabilization of retinal thickness, and OCT revealed complete or
partial resolution of SRF, PED and SRH. Among those whose VA
improved, 4 patients remained stable over mean 18 months (range 436) after mean 9.5 total injections (range 2-25). One patient who
failed to respond to a series of 3 injections showed evidence of tumor
growth 6 months after the first injection. The patient was treated with
radioactive plaque, which resulted in tumor regression, resolution of
macular SRF and improvement in VA. The remaining 8 tumors were
stable, and there was no other adverse effect from bevacizumab
injections.
Conclusions: Intravitreal bevacizumab may be an effective
management option for symptomatic macular melanocytic lesions
with or without angiographic evidence of CNV. Decrease in large
fluctuation of VA and stabilization of retinal thickness was a
consistent treatment outcome. Further study will be required to
determine a need for an adjuvant consolidation therapy, and a close
follow-up and vigilance for tumor growth during and after the
therapy will be warranted.
Commercial Relationships: Jella A. An, None; Christine
Corriveau, None; Mikael Sebag, Alcon (R); Sonia A. Callejo,
None
Minimally invasive therapy of exudative retinal detachment
(ERD) due to choroidal or ciliary body melanoma after
irradiation therapy compared to endoresection
Ira Seibel1, Dino Cordini2, Gregor Willerding-Beaucamp1, Jens
Heufelder2, Antonia M. Joussen1. 1Department of Ophthalmology,
Charité University Medicine, Berlin, Germany; 2Proton beam
therapy, Helmholtz-Zentrum Berlin für Materialien und Energie Lise-Meitner-Campus, Berlin, Germany.
Purpose: Choroidal melanoma is often accompanied by an ERD.
This retrospective study investigates if minimally invasive surgery
consisting of vitrectomy with endodrainage,-photocoagulation and
silicone oil filling leads to permanent reattachment and if there are
differences in visual outcome, tumor control or retinal attachment
compared to endoresection.
Methods: 28 patients who presented with a persistent ERD due to
choroidal (n=23) or ciliary body (n=5) melanoma between 2003 and
2011 were included. After irradiation vitrectomy was performed. As
comparison group we identified 183 patients (174 with choroidal
melanomas and 9 with ciliary body melanomas) with a persistent
ERD who delivered secondary endoresection between 2003 and
2011.
Results: Endodrainage- group: Mean follow-up was 27.3 months
(4.2-95.1). At follow-up retina was attached in 25 patients (90%).
Mean visual acuity (VA) before irradiation was 0.5 logMar (20/60
sn) and 0.8 (20/120) at time of follow-up. Initial tumor thickness was
6.49 mm (1.91-10.2; ± 2.17) and was reduced to 3.45 (1.18-5.66; ±
1.28) at follow-up. No patient showed local recurrence. Metastasis
appeared in 2 patients.
Endoresection-group: Mean follow-up was 45.4 months (2.8 -123.5).
At follow-up retina was attached in 171 (93%) patients. Initial VA
was 0.35 logMar (20/45), VA in follow-up was 1.0 (20/200). Local
recurrence was found in 5 (2.7%) patients. Metastasis occurred in 38
patients.
Retinal reattachment was equally successful in both groups. Both had
a significant visual impairment and a decrease of tumor thickness in
follow-up. Patients with endoresection showed more frequent
radiation retinopathy, radiation optical neuropathy and cataract than
patients of the other group. These complications mentioned above
depended on time of follow-up. Due to differing time of follow-up
these results must be considered critically. Related to local or
systemic tumor control there was no significant difference between
both groups.
Conclusions: The minimally invasive method for ERD is a
successful treatment with an equal outcome compared to
endoresection. We recommend this treatment modality in patients
with expected systemic and ocular complications (large LTD,
cardiovascular disease, hypertension, diabetes, anticoagulation
therapy). Sufficient local anti-inflammatory therapy is relevant during
follow-up.
Commercial Relationships: Ira Seibel, None; Dino Cordini, None;
Gregor Willerding-Beaucamp, None; Jens Heufelder, None;
Antonia M. Joussen, None
Detection of Extrascleral Extension in Uveal Melanoma
Christopher Burris1, Vasilios Papastefanou2, 3, Caroline Thaung4,
Mandeep S. Sagoo2, 3, Victoria Cohen2, 3. 1Ophthalmology, Howard
University College of Medicine, Washington, DC; 2Ocular Oncology,
St. Bartholomew's Hospital, London, United Kingdom; 3Ocular
Oncology, Moorfields Eye Hospital, London, United Kingdom; 4Eye
Pathology, UCL Institute of Ophthalmology, London, United
Kingdom.
Purpose: Uveal melanoma is the most common primary intraocular
malignancy. Extrascleral extension (ESE) by this tumor is rare, but
has been associated with an increased rate of orbital recurrence, as
well as an overall poor prognosis. Studies looking at clinical
detection show low rates when compared with those focusing on
histological detection. Due to the prognostic importance of ESE, we
seek to compare our clinical, intraoperative, and histological
detection rates.
Methods: A retrospective cross-sectional case series of eyes
enucleated for uveal melanoma was compiled from the admissions
records of the London Ocular Oncology Service during the 28-month
period between January 2010 and April 2012. Histopathology reports
were searched for ESE. If a case was found to have ESE, the surgical,
and clinical notes were obtained to determine when it was first
diagnosed or suspected (clinically, intraoperatively, or on histological
examination). The patients’ ages, genders, treatments prior to
enucleation, and adjuvant therapies were recorded. Tumor locations,
shapes, sizes, routes of ESE, pigmentation, cellular morphology, and
chromosome 3 status were extracted from the clinical notes,
histopathologic, and cytogenetic reports when available.
Results: Of 174 eyes enucleated for uveal melanoma, 16 (9%) had
histologically proven extrascleral extension. Eight (50%) of these 16
cases were diagnosed clinically, 3 (19%) were discovered
intraoperatively, and 5 (31%) were first detected during microscopic
examination. 7/7 (100%) of the cases with anterior ESE vs. 1/9 (11%)
of cases with posterior ESE were detected clinically.
Conclusions: ESE was clinically undetectable in a significant
percentage (50%) of cases, and clinical detection correlated with the
location of the extension (anterior or posterior). Although ultrasound
is the best clinical modality for detecting posterior ESE, most of our
cases were microscopic. This series highlights the importance of a
high level of suspicion for ESE in posterior uveal melanoma, as well
as good communication between the surgeon and the pathologist.
Commercial Relationships: Christopher Burris, None; Vasilios
Papastefanou, None; Caroline Thaung, None; Mandeep S. Sagoo,
None; Victoria Cohen, None
Extraocular extension in uveal melanoma
Jackelien van Beek1, Anna E. Koopmans1, 2, Jolanda Vaarwater2,
Annelies de Klein2, Robert M. Verdijk3, Emine Kilic1, 2.
1
Ophthalmology, Erasmus MC Rotterdam, Rotterdam, Netherlands;
2
Clinical Genetics, Erasmus MC Rotterdam, Rotterdam, Netherlands;
3
Pathology, Erasmus MC Rotterdam, Rotterdam, Netherlands.
Purpose: In approximately 13% of all enucleated uveal melanoma
patients, extraocular spread is found. It is reported that the size of the
intraocular tumour is related to extraocular extension and to
metastatic death. Extraocular spread, irrespective of which route, is
correlated with histopathologic and cytogenetic factors of increased
malignancy. Hence in this study we have identified all cases of uveal
melanoma with extraocular extension and investigated prognostic
factors and correlation with survival.
Methods: In this retrospective study, initiated by the Rotterdam
Ocular Melanoma Study (ROMS) group, the Netherlands, patients
were included if they were referred with a choroidal or ciliary body
uveal melanoma from 1987 until 2011. Only patients were included
who underwent primary or secondary enucleation due to a choroidal
of ciliary body melanoma.
Results: In total 356 patients were included, with a mean age of 60.6
years at moment of diagnosis (range 21-91 years). Extraocular
extension was confirmed by revision of the histopathological coupes
in 43 patients (67.4% male, mean age: 63.7 years). Extraocular
spread occurred in 34 patients with choroidal melanoma, in 8 with
ciliary body melanoma and in 1 patient with a ciliary body and
choroidal melanoma localisation. The mean basal diameter was 14.2
mm (range: 6-22 mm) with a mean prominence of 7.7 mm (range: 1 22 mm). Five times an epithelioid cell type, 22 times a mixed cell
type and 16 times a spindle cell type was observed. Extraocular
extension occurred via perivascular invasion (41 patients), direct
scleral invasion (33 patients), perineural invasion (29 patients) and
invasion through Schlemm’s canal (6 patients). Most of the
extraocular extensions followed a combination of these routes, 4
patients had only vascular spread and 1 patient had only neural
spread. The (largest) periscleral diameter of the extraocular extension
ranged from 0.1 mm to 40 mm, with a mean of 2.86 mm. In 19 out of
37 tumors with extraocular extension, monosomy 3 was observed.
Of the 43 patients with extraocular extension, 20 are alive, of which 4
with metastases. There were 19 patient deaths, of which 16 had
metastases.
Conclusions: Presence of extraocular extension was not related with
histological cell type. Even though 44% of the tumors with
extraocular extension had monosomy 3, metastasis was observed in
only 47%. Nevertheless, extraocular extension was associated with a
worse survival (p=0.019).
Commercial Relationships: Jackelien van Beek, None; Anna E.
Koopmans, None; Jolanda Vaarwater, None; Annelies de Klein,
None; Robert M. Verdijk, None; Emine Kilic, None
Ciliary Body and Choroidal Pseudomelanoma from
Hypermature Cataract in 20 Cases
Marco Pellegrini, Carol L. Shields, Brad E. Kligman, Carlos G.
Bianciotto, Jerry Shields. Ocular Oncology Service, Wills Eye
Institute, Philadelphia, PA.
Purpose: To describe pseudomelanoma of the ciliary body and
choroid in eyes with opaque media from oblique ultrasonographic
imaging through hypermature cataract.
Methods: Retrospective chart review of 20 eyes.
Results: All eyes had opaque media from hypermature cataract with
no view of the fundus. All were referred with uveal melanoma, based
on ultrasonographic imaging. The melanoma was located in the
ciliary body (n=17) or choroid (n=3). The median patient age was 54
years (range 17-86 years). Most patients were Caucasian (n=13) or
African American (n=4). There was a history of eye trauma (n=3)
and ocular surgery (n=1). The brunescent cataract was in anatomic
position (n=18) or subluxated (n=2). On B-scan ultrasonography, the
mass was dome (n=10) or elliptical (n=10) shape, acoustically hollow
center with dense rim (n=20), located in the ciliary body (n=17) or
choroid (n=3). The mean thickness was 7.2 mm and mean base was
9.3 mm. All 20 cases were diagnosed with pseudomelanoma from
hypermature cataract. Features that suggested cataract rather than
melanoma included lack of contiguity with the uvea (n=20) on
videoimaging using standard ultrasonography and ultrasound
biomicroscopy, lack of transillumination defect, and lack of sentinel
vessel. For those in the ciliary body, an additional feature was the
ultrasonographic presence of pseudomelanoma in all 4 quadrants
(n=17), representing oblique imaging of the lens equator. For those in
the choroid, the pseudomelanoma shifted when imaged reclined
compared to upright positions. Following cataract surgery, the lack of
melanoma was confirmed in all 20 cases.
Conclusions: Hypermature cataract can preclude a fundus view,
necessitating ultrasonography for imaging the posterior segment of
the eye. Ultrasonographic confusion with ciliary body and choroidal
melanoma can occur as the dome-shaped cataract can simulate a
dome-shaped melanoma. Consultation with an ocular oncologist is
advised.
Commercial Relationships: Marco Pellegrini, None; Carol L.
Shields, None; Brad E. Kligman, None; Carlos G. Bianciotto,
None; Jerry Shields, None
Prevalence of Asteroid Hyalosis in Human and Canine
Melanoma Eyes
Heather Potter1, Mozhgan Rezaei Kanavi1, 2, Amir Azari1, Richard R.
Dubielzig1, Daniel M. Albert1. 1Ophthalmology, University of
Wisconsin, Madison, WI; 2Ophthalmic Research Center, Shahid
Beheshti University of Medical Sciences, Tehran, Islamic Republic
of Iran.
Purpose: To explore a possible association between presence of
asteroid hyalosis (AH) and uveal melanoma in human and animal
subjects.
Methods: The histopathologic slides of enucleated melanoma eyes in
the collection of the eye pathology laboratory of University of
Wisconsin School of Medicine and Public Health were reviewed for
the presence of AH. The histopathologic data of the Comparative
Ocular Pathology Laboratory of Wisconsin (COPLOW) were also
reviewed for incidence of AH in canine eyes with the diagnosis of
uveal melanoma. For human control group, the incidence of AH in
patients seen at the University of Wisconsin Eye clinic was
established based on the records provided to us by the retina service.
The control group for the dog eyes consisted of those enucleated for a
common non-neoplastic eye disease (goniodysgenesis). Then the
association of AH with uveal melanoma in human and dogs was
evaluated.
Results: AH was found in 12 out of 1759 human eyes (0.7%) with
uveal melanoma (Figure 1). Forty three out of 5218 patients (0.82%)
seen at the eye clinic were shown to have AH. The prevalence of
asteroid hyalosis in human melanoma eyes was comparable with
those of age-matched patients seen at the eye clinic (control group).
The rate of AH in dogs with the diagnosis of uveal melanoma was
6.2% (46 out of 746 eyes) while it was 1.9% (45 out of 2309 eyes) in
the control group (eyes with goniodysgenesis).
Conclusions: Although we observed a three-fold increase in the rate
of AH in melanoma eyes in dogs compared to the control, no similar
association was found between AH and uveal melanoma in humans.
Figure 1. Image depicts the round spherules of asteroid hyalosis in
vitreous of a human eye with uveal melanoma (Alcian blue X400).
Commercial Relationships: Heather Potter, None; Mozhgan
Rezaei Kanavi, None; Amir Azari, None; Richard R. Dubielzig,
OSOD, LLC (I), Allergan (C); Daniel M. Albert, None
Pain as presenting feature of uveal melanoma
Pukhraj Rishi1, 2, Carol L. Shields1, Kaitlin A. Patrick1, Mohammed
A. Khan1, Jerry Shields1. 1Ocular Oncology Service, Wills Eye
Institute, Philadelphia, PA; 2Sankara Nethralya, Chennai, India.
Purpose: To describe the clinical features and outcomes of patients
with pain from underlying uveal melanoma.
Methods: Retrospective case series of 9 patients.
Results: The mean patient age at presentation was 62 years (median
62 years, range 41-83 years). The pain manifested in the periocular
region (n=9) and additionally as diffuse ipsilateral headache (n=5).
On a scale of 0-10, the average pain score was 7.5 (median 9, range
2-10) and four patients reported pain severity at maximum (10/10).
The average duration of pain was 27 days (median 7, range 2-180
days). Visual acuity was reduced in 8 of 9 eyes. The melanoma was
located in the ciliary body (n=3) or choroid (n=6). The mean tumor
basal diameter was 18 mm (median 18 mm, range 14-24 mm) and
mean tumor thickness was 7 mm (median 6 mm, range 5-11 mm).
Clinical features of spontaneous melanoma regression was evident in
all cases (n=9). Treatment included plaque radiotherapy (n=5),
enucleation (n=3) and one patient refused therapy (n=1). Following
therapy, all patients were relieved of pain.
Conclusions: Ocular pain or headache can be the initial
manifestation of uveal melanoma. Following ocular therapy, pain
symptoms are relieved.
Commercial Relationships: Pukhraj Rishi, None; Carol L.
Shields, None; Kaitlin A. Patrick, None; Mohammed A. Khan,
None; Jerry Shields, None
Analysis of the quality of life of patients receiving conservative
treatment for uveal melanoma
Federica Genovesi-Ebert1, Clara Meo2, Franco Perrone3, Clara
Meo2, Federica Cresti1, Patrizia Ferrazza2, Stanislao Rizzo1. 1U.O.
Chirurgia Oftalmica, Azienda Ospedaliera Universitaria Pisana,
Livorno, Italy; 2Radiotherapy Department, Azienda Ospedaliera
Universitaria Pisana, Pisa, Italy; 3Sanitary Physics, Azienda
Ospedaliera Universitaria Pisana, Pisa, Italy.
Purpose: Patients (pts) affected by ocular malignant melanoma
(MM) may experience distress or changes in their quality of life
following treatment . In order identify areas of primary concern, we
conducted a crosssectional survey to describe the experiences of ps
treated for MM with different therapeutic options .
Methods: A total of 130 pts (median age 71 years,range 35-90 y,)
treated at Azienda Ospedaliera Universitaria Pisana for MM by using
brachitherapy (BRT) with ruthenium 106 plaques and Iodine plaques,
stereotatic radiation therapy (SRS) and enucleation participated in
this study.
We examined quality of life, levels of distress, appearance
perceptions, body image, fear of recurrence by using The Quality of
Life Scale (QoL) test and the Hospital Anxiety and Depression scale.
The results have been analized considering tumor stage, type of
treatment, visual and anatomic outcome.
Results: Depressive symptomatology occurred in 6 % of pts
analyzed, and 24 % reported a clinically established high level of
intrusive thoughts related to melanoma.
Quality-of-life scores indicated more disruption on psychological
side, compared with social and physical functioning.
There are no significant differences at 95% C L with respect to QoL,
anxiety, depression and other indices tested among pts who have
preserved the eye even though they had lost visual function
We appreciated significant differences between pts enucleated and
treated conservatively, with regard to anxiety (p = 0.006), QoL (p =
0.012), emotional (p = 0.001) and social functioning (p = 0.05). No
statistically significant differences were highlighted between groups
of patients treated conservatively by using different therapeutic
modalities (BRT vs. SRS) (fig 1)
Conclusions: The pts treated conservatively for ocular MM reported
a low level of distress associated with impairment of visual function.
Moreover in these subjects anxiety, emotional disorders, fear of
recurrence and social functioning deterioration were lower than in the
enucleated pts. Therefore eye preservation may play a role in order to
achieve a better quality of life.
Improvements in patient education prior to surgical intervention may
reduce the distress associated with the diagnosis and treatment of
melanoma. Psycological preoperative and postoperative support
should be useful in patients affectd by MM, especially in patients
who will undergo enucleation.
Commercial Relationships: Federica Genovesi-Ebert, None;
Clara Meo, None; Franco Perrone, None; Clara Meo, None;
Federica Cresti, None; Patrizia Ferrazza, None; Stanislao Rizzo,
None
428 Uveal Melanoma: Experimental Therapeutics
Wednesday, May 08, 2013 11:00 AM-12:45 PM
608 Paper Session
Microphthalmia-associated transcription factor (MITF) and
Steroid Receptor Coactivator (SRC)-3 cooperate to promote
proliferation, survival and metabolism in Gα-mutant uveal
melanoma (UM) cells
Vassiliki Poulaki1, 2, Sue Anne Chew3, 4, Bin He3, 4, Vijay Kumar
Eedunuri5, Martine M. Jager6, Bert W. O'Malley4, Nicholas
Mitsiades3, 4. 1Ophthalmology, Boston VA Healthcare System,
Boston, MA; 2Ophthalmology, Boston University, Boston, MA;
3
Medicine, Baylor College of Medicine, Houston, TX; 4Molecular
and Cellular Biology, Baylor College of Medicine, Houston, TX;
5
Adrienne Helis Malvin Medical Research Foundation, New Orleans,
LA; 6Ophthalmology, Leiden University Medical Center, Leiden,
Netherlands.
Purpose: We recently reported that somatic mutations in Gαq and
Gα11 (encoded by GNAQ and
GNA11, respectively), found in most UMs, activate protein kinase C
(PKC) and protein kinase D (PKD), which then phosphorylate and
stabilize SRC-3. SRC-3 expression is critically important for UM
cells, but its exact function remains unknown.
Methods: We screened 359,199 compounds in vitro for SRC-3 small
molecule inhibitors (SMIs) by a high-throughput cell-based reporter
assay. Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) for
SRC-3 and MITF (a critical transcription factor for melanocytes) was
performed in Gαq-mutant (mt) Mel202 cells. We explored the role of
SRC-3 and MITF in Gα-mt UM cell lines using RNA interference
(RNAi) and adenoviral-mediated gene transfer.
Results: Our screen for SRC-3 SMIs yielded 620 hits, with
significant overlap to a previous assay for
MITF SMIs (406 overlapping compounds, P<0.0001, by Fisher's
exact test). Representative MITF/SRC-3 SMIs exerted greater
anticancer activity against Gα-mt than wild type cells, which was
attenuated by SRC-3 overexpression. Co-immunoprecipitation
revealed a direct physical interaction of MITF with SRC-3. Per ChIPSeq, 96.7% of genes recruiting SRC-3 also recruit MITF. The
consensus binding motifs for MITF and SRC-3 overlapped
significantly, supporting their cooperative interaction. Silencing
either MITF or SRC-3, by RNAi, had profound anticancer activity in
Gα-mt cells, and the resulting gene expression profiles revealed
significant overlap, that involved suppression of transcripts for
kinases, nutrient transporters and metabolic enzymes. The promoters
of those genes directly recruited SRC-3 and MITF, as documented by
ChIP-Seq and confirmatory ChIP-PCR. Inhibition of the
Gα/PKC/SRC3 pathway in Gα-mt cells suppressed metabolic activity
and oxygen consumption and led to autophagy.
Conclusions: SRC-3 is stabilized post-translationally in Gα-mt UM
cells by the Gα/PKC pathway and colocalizes on chromatin with MITF to cooperatively drive
transcription of metabolism, proliferation and survival genes. This
direct physical and functional interaction of SRC-3 with MITF
highlights both of them as effectors of Gα-induced oncogenic
signaling and as therapeutic targets in UM. The 406 SMIs found to
target both SRC-3 and MITF represent potential drug candidates for
UM.
Commercial Relationships: Vassiliki Poulaki, None; Sue Anne
Chew, None; Bin He, None; Vijay Kumar Eedunuri, None;
Martine M. Jager, None; Bert W. O'Malley, None; Nicholas
Mitsiades, None
Support: The authors acknowledge the joint participation by
Adrienne Helis Malvin Medical Research Foundation through its
direct engagement in the continuous active conduct of medical
research in conjunction with Baylor College of Medicine. B.H. is
supported by NIH/NIDDK grant 5K01DK081446. N.M. is a Dan L.
Duncan Scholar and a Caroline Wiess Law Scholar at Baylor College
of Medicine and his work is also supported by the Conquer Cancer
Foundation of the American Society of Clinical Oncology (ASCO)
Career Development Award and by the Prostate Cancer Foundation.
Uveal Melanoma Cells Are Inhibited by AICAR Partially
Through Activation of AMP-Dependent Kinase
Ahmad Al Moujahed1, Fotini Nicolaou2, Thanos D. Papakostas1,
Joan W. Miller1, Evangelos S. Gragoudas1, Demetrios Vavvas1.
1
Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard
Medical School, Boston, MA; 2Pediatric Surgery Laboratories,
Massachusetts General Hospital, Boston, MA.
Purpose: 5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside
(AICAR), an analog of AMP is widely used as an activator of AMPkinase (AMPK), a protein that regulates the cell energy homeostasis
and response to metabolic stress. The purpose of this study is to
evaluate the effects of AICAR, AMPK activator, on the growth of
uveal melanoma cell lines (OCM3 and MEL92.1)
Methods: Two different cell lines (OCM3, MEL92.1) were treated
with AICAR (1-4 mM) for 3-5 days. Cell growth was assessed by
MTT assay. Cell cycle analysis was assessed with propidium iodide
staining and flow cytometry. Expression of cell cycle regulators
(Cyclins, p21, p27, PCNA , CDK2 and CDK4), Acetyl-CoA
Carboxylase, S6, 4E-binding protein 1 and the autophagy marker
LC3 was assessed by RT-PCR and Western blots.
Results: Cell treatment with AICAR inhibited cell growth, induced
S-phase cell cycle arrest and led to activation of AMPK. These
effects were abolished by treatment with dypiridamole, an inhibitor
of AICAR entrance into cells. Treatment with adenosine kinase
inhibitor 5-iodotubericidin to inhibit the conversion of AICAR to
ZMP (the direct activator of AMPK) reversed a significant part of the
growth inhibiting effects of AICAR indicating that some of AICAR’s
anti-proliferative effect are mediated through AMPK activation. In
addition, AICAR treatment was associated with upregulation of the
autophagy marker LC3, downregulation of 4EBP1 phosphorylation
via mammalian target of rapamycin (mTOR) independent
mechanism, and down-regulation of cyclins A and D.
Conclusions: Our results indicate that AICAR-induced activation of
AMPK inhibits uveal melanoma cell growth. This is one of few
descriptions of low toxicity drug that may be effective in treating
uveal melanoma patients.
Commercial Relationships: Ahmad Al Moujahed, None; Fotini
Nicolaou, None; Thanos D. Papakostas, None; Joan W. Miller,
Massachusetts Eye and Ear Infirmary (P), Novartis (I), Alcon (C),
KalVista Pharmaceuticals (C); Evangelos S. Gragoudas, QLT
Phototherapeutics, Inc. (P); Demetrios Vavvas, MEEI (P), Kala
pharmaceuticals (C), Roche (C), Genentech (C)
Support: RPB unrestricted fund
SRPK1 Inhibition, As A Way Of Targeting Pro-Angiogenic
VEGF-A Production In Ocular Tumours
Melissa V. Gammons1, Sarah E. Coupland3, Andrew D. Dick2, David
O. Bates1. 1Department of Physiology and Pharmacology, University
of Bristol, Bristol, United Kingdom; 2School of Clinical Sciences and
School of Cellular and Molecular medicine, University of Bristol,
Bristol, United Kingdom; 3Department of Molecular and Clinical
Cancer Medicine, University of Liverpool, Liverpool, United
Kingdom.
Purpose: Uveal melanoma (UM), although rare, is the most common
type of intraocular tumour. Approximately 50% of UM are fatal due
to their tendency to metastasise to the liver. Angiogenesis is regulated
by vascular endothelial growth factor-A (VEGF-A), a growth factor
that is expressed by UM. VEGF-A is alternatively spliced to form
two families of isoforms: those that are pro-angiogenic and those that
are anti-angiogenic. Splicing of the pro-angiogenic isoforms is
mediated by serine-rich protein kinase-1 (SRPK1), a protein kinase
that results in the phosphorylation and nuclear localization of serinerich splicing factor 1 (SRSF1). SRSF1 is a promoter of proximal
splice site selection, thus selectively upregulating pro-angiogenic
VEGF-A production. Activation of this pathway can be achieved by
administration of insulin-like growth factor-1 (IGF-1), a promoter of
VEGF mediated tumour growth. We investigated the effect of
SRPK1 inhibition on VEGF-A splicing in UM cell lines.
Methods: UM cell lines Mel270, Omm2.5 and 92.1 were grown in
culture medium. Both before and after 24hours exposure to 1, 5, 10
or 20μM SRPIN340 or combined with 100nM IGF-1 mRNA was
extracted, cDNA was synthesised and used in q-PCR and RT-PCR
for SRPK1, VEGF165 and VEGF165b expression.
Immunofluorescence determined SRSF1 and SRPK1 localisation
within the cells, and the protein assessed for VEGF-A expression by
Western blot.
Results: SRPK1 was expressed in the UM cytoplasm and
translocated to the nucleus during IGF-1 treatment. IGF-1 dose
dependently increased pro-angiogenic VEGF-A mRNA expression
whereas inhibition of SRPK1 with SRPIN340 reduced proangiogenic VEGF-A expression, maintaining the production of antiangiogenic VEGF-A isoforms.
Conclusions: Targeting SRPK1 reduces the expression of proangiogenic VEGF-A in UM cell lines. Selective blocking of proangiogenic isoforms may reduce UM metastasis rates whilst
maintaining the production of cytoprotective anti-angiogenic
isoforms. We wish to validate these results in fresh UM specimens
where clinical outcome and genetic status is known.
Commercial Relationships: Melissa V. Gammons, None; Sarah E.
Coupland, None; Andrew D. Dick, Novartis (C), Novartis (F), GSK
(F), Abbott (F); David O. Bates, University of Bristol (P)
Targeting Uveal Melanoma Response to Hypoxia
Dudi Shneor1, 2, Alik Honigman2, Jacob Pe'er1, Shahar Frenkel1.
1
Ophthalmology, Hadassah-Hebrew University Medical Center,
Jerusalem, Israel; 2Biochemistry and Molecular Biology, IMRIC, The
Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Purpose: Tumor hypoxia is considered to be a potential therapeutic
problem because it renders solid tumors more resistant to ionizing
radiation and chemotherapeutic drugs. We describe a recombinant
Murine leukemia virus (MuLV)-based replication-competent
retroviruses (RCR) delivery system that infects only growing cells.
This results in a persistent viral knockdown of the expression of the
regulators of the hypoxia responding genes CREB, HIF1, and HIF2,
via shRNA targeting of these genes separately and all together from a
single polycistronic RNA.
Methods: C918 uveal melanoma cells were stably infected with RCR
expressing shRNA targeting CREB, HIF1, and HIF2 separately and
all together from a single polycistronic RNA. Knockdown of the
target genes was analyzed using qRT-PCR and Western blot. Infected
cells co-transfected with either CRE or HRE mediated luciferase
(luc) gene expression vector, pCREluc or pHREluc, respectively,
served for functional analyses of the transcription factors. Cell
viability and caspase 3 activity were determined using the
Fluorescent Cell Viability and Caspase-Glo 3/7 Assays (Promega)
after 0-72 hours under normoxic and hypoxic (0.5% O2) conditions.
Results: The different RCRs efficiently infect the C918 cells and
efficiently knockdown the mRNA and protein levels of CREB, HIF1
and HIF2. Knockdown of these genes by the recombinant RCRs
reduced VEGF secretion, reduced tumor cell proliferation, and
increased Caspase 3 activity in hypoxia. We found that CREB, more
than HIF1 and HIF2, plays a pivotal role in the survival of C918
under hypoxia in vitro while HIF1 affects VEGF in vitro.
Conclusions: MuLV-based RCRs affecting the response of UM to
hypoxia, specifically affecting CREB and HIF1, have potential as a
novel therapeutic approach for metastatic UM.
Commercial Relationships: Dudi Shneor, None; Alik Honigman,
None; Jacob Pe'er, None; Shahar Frenkel, None
Support: Israeli Ministry of Health Chief Scientist grant, and the
Israel Science Foundation's grant for physician-researchers, both to
Shahar Frenkel
Efficacy of A Novel Anti-tumor Agent KCN1 in the Control of
Ocular Melanoma In vitro and In vivo
Qing Zhang1, 2, Hua Yang1, Stefan Kaluz3, Erwin G. Van Meir3, 4,
Hans E. Grossniklaus1, 5. 1Ophthalmology, Emory University School
of Medicine, Atlanta, GA; 2Ophthalmology, Central South University
Xiangya School of Medicine, Changsha, China; 3Neurosurgery,
Winship Cancer Institute, Emory University School of Medicine,
Atlanta, GA; 4Hematology and Medical Oncology, Emory University
School of Medicine, Atlanta, GA; 5Pathology, Emory University
School of Medicine, Atlanta, GA.
Purpose: To evaluate the effects of a novel small molecule KCN1 in
suppressing of ocular melanoma in vitro and in vivo.
Methods: Human uveal melanoma Mel270 and Mel290, and mouse
melanoma B16F10, B16LS9 cell lines were cultured with KCN1 or
DMSO. SRB cell cytotoxicity assay was performed. Total RNA was
extracted and RT-PCR analysis was performed. The protein
expression of p-met, p-MAPK, p-STAT3, MMP2, MMP9, CA9 and
Bcl-2 was detected by Western Blot. The level of VEGF protein in
the culture media was measured by ELISA. Melanoma cells were
then transfected with reporter constructs driven by V6R, VEGF and
CA9 promoters. 220 C57Bl/6 mice were inoculated into their right
eyes with 5X105 B16LS9 melanoma cells, and randomly assigned
into 22 groups (n=10). Timing and dosing experiments were
performed using 30 mg/kg or 60mg/kg KCN1 i.p. starting on days 1,
4, or 7 after inoculation. Tumor-burden eyes were enucleated on the
7th day after inoculation for size measurement, and livers were
collected on the 28th day for number count. A longitudinal
experiment was also performed to determine the hepatic
micrometastases and serum VEGF level at weeks 1, 2, 3, 4 and 5.
Proliferation, apoptosis and angiogenesis markers were evaluated in
the ocular melanoma by immunostaining.
Results: KCN1 inhibited melanoma cell growth dose-dependently
under both normoxic and hypoxic conditions. The level of CA9 and
VEGF mRNAs decreased in the melanoma cells when treated with
KCN1. The activity of reporter constructs were inhibited in the
presence of KCN1 by 30-80%. KCN1 suppressed the protein
expression of p-met, p-MAPK and p-STAT3 dose-dependently, and
also significantly reduced the level of VEGF in the culture media
from melanoma cells. In a mouse model of melanoma, KCN1
decreased the growth of primary ocular melanoma by up to 70% and
reduced the formation of hepatic micrometastases by up to 50% in a
dose-dependent manner. Furthermore, immunostaining showed
decreased Ki67 and VEGF expression after treatment with KCN1.
Conclusions: Anti-tumor small molecule KCN1 reduced both in
vitro growth and in vivo hepatic micrometastases of ocular
melanoma. KCN1 is a potential therapeutic agent for the treatment of
uveal melanoma. Further preclinical evaluation of KCN1 and related
complexes is warranted.
KCN1 effects on the growth of melanoma cell lines in vitro
KCN1 effects on the growth of primary ocular melanoma and hepatic
micromestasis in vivo
Commercial Relationships: Qing Zhang, None; Hua Yang, None;
Stefan Kaluz, None; Erwin G. Van Meir, Emory University (P);
Hans E. Grossniklaus, None
Support: NIH R01 CA126447, CA116804, R24 EY017045,
National Natural Science Funds (NNS81201808) for Young Scientist
in China, Fight For Sight Postdoctoral Award, Emory Melanoma
Prevention and Research Discovery Fund, a Winship Cancer Institute
pilot grant, a Central South University Lieying pilot grant, and an
unrestricted grant from Research to Prevent Blindness, Inc.
NFkB Inhibition in Uveal Melanoma - Contrariety of in vitro vs.
in vivo Results
Shahar Frenkel1, Dudi Shneor2, 1, Jacob Pe'er1, Alik Honigman2.
1
Ophthalmology, Hadassah-Hebrew University Medical Center,
Jerusalem, Israel; 2Biochemistry and Molecular Biology, IMRIC, The
Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Purpose: To describe the differential response of uveal melanoma to
NFkB inhibition in vitro vs. in vivo.
Methods: The C918 uveal melanoma (UM) cell line was grown in
culture without and with 1μg/ml of the BMS-345541 NFkB inhibitor.
Cells were grown under normoxic and hypoxic conditions (0.5%O2)
for 72 hours with viability and Caspase3 measurements every 24
hours using the Fluorescent Cell Viability and Caspase-Glo 3/7
Assays (Promega, Madison, WI, USA). Later, C918 cells were
transfected with the luciferase (luc) gene and cells that stably
expressed luc were selected. The new C918-Luc cell line was
trypsinized, washed in 1xPBS and injected either subcutaneously or
directly into the livers of SCID mice through a small (1 cm)
abdominal wall incision via a 0.5 cc insulin syringe (29 gauge needle)
in a non-reflux technique. Injected cells were visualized with an IVIS
bioluminescence (BioL) camera (Caliper Life Sciences, Hopkinton,
MA, USA) after injecting the mice with luciferin (IP 3 mg/ 0.3 cc).
Cells were allowed to settle for 1 week before BMS-345541 (0, 2, 10,
20, and 50 mg/kg) was administered IP 3 times / week for 3 weeks,
and tumor growth was monitored via BioL twice weekly. The
experiment was terminated following euthanasia and the livers were
harvested for histopathologic evaluation. The research reported
herein was conducted in compliance with the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research.
Results: Cell viability diminished similarly either with addition of
BMS in normoxia or without it in hypoxia, and was abolished with
BMS in hypoxia. A similar increase in Caspase3 was noted at 72
hours. In SCID mice, bioluminescence started decreasing after
administration of BMS, but then turned into a remarkable growth
spurt which increased with higher doses of BMS. Histopathology of
treated tumors showed a central area of necrosis surrounded by viable
tumor vs. a completely viable tumor in untreated animals.
Conclusions: Inhibition of NFkB reduces cell viability and increases
apoptosis in vitro and in vivo. However, this effect is reversed by yet
unknown mechanisms in vivo, possibly triggered by the necrotic
tumor. These mechanisms may lie behind UM’s unresponsiveness to
chemotherapeutics.
Commercial Relationships: Shahar Frenkel, None; Dudi Shneor,
None; Jacob Pe'er, None; Alik Honigman, None
Support: Israeli Ministry of Health Chief Scientist grant, and the
Israel Science Foundation's grant for physician-researchers
Identification of regulators of uveal melanoma metastasis
through whole exome sequencing
Sarah Lake1, Bertil E. Damato2, Helen Kalirai1, Lisa Olohan3, Xuan
Liu3, Sarah E. Coupland1. 1Molecular And Clinical Cancer Medicine,
University of Liverpool, Liverpool, United Kingdom; 2Royal
Liverpool University Hospital, Liverpool, United Kingdom; 3Centre
for Genomic Research, University of Liverpool, Liverpool, United
Kingdom.
Purpose: The drivers of uveal melanoma (UM) metastasis are not yet
fully understood despite studies identifying a potential function for
genes including BAP1, MDA-9, DDEF1 and ID2. This study aimed to
identify genes that are mutated in primary, metastasising, UM and
which may therefore be additional, key regulators of UM metastasis.
Methods: DNA from 12 clinically and histologically well-defined
UM was sequenced following exome capture (50Mb SureSelect,
Agilent) using the SOLiD system (Applied Biosystems). Tumours
were stratified according to their risk of developing metastasis using
an accelerated failure time model and included six monosomy 3 UM
with high-risk of metastasis, and six disomy 3 UM with low
metastatic risk.1 A systems biology approach (GeneGo, Metacore)
was used to identify mutated genes that occurred in all high-risk UM
and which were therefore likely to influence metastatic cell
behaviour. Bidirectional, chain-termination, sequencing was used to
confirm the presence of mutations. Expression of the encoded
proteins was assessed by immunohistochemistry.
Results: Seven genes had non-synonymous mutations in their coding
regions. These were: LAMB2 (chr 3), USP19 (chr 3), ULBP3 (chr 6),
ARFGEF1 (chr 8), EPHA1 (chr 7), FOS (chr 14) and KIF23 (chr 15).
A statistically-significant difference in the frequency of mutations in
the high-risk UM compared to the low-risk UM was observed
(p<0.003-0.046). These mutations were not present in dbSNP
(http://www.ncbi.nlm.nih.gov/projects/SNP/). No mutations of BAP1
were identified in any UM. Copy number alterations and/or
insertion/deletions of genes were not consistently seen in the highrisk UM. Protein expression of the mutated genes was detected in
UM, regardless of metastatic risk, for USP19, ULBP3, ARFGEF1,
EPHA1 and KIF23. No expression of FOS or LAMB2 protein was
detected in UMs.
Conclusions: Investigations are underway to determine whether the
mutations detected in the coding regions of USP19, ULBP3,
ARFGEF1, EPHA1 and KIF23 alter the biological function of the
encoded proteins to promote metastatic progression.
References
1. Eleuteri A DB, Coupland SE and Taktak AFG. Enhancing survival
prognostication in patients with choroidal melanoma by integrating
pathologic, clinical and genetic predictors of metastasis. Int J
Biomedical Engineering and Technology 2012;8:18-35.
Commercial Relationships: Sarah Lake, None; Bertil E. Damato,
None; Helen Kalirai, None; Lisa Olohan, None; Xuan Liu, None;
Sarah E. Coupland, None
Support: Cancer Research UK
zebrafish.
Megalin is a multi-ligand endocytic receptor and extensive studies of
renal proximal tubular epithelium have shown that megalin is crucial
for conservation of filtered nutrients including vitamin D, vitamin A,
vitamin B12 and iron. Megalin has previously been localized to the
retina pigment epithelium (RPE) and ciliary body epithelium of the
adult eye, however, the physiological role of megalin in these
epithelia has not been clarified.
The purpose of this study was to determine subcellular localization of
megalin in the adult eye and, prospectively, also the physiological
role.
Methods: Eye tissue from normal and megalin-deficient mice as well
as normal human eye was examined with immunological techniques
using confocal and electron microscopy to determine the
topographical and subcellular localization of megalin.
Results: Megalin was identified in RPE cells and ciliary body nonpigmented epithelium (CBNPE) in both human and mouse adult eye.
Immunocytochemical investigations of ultrathin cryosections of
mouse eyes displayed a disperse, vesicle-like, cytoplasmic
localization of megalin in RPE cells but a predominantly apical
localization in CBNPE cells. Furthermore, histological cross sections
of eyes from normal and megalin-deficient mice showed a severe
increase in overall eye size and axial length (See figure) as well a
thinning of the neural layers of the retina in mice deficient of
megalin.
Conclusions: The topographical localization of megalin in the adult
eye corresponds to the blood-aqueous humor and blood-retina
barriers respectively. Based on our findings, megalin seems to be a
prime candidate to mediate selective transport of nutrients to the
inner structures of the eye. Finally, the increased axial length
observed in the megalin-deficient mice supports findings from
patients and zebrafish and suggests that the megalin-deficient mice
may prove a valuable model for future studies of myopia.
472 Myopia II, AP
Wednesday, May 08, 2013 2:45 PM-4:30 PM
Megalin and myopia
Tina Storm1, Steffen Heegaard2, 3, Erik I. Christensen1, Rikke
Nielsen1. 1Department of Biomedicine, Aarhus University, Aarhus,
Denmark; 2Eye Pathology Institute, Department of Neuroscience and
Pharmacology, University of Copenhagen, Copenhagen, Denmark;
3
Department of Opthalmology, Glostrup Hospital, University of
Copenhagen, Copenhagen, Denmark.
Purpose: Myopia is the most common human eye disorder
worldwide and high-grade myopia is one of the most frequent causes
of blindness due to associated complications such as glaucoma.
In man, mutations of the megalin encoding gene causes the extremely
rare Donnai-Barrow/ Facio-Oculo-Acoustico-Renal Syndrome that is
characterized by diverse clinical manifestations of which high-grade
myopia has been consistently observed. Additional evidence
supporting that megalin may play a role in development of myopia
includes observations of adult-onset myopia in megalin-deficient
Commercial Relationships: Tina Storm, None; Steffen Heegaard,
None; Erik I. Christensen, None; Rikke Nielsen, None
Support: Velux Foundation, NOVO-Nordisk Foundation, Danish
Medical Research Council, and King Christian X's Foundation
Refractive Development and Form-Deprivation in Dopamine D4
Receptor Knock-Out Mice
Han na Park1, Christopher C. Tan1, Jacob G. Light1, Fazila Aseem1,
P M. Iuvone1, 2, Machelle T. Pardue1, 3. 1Ophthalmology, Emory
University, Atlanta, GA; 2Pharmacology, Emory University, Atlanta,
GA; 3Rehabilitation Research & Development Center, Atlanta
Veterans Affairs Medical Center, Decatur, GA.
Purpose: Dopamine has been implicated in the regulation of ocular
growth and development of myopia. Furthermore, recent studies have
shown that dopamine receptors have selective effects on contrast
sensitivity and visual acuity. Here, we explore the contribution of
dopamine D4 receptors (D4) in refractive development and its
possible environmental interactions during form-deprivation.
Methods: Refractive development of D4 knock-out (KO) and agematched C57BL/6 wild-type (WT) mice were monitored from
postnatal day (P) 28 to 112. Biweekly measurements of refractive
error were made with a photorefractor and corneal radius of curvature
(CRC) with keratometry. Ocular parameters were measured using
cross-sectional images from 1310nm SD-OCT, including corneal
thickness (CT), anterior chamber depth (ACD), posterior chamber
depth (PCD), lens thickness (LT), retinal thickness (RT), and axial
length (AL). A separate cohort of D4 KO mice received headmounted diffuser goggles over the right eye at P28 to induce FD
myopia. These mice were measured with the same instruments listed
above. At end of the experiment, retinas were collected for
quantification of dopamine using HPLC.
Results: The D4 KO mice (n=4-13) were relatively more myopic
compared to WT mice (n=14-34) with an average diopter (D)
difference of -3.15±1.20 across the age range of P28 to P112,
respectively (RM ANOVA, p <0.001). Across the same ages, D4 KO
mice had significantly longer CRC (0.03 ±0.01mm, RM ANOVA,
p=0.03) and a trend for longer axial lengths (12 ± 2µm) and thicker
lenses (26 ±4µm) than WT mice. After 2 weeks of goggling, D4 KO
and WT FD mice showed a significant myopic shift of ~2D
compared to contralateral and naïve control eyes (RM ANOVA, main
effect p=0.016 and p=0.005, respectively). Goggled D4 KO mice
showed trends towards shorter CRC (14µm), longer ACD (10µm),
longer PCD (31µm), and longer AL (32µm) compared to opposite
and naïve eyes, while WT mice did not have measureable changes at
this time point.
Conclusions: The absence of the D4 resulted in more myopic
refractions with normal refractive development. However, the
response to form deprivation was not significantly altered. At 2
weeks post-FD, the ocular parameters in D4 KO mice showed trends
that correspond to the refractive changes, but did not reach
significance with the current animal numbers and the resolution of
the current instruments.
Commercial Relationships: Han na Park, None; Christopher C.
Tan, None; Jacob G. Light, None; Fazila Aseem, None; P M.
Iuvone, None; Machelle T. Pardue, None
Support: NIH EY016435 (MTP), NIH P30 EY006360,
Departmental grant from Research to Prevent Blindness, and the
Department of Veterans Affairs
The effect of 2% homatropine on the choroidal thickness of
young healthy adults
Beata P. Sander, Michael J. Collins, Scott A. Read. School of
Optometry and Vision Science, QUT, Kelvin Grove, QLD, Australia.
Purpose: Changes occurring in the choroid are associated with in
ocular growth in many studies with animals. Since the cholinergic
system may be involved in regulating choroidal thickness, we
assessed whether the anticholinergic agent- homatropine alters
choroidal thickness in humans.
Methods: Fourteen young healthy adults (27.92 ± 4.05 years of age)
participated in this randomized, placebo-controlled crossover study.
Choroidal thickness was measured with a spectral domain OCT
(SOCT- Copernicus HR) prior to, as well as 30 and 60 minutes
following the instillation of one drop of 2% homatropine
hydrobromide or placebo (0.3% hydroxypropyl methylcellulose). All
measurements were carried out on the right eye only. The pre- and
post-drop OCT images (6 mm width horizontal and vertical line scans
centred on the fovea) were manually segmented to calculate
subfoveal and parafoveal (1.5 mm from the center of the fovea)
choroidal thickness.
Results: The subfoveal choroidal thickness in the OCT scans
underwent a small but significant thickening after instillation of
topical homatropine when compared to the placebo (ANOVA,
p<0.001). The mean change in choroidal thickness following
homatropine instillation was 8 ± 4 μm and 12 ± 6 μm after 30 and 60
minutes respectively. The parafoveal choroid also exhibited increase
with time after homatropine instillation in all quadrants (p<0.01).
However, only the temporal and nasal parafoveal choroid showed
significant change in thickness after 60 min when compared to the
placebo (both p<0.05).
Conclusions: Instillation of homatropine results in an increase in
choroidal thickness. These data provide insights into the mechanism
regulating choroidal thickness in human eyes and suggests that the
cholinergic signalling may be involved in this process.
Commercial Relationships: Beata P. Sander, None; Michael J.
Collins, None; Scott A. Read, None
Support: None in the Support filed below
In Vivo Crosslinking of Scleral Collagen in the Rabbit Using
Sub-Tenon Injection of Nitroalcohol
Quan V. Hoang1, 2, Quan Wen1, Stanley Chang1, Stephen L. Trokel1,
Ronald H. Silverman1, David C. Paik1. 1Ophthalmology, Harkness
Eye Institute, Columbia Univ, New York, NY; 2Vitreous, Retina,
Macula Consultants of New York, New York, NY.
Purpose: Myopic progression occurs in up to 50% of myopic
patients. Scleral weakness and abnormal scleral collagen are possible
etiologic factors. We evaluate the efficiency of non-light-activated
collagen crosslinking to increase biomechanical strength and stunt
axial length growth in vivo.
Methods: Ten (~35-51 day-old) New Zealand White rabbits
underwent baseline B-scan ultrasonography (US) axial length
recordings. Rabbits then received 5+ posterior sub-tenon injections
over 2-4 weeks composed of 0.9% NaCl in the left eye (OS) and
collagen crosslinking agents in the right eye (OD). Nine rabbits
received 2-hydroxymethyl-2-nitro-1,3-propanediol (Triol) and 1
rabbit received glyceraldehyde (GLY) dissolved in 0.9% NaCl. 8
rabbits were sacrificed immediately after the treatment period. A pair
of rabbits (1 treated with Triol, and 1 with GLY) were followed with
weekly US axial length measurements until sacrifice at 105 days of
life.
Results: Baseline axial length ranged from 13.82 +/- 0.2 (mean +/standard deviation, in mm) OD and 13.76 +/- 0.2 OS (n = 2) among
35 day-old rabbits to 14.62 +/- 0.4 OD and 14.62 +/- 0.4 OS (n = 8)
among ~51 day-old rabbits. Postmortem biochemical testing showed,
in terms of half-maximal shrinkage temperature, Triol-treated eyes
had a shift ranging from negligible to 2.6 degrees, with a mean shift
of 0.9 (n = 9). A 0.9 degree shift was also seen in the GLY-treated
rabbit. Also, Triol-treated eyes had 28% resistance to enzymatic
digestion compared to 37.8% in GLY-treated eyes. Histologic studies
did not show toxicity in either Triol- or GLY-treated eyes. In the
longitudinally-followed rabbits, 1 week after completion of Triol
treatment, OD AL had grown 0.15 mm over baseline, which was 43%
of the 0.35 growth in the contralateral control OS. This difference
diminished. By 105 days of life, axial growth (1.1 mm) was
equivalent among treated and control eyes. In contrast, 1 week after
completion of GLY, OD axial length had grown 0.58 mm over
baseline, which was 90.6% of the 0.64 growth in the control OS.
Difference was maintained at 105 days of life (axial length growth
OD was 1.0 mm, 91.8% of the 1.1 mm growth OS).
Conclusions: Sub-Tenon delivery of Triol increased scleral
biomechanical rigidity in vivo and delayed axial length growth
transiently with no noted side effects on the retina. The induced
scleral crosslinking may become a treatment modality to prevent
myopic progression.
Commercial Relationships: Quan V. Hoang, None; Quan Wen,
None; Stanley Chang, Alcon Laboratories (C), Alimera Sciences
(C); Stephen L. Trokel, Avedro (C); Ronald H. Silverman, None;
David C. Paik, 12/517,382 and PCT/US2007/025126 (P)
Support: NIH Grant EY020495
Cross-linking of rabbit sclera using riboflavin and UVA for the
prevention of progressive myopia
ASSAF DOTAN1, Israel Kremer1, Arie Zigler2, Dan Bourla1.
1
Ophthalmology, RABIN MEDICAL CENTER, Petach Tikva, Israel;
2
Racah Institute of Physics, The Hebrew University, Jerusalem,
Israel.
Purpose: To demonstrate the effect of scleral crosslinking by the
photosensitizer riboflavin and ultraviolet A on the development of
axial myopia in a rabbit eye model.
Methods: Twenty two, 13 day-old new zealand white rabbits were
divided equally to control and study groups. The eyes axial lengths
were determined by A-scan ultarsonography.
Only the right eye in each rabbit had surgery. Eleven eyes in the
control group had 360 degrees conjunctival peritomy followed by
tarrsorhaphy.
Eleven eyes in the study group had 360 degrees conjunctival
peritomy and scleral crosslinking followed by tarrsorhaphy: 0.1%
riboflavin-5-phosphate dextran free (Concept for pharmacy Ltd.
Kfar-Saba, Israel) was dropped onto the scleral irradiation zone 20
seconds before the irradiation and every 20 seconds during the 200
sec. irradiation time.
Each eyeball was divided to four quadrants and every quadrant had
two or six scleral irradiation zones ( 8 eyes had two and 3 eyes had
six) - each zone had a radius of 2 mm and an area of 0.2 cm2. UVA
irradiation (370 nm) was applied over the sclera, which was
irradiated at 570 mW/cm2 for an area of 0.2 cm2. This method
provides a total UVA light dose of 5.7 J/cm2.
Fifty five days later, the tarrsorhaphies were removed and the eyes
had a second A-scan ultrasonography for axial length measurement.
Results: In the control group the mean right eye axial length was
10.50+ 0.67 mm before eyelid suture and 15.69+ 0.39 mm 55 days
later, with a mean difference of 5.19+ 0.85 mm.
In the experimental group the mean right eye axial length was
10.68+ 0.74 mm before eyelid suture and 14.29+ 0.3 mm 55 days
later, with a mean difference of 3.61+ 0.76 mm.
This difference was statistically significant (p<0.001, Mann-Whitney
non-parametric test).
Conclusions: Scleral crosslinking by the photosensitizer riboflavin
and ultraviolet A is an effective procedure to prevent axial elongation
induced by occlusion in a rabbit eye model.
Commercial Relationships: ASSAF DOTAN, None; Israel
Kremer, None; Arie Zigler, None; Dan Bourla, None
Vascular endothelial growth Factor A, C, D and Vascular
Endothelial Growth Factor Receptor 1, 2, 3 mRNA Expression in
the Chicken Retina, RPE and Choroid
Marita P. Feldkaemper, Frank Schaeffel, Lena Fuchs. Centre for
Ophthalmology, Institute for Ophthalmic Research, Tuebingen,
Germany.
Purpose: Several vertebrate species have been shown to be able to
change the rate of axial eye growth, as well as of choroidal thickness
to compensate for retinal image defocus. Hyperopic defocus induces
choroidal thinning in vivo. Vascular Endothelial Growth Factor A
(VEGFA) was suggested as one of the signals that induce choroidal
thinning in vitro (Sheng et al., 2012). The concentration of Vascular
Endothelial Growth Factor A (VEGFA) in the aqueous of highly
myopic patients was found to be lower than in emmetropes (Shin,
Nam et al., 2012), but the time courses of the changes in VEGFA
during myopia development is not known. We are planning to study
the role of VEGF in the chicken model but first it is necessary to
describe the expression of the different members of the VEGFs and
their receptors in the fundal layers of the chick eye.
Methods: Eyes from 3 weeks old chicks (n = 5) were hemisected and
the retinas, retinal pigment epithelium and choroid were carefully
dissected. Semi-quantitative real-time PCR was used to quantify
mRNA level of VEGFA (transcript variant 1 and 2), VEGFC,
VEGFD, VEGFR1, VEGFR2 and VEGFR3 in the fundal layers using
beta-actin as reference gene. PCR products were verified by
automated sequencing.
Results: In animals with normal visual exposure, VEGFA, VEGFC,
VEGFD, VEGFR1, VEGFR2 and VEGFR3 mRNA were expressed
in retina, retinal pigment epithelium and choroid, although the mean
normalised expression of VEGFR1 was very low in all tissues.
VEGFR3 mRNA expression was significantly higher in the choroid
compared to retina and RPE (ANOVA, p < 0.05).
Conclusions: This study shows that the mRNA of VEGF and VEGF
family receptors is expressed in the avascular retina of the chicken, as
well as retinal pigment epithelium and choroid. Furthermore,
quantification of its abundance is possible. Therefore, the regulation
of the VEGF signaling system can now be studied during myopia
development and pharmacological treatment.
Commercial Relationships: Marita P. Feldkaemper, None; Frank
Schaeffel, None; Lena Fuchs, None
Effects of dual-focus, multizone lenses on refractive development
in macaques
Baskar Arumugam1, 2, Li-Fang Hung1, 2, Juan Huang1, 2, Earl L.
Smith1, 2. 1College of Optometry, University of Houston, Houston,
TX; 2Vision CRC, Sydney, NSW, Australia.
Purpose: In several species, positive-lens-induced defocus has a
greater effect on early refractive development than unrestricted vision
or negative-lens-induced defocus, regardless of whether the two focal
conditions are viewed simultaneously or sequentially. The aim of this
study was to investigate how simultaneous, dual-focus lenses
influence eye growth and refractive development in infant rhesus
monkeys.
Methods: Starting at 3 weeks of age, infant monkeys were reared
with dual-focus, multizone spectacle lenses over both eyes. The
treatment lenses had central 2 mm zones of zero power and
concentric annular zones (Fresnel design) that had alternating powers
of +3.0 D and 0 D (n=7; +3D/pl) or -3.0 D and 0 D (n=7; -3D/pl).
The lenses were worn continuously until 150 ± 4.4 days of age.
Retinoscopy and A-scan ultrasonography were performed every two
weeks throughout the treatment period. Control data were obtained
from 33 normal monkeys.
Results: The +3D/pl lenses produced axial hyperopic shifts in all
seven of the treated monkeys. At the end of the lens-rearing period,
the +3D/pl monkeys were significantly more hyperopic than agematched normal monkeys (right eye medians: +5.25 D vs +2.41 D,
p=0.0002) and they had significantly shorter vitreous chamber depths
(average right eye: 9.31 ± 0.34 mm vs 9.84 ± 0.29 mm, p=0.0001).
On the other hand, at the end of treatment period, only one of the 3D/pl monkeys had developed a relative axial myopia. The other six 3D/pl monkeys exhibited ametropias that were either within the
normal range or slightly more hyperopic than normal. The refractive
errors (right eye median: +3.13 D, p=0.15) and vitreous chamber
depths (9.90 ± 0.60 mm, p=0.71) for the -3D/pl monkeys were not
different from those in normal monkeys.
Conclusions: In both groups of treated monkeys, refractive
development was dominated by the most positive-powered / least
negative-powered components of the dual-focus lenses. This pattern
of results is in agreement with previous findings from cylinder-lensreared monkeys and from chicks, guinea pigs and marmosets reared
with dual-focus lenses. Overall, the results of this study support the
idea that imposing relative myopic defocus over a large extent of the
retina would be an effective means for slowing ocular growth.
Commercial Relationships: Baskar Arumugam, None; Li-Fang
Hung, None; Juan Huang, None; Earl L. Smith, Ziess (P)
Support: NIH Grants EY03611 and EY07551; Vision CRC
Histo-pathological and functional degenerative changes of
Bruch’s membrane in form-deprivation myopia in chicks
Tzyy-Chang Ho. Ophthalmology, National Taiwan Univ Hospital,
Taipei, Taiwan.
Purpose: To determine the histo-pathological and functional changes
of Bruch’s membrane and adjacent retinal pigment epithelium(RPE)chorioretinal complex in a chick animal model.
Methods: White Leghorn chicks were raised for 2, 4, 6 and 8 weeks.
Myopia was induced in the right eye of first-day chick by wearing
translucent goggles. Histopathologic studies were performed on
equatorial and posterior pole areas by light microscopy and electron
microscopy. Immunohistochemical study was performed to detect the
elastic membrane. Cultured chick RPE cell binding to trephined
Bruch’s membrane-choroid-sclera eye cup buttons were measured by
MTT essay to determine the reattachment rates.
Results: There were no morphological changes in the equatorial and
posterior pole areas in the 2-, 4-, and 6-weeks groups. Elastic
membrane rupture was found in six out of eight chicks by 8 weeks in
the equatorial area but not the posterior pole. Immunohistochemical
study showed faint staining of the elastic membrane in the 8-weekold eyes compared to that in the control group. The reattachment
rates of chick RPE to equatorial Bruch’s membrane were 82.3 ± 4.5%
in the control group, and 78 ± 4.1%, 52.4 ± 8.3% ( P < 0.05), 43.8
±3.4 (P<0.05), and 28.6 ± 6.5% (P<0.05) respectively in 2-, 4-, 6- and
8-weeks groups.
Conclusions: Rupture of the elastic membrane, the first sign of
Bruch’s membrane degeneration was seen at 8 weeks in White
Leghorn chicks, and the reattachment efficiency of RPE to Bruch’s
membrane was decreased beginning 4 weeks after deprivation. Our
results indicated that the degenerative changes in the RPE binding
capacity-related extracellular matrix proteins on Bruch’s membrane
may serve as a chick animal model in the study of complications of
pathological myopia.
Commercial Relationships: Tzyy-Chang Ho, None
Effect of bright light on choroidal thickness in chickens as
measured with OCT
Weizhong Lan1, 2, Marita P. Feldkaemper1, Frank Schaeffel1.
1
Section of Neurobiology of the Eye, Ophthalmic Research Institute,
University of Tuebingen, Tuebingen, Germany; 2Zhongshan
Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Purpose: Bright light was found to be a powerful inhibitor of myopia
development in some animal models. We have studied whether its
effect may involve changes in choroidal thickness (CT).
Methods: Three day old male white leghorn chickens were raised in
temperature-controlled facilities under a 10/14 hour light/dark cycle
either in “normal light”( 500 human lux, from 8AM to 6PM, n=14),
or “bright light” (15,000 lux, 10AM to 4PM, n=14) for 5 days. CT in
the posterior pole of right eyes was measured in alert, hand-held
animals at 10AM, 4PM and 8PM every day, using small animal
optical coherence tomography (Spectralis HRA+OCT, Heidelberg
Engineering, Germany). Within-subject repeatability was high (SD
7.3µm or ± 4.4%; n=168, average CT=168.6µm). To uncover shortterm effects of bright light, data collected at 4PM and 8PM were
normalized to the individual CT data at 10AM every day. To
determine long-term effects, individual CT at 10AM at day 1 was
taken as baseline and changes between 10AM in day 1 and 10AM at
day 5 were determined. Changes were analyzed using one-way
repeated measures ANOVA and unpaired t-tests, respectively.
Results: Among 28 chickens, 2 could not be properly measured and
were excluded. In 3, one data point was lacking due to poor
cooperation. Thus, complete data were available only for 21 chickens
(9 in normal light and 12 in bright light). Immediately after bright
light was switched off at 4PM, CT decreased by -5.2%±4.0%
(mean±SEM). In contrast, in the normal light group, CT increased by
15.4%±4.7% (ANOVA: p=0.003). After further four hours, CT in the
bright light group increased again by 17.8%±3.5%, but showed little
further change in the normal light group (0.6%±4%; ANOVA,
p=0.004). After 4 days in bright light, CT was thicker than the normal
light group (7.6%±26.0% vs. -18.6%±26.9%, t-test: p=0.036),
indicating that CT became thinner over time in normal light.
Conclusions: CT responds to ambient light intensity. Bright light
modulated the diurnal CT cycling and increased CT over the 4 day
period as a long-term effect. Since thicker choroids were previously
shown to be associated with eye growth inhibition, it could be that
the increase in CT induced by bright light also mediates the inhibition
of myopia.
Commercial Relationships: Weizhong Lan, None; Marita P.
Feldkaemper, None; Frank Schaeffel, None
Support: Supported by DAAD PhD scholarship and National
Natural Science Foundation of China (Grant No. 81200714)
The efficacy of brief periods of “vision” in chick eyes wearing
negative lenses is dependent on time of day
Debora L. Nickla, Kristen Totonelly. Biosciences, New England
College of Optometry, Boston, MA.
Purpose: The influence of light cycles on eye growth has long been
of interest in the field of myopia, but the crucial parameters, and the
ocular mechanisms on which they impact, are as yet unknown. To
elucidate effects of phase, we exposed eyes to brief myopic defocus
at different times of day, and examined the effects on eye growth and
on the rhythms in axial length and choroidal thickness.
Methods: Chicks, aged 12-16 d, underwent one of the following
conditions for 5 -7 d. The light cycle was 12L/12D. Negative lenses:
Birds wore monocular -10 D lenses that were removed for 2 hrs daily
at 12 pm (n=6), 12 am (n=6), 5:30 am (“dawn”; n=5) or 7:30 pm
(“dusk”; n=6). Positive lenses: Birds wore +10 D lenses for 2 hrs at
12 pm (n=11) or 12 am (n=12). Normal eyes: Lights went on for 2
hrs at 12:00 am; controls were in normal 12L/12D. Ultrasonography
was done at the start and end for all. In some groups, eyes were
measured every 6 hrs over the course of 24 hrs on the last day.
Results: Negative lenses: 2 hrs of lens-removal at dusk was more
effective at growth inhibition than 2 hrs at dawn (830 vs 988 µm/7d;
p<0.05). There were no differences between the “mid day” and “mid
night” groups in growth rates, but the night exposure (12 am) had an
acute effect immediately following lens removal: growth rate
increased from 12 am to 6 am (X night vs controls and X day: 127 vs
0 and 56 µm; p<0.05 for both). The choroidal rhythm was phasedelayed (5 am vs 9:30 pm). Positive lenses: There was no difference
in eye growth between day vs nighttime myopic defocus (246 vs 251
µm/5d); both grew slower than fellow controls (p<0.001 for both).
Night defocus had similar effects on the rhythms in axial length and
choroid thickness as in the night “negative lens removal” group.
Normal eyes: 2 hrs of light at night inhibited ocular growth (615 vs
780 µm/7d; p=0.0001) and disrupted the choroidal rhythm; there
were no changes in thickness at any interval except 6 am-12 pm,
when choroids showed normal thinning.
Conclusions: Myopic defocus has a time-of-day-dependent efficacy
in inhibiting eye growth, and when it occurs at mid-night it alters the
rhythms in axial length and choroid thickness. These results have
implications for the current controversy over what attributes are
important in the myopia-inhibiting effects in children of time spent
outdoors.
Commercial Relationships: Debora L. Nickla, None; Kristen
Totonelly, None
Support: NIH-EY013636
The effect of two-zone concentric bifocal lenses on refractive
error and eye shape in guinea pigs
Hannah E. Bowrey1, Guang Zeng1, Amelia J. Leotta1, Christine F.
Wildsoet2, Sally A. McFadden1. 1The University of Newcastle,
Callaghan, NSW, Australia; 2The University of California, Berkeley,
Berkeley, CA.
Purpose: The phenomena of relative peripheral hyperopia in myopic
eyes has led to the idea that lenses which correct the peripherial
hyperopia may inhibit the progression of myopia. We studied the
changes in eye shape which occur in mammalian eyes wearing a
concentric bifocal lens which contained either plus or minus defocus
in its periphery.
Methods: Guinea pigs wore one of 5 lens types on one eye from 8
days of age for 12 days. Lenses were 14mm in diameter and
contained either a single power (-4D, 0D, or +4D, n=85) or dual
power with a 4mm plano center and minus or plus power in the
surround (0/-4D or 0/+4D, n=31). At the end of the lens-wear period,
refractive error was measured centrally and 30° off-axis in the
temporal and nasal fields under cycloplegia . Eyes were enucleated
immediately after death and rapidly frozen and sectioned in the
horizontal plane. Eye shape was analyzed from high-resolution
images of the section with maximum lens thickness and distances
calculated relative to the center of the lens axes.
Results: Eye shape analysis showed that -4D lens wear caused
elongation around the optic nerve (peri-papilliary zone, PPZ) with
relatively little change in the periphery. A similar reduced pattern
occurred with 0/-4D lens wear, showing that lenses containing
peripheral hyperopic defocus induce growth in the PPZ. +4D lenses
inhibited peripheral growth, particularly in nasal retina, but little
change occurred in the PPZ, suggesting that myopic defocus
predominantly influences peripheral eye shape. 0/+4D lenses caused
an identical inhibition in nasal retina, but there was significant
expansion in the PPZ, very similar to that seen with 0/-4D lenses.
Since single vision 0D lenses did not significantly change posterior
eye growth, it suggests that the PPZ is sensitive to peripheral lens
defocus of either sign when combined with a 0D central lens zone.
Conclusions: Regional sensitivity to defocus is differentially affected
by the sign of defocus. Minus lenses caused elongation in the PPZ
and plus lenses caused peripheral inhibition in eye shape. However,
when the center of an imposed lens contained no additional defocus,
the remaining peripheral power induced elongation in the PPZ
regardless of its sign. If human eyes act like guinea pig eyes, it
suggests that lenses with peripheral defocus will change eye shape
asymmetrically in the zone surrounding the optic disk.
Commercial Relationships: Hannah E. Bowrey, None; Guang
Zeng, None; Amelia J. Leotta, None; Christine F. Wildsoet, None;
Sally A. McFadden, None
CG120160 (SAM), NIH R01 EY12392 (CFW, SAM)
Comparing Rates of Emmetropization and Diurnal Rhythms
Before and After Goggle Removal in Chick
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 shown, in chicks recovering from either negative
or positive lenses, that the rapid reduction in spherical refractive error
is due in part to an increase in the amplitude of its sinusoidal
variation. Here we investigate whether this alteration occurs before or
after goggle removal. Short term changes in mean ocular refraction
(MOR) are measured immediately before and after goggle removal
following 6 days of emmetropization to lens induced myopia.
Methods: Twelve birds, unilaterally treated with a -15D goggle on
the day of hatching, were raised on a 14h/10h light dark cycle. On
day 6, at 8:30 am, 4 hourly Hartmann Shack and A scan ultrasound
axial length measurements began, ending on day 9. The goggle was
permanently removed after the day 7 830am measurement. MOR was
analyzed for the largest common pupil and slopes were taken over the
first 4 hours on days 6 and 7. Linear variations were subtracted and
residual sinusoidal variations were fitted before and after goggle
removal. Comparisons were made to previously presented results of
control birds. Paired t tests were used, p≤0.05 for significance.
Results: On day 6, sinusoidal amplitudes and periods of control and
goggled eyes did not differ significantly and goggled eyes were not
different from eyes of control birds on day 7. Acrophases on day 6
for control eyes were significantly clustered, and significantly
different from those of goggled eyes that were not significantly
clustered (Rayleigh test). On day 6, average MOR slopes for each of
goggled and control eyes did not differ from 0 or from each other.
Average initial slopes for day 7 (-0.08 D/Hr and 0.84 D/Hr for
control and treated eyes) were significantly different from each other.
Values for the control eyes did not differ from 0 or from day 6. The
immediate rate of emmetropization of the previously goggled eye
differs significantly from that prior to goggle removal.
Conclusions: After 6 days of unilateral emmetropization to a -15D
goggle, the rate of change of MOR approached 0 in both eyes. The
amplitude, period and phase of the diurnal variation of MOR in the
goggled eye did not differ from the control eye. The timing of the
first peak of the diurnal variation differed. On goggle removal, within
the first four hours, the direction of emmetropization changed
significantly. Sinusoidal changes appear to occur following goggle
removal.
Commercial Relationships: Melanie C. Campbell, CanCog
Technology (F), University of Waterloo (P); Kaitlin Bunghardt,
None; Marsha L. Kisilak, None; Elizabeth L. Irving, None
Support: NSERC Canada, CFI, CRC Canada
Temporal Frequency Sensitivity of the Emmetropization
Mechanism in Chicks to Color and Luminance Flicker without
Blue Light
Stephanie Britton, Molly Fellows, Frances J. Rucker. Biomedical
Science, New England College of Optometry, Boston, MA.
Purpose: Chicks were used to investigate the temporal sensitivity of
the emmetropization mechanisms to color and luminance flicker.
Previous experiments showed that exposure to 2 Hz luminance
flicker produced a hyperopic refractive shift associated with a
decrease in eye length and choroidal thinning, which disappeared
when the light source did not contain blue light. Color flicker caused
a myopic refractive shift, towards emmetropia, associated with an
increase in eye length and less choroidal thinning.
Methods: 4-5 day old chicks were exposed daily for three days (9am
to 5pm) to sinusoidal luminance (LUM) or color modulation (R/G) at
80% contrast, at one of six frequencies: 0, 0.2, 1, 2, 5, 10 Hz. Mean
illumination was 680 lux. Chicks were kept in the dark overnight.
LUM was created with in-phase modulation and R/G with counterphase modulation of red (615 nm) and green (515 nm) light. 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.
Results: LUM: With exposure to luminance flicker, there were
changes in refraction and eye length with temporal frequency. There
was a pronounced hyperopic shift in refraction at 5 Hz (1.38 D) and
10 Hz (1.34 D), but not at ≤2 Hz (2 Hz: -0.66 D; 0.2 Hz: -0.48 D;
Both p=0.03). The hyperopic shift in refraction at 10 Hz was
associated with a 191 μm increase in eye length compared to a 354
μm increase in eye length at 0.2 Hz; p=0.004). Choroids thinned
(range: -43 μm to -82 μm), but there was no significant change with
frequency.
R/G: With exposure to color flicker, there were changes in choroidal
thickness with temporal frequency. There was less choroidal thinning
at 0.2 Hz (-44 μm) than at 5 Hz (-82 μm; p=0.02). There was no
significant variation in eye length (range: 209-298 um) or refractive
error (± 0.50 D) with temporal frequency.
NF: With no flicker there was a 0.1 D change in refraction associated
with 346 ± 48 μm increase in eye length and 38 ± 12 μm choroidal
thinning.
Conclusions: The emmetropization mechanism shows a temporal
sensitivity to changes in color and luminance contrast that
differentially affect choroidal thickness and eye length. Without blue
light, higher temporal frequencies are necessary to produce the
hyperopic shift seen with luminance flicker.
Commercial Relationships: Stephanie Britton, None; Molly
Fellows, None; Frances J. Rucker, None
The effect of induced myopia on the scleral creep response of
whole chick eyes
Jacob A. Lewis1, Mariana Garcia2, Christine F. Wildsoet2.
1
Bioengineering & Materials Science and Engineering, UC Berkeley,
Berkeley, CA; 2Vision Science, University of California Berkeley,
Berkeley, CA.
Purpose: To determine the effect of induced form deprivation
myopia on the scleral creep response of intact chick eyes. Testing
involved pressurized intact globes as a more physiologically
representative model for testing the mechanical properties of the
sclera than traditional uniaxial tensile testing of scleral strips.
Methods: Diffusers were applied to one eye of 6 one-day old chicks,
while the fellow eye was left uncovered as a contralateral control.
Retinoscopy and A-scan ultrasonography were performed on days 1
and 5. After enucleation on day 5, each eye was cannulated via a 25G
needle passed through the cornea and lens into the vitreous chamber
of the eye, and connected externally to a column of phosphate
buffered saline (PBS) solution; the eye was also immersed in PBS.
Intraocular pressure (IOP) was adjusted by altering the height of the
column stepwise to 80 mmHg where it was held for one hour during
which time changes in eye shape were recorded at a sampling
frequency of 0.1 hz. Four 2 mm beads were glued to the sclera at the
equator and posterior pole, as an aid to visualizing scleral changes.
Image processing software was subsequently used to determine the
position of the beads in each photograph, to measure the deformation
of the posterior sclera over the course of the experiment and thus its
creep.
Results: After 5 days of form deprivation, the axial lengths of
deprived eyes had increased by an average of 0.44 mm compared to
0.04 mm for control eyes. The form-deprived eyes, which were also
myopic, exhibited significantly larger sclera creep compared to that
of control eyes. With IOP held at 80 mmHg, form-deprived eyes
elongated by 0.33% compared to 0.21% for control eyes (p < 0.05).
The scleral creep rates determined for the final half hour of testing
were 0.26 and 0.13 %/hour for myopic and control eyes respectively,
averaged across eyes (p < 0.05).
Conclusions: Myopic chick eyes exhibit a higher scleral creep rate
than control eyes when tested as intact globes. These results are
consistent with results from earlier studies involving uniaxial loading
of scleral strips.
Commercial Relationships: Jacob A. Lewis, None; Mariana
Garcia, None; Christine F. Wildsoet, None
Support: Rose Hills Foundation Summer Undergraduate Research
Fellowship, NIH Grant EY012392 to CFW
Inhibition in peripheral scleral lengthening during the
development of myopia in the guinea pig
Guang Zeng, Sally A. McFadden. School of Psychology, the
University of Newcastle, Callaghan, NSW, Australia.
Purpose: Human myopic eyes elongate posteriorly and show relative
peripheral hyperopia. Since hyperopic defocus induces myopia in
animal models, these peripheral changes in myopic eyes are thought
to cause the progression of central myopia. Since the sclera is
remodelled and thins in myopic eyes and its structural integrity
determines the shape of the eyeball, we studied the changes in the
length of the sclera during the development of form deprivation
myopia, to determine the relationship between central and peripheral
changes in ocular growth.
Methods: 31 guinea pigs were monocularly form deprived (FD) from
6 days of age for periods of 8, 14, 23 or 63 days in 4 different groups.
At the end of the FD period, eyes were enucleated, quickly frozen,
and cut on a freezing microtome in the horizontal plane 1. Eye shape
was analyzed from digital images of the frozen sections and the
perimeter lengths of the sclera were calculated for every degree.
1. Zeng G, McFadden SA. The development of eye shape and the
origin of lower field myopia in the guinea pig eye. Vision Res.
2012,76:77-88.
Results: Myopic ocular elongation was specifically centered on
either side of the optic nerve exit (the peripapillary zone or PPZ) with
rapid expansion in the length of the sclera after 8 days of FD in the
PPZ (increase of +5.3 μm/deg) accompanied by smaller increases in
the length of the sclera in the periphery, particularly in nasal retina
(+1.5 μm/deg). Absolute peripheral inhibition in scleral length started
in the second week of FD, after the initial elongation in the PPZ, and
was present throughout the periphery. After 3 weeks of FD, shrinkage
was present in 6/8 animals (mean, -1.3 μm/deg). However, the
relative difference between center and periphery increased over time.
By 9 weeks of FD, the sclera length had increased further in the PPZ
(to +9.0 μm/deg ) so that the difference between the PPZ and the
periphery was now maximised.
Conclusions: During the development of myopia, the sclera in the
peri-papillary zone surrounding the optic nerve rapidly expanded, and
this expansion occurred prior to inhibition in the length of the sclera
in the periphery, suggesting that a weakened sclera in the PPZ may
induce the peripheral changes. Conversely, the subsequent inhibition
in peripheral sclera growth was followed by lengthening in the sclera
in the PPZ and this may be the source of the subsequent progression
of myopia.
Commercial Relationships: Guang Zeng, None; Sally A.
McFadden, None
CG120160 (SAM)
Luminance variations can reduce or reverse plus lens
compensation in chicks
Alan G. Busby. Biology, City College of New York, New York, NY.
Purpose: Luminance varies significantly in the natural environment,
often by 5-6 log units or more. Published experiments have shown
that bright lights can alter the expected emmetropization responses in
animals (Ashby & Schaeffel, IOVS, 2010; Smith et al, IOVS, 2012).
It was hypothesized that a visual scene’s brighter areas would affect
emmetropization more than its dimmer areas. The experiments below
tested whether an environment’s luminance variations could alter
chick lens compensation.
Methods: 1-week-old Chicks (8 birds/group with 2 birds/cage) wore
a +3D lens on one eye and an occluder on the other in a cylindrically
shaped cage (18cm x 12cm high), so that all objects inside the cage
had hyperopic defocus while objects outside had myopic defocus.
50% of the cage’s walls were covered with 4 equidistant 7cm wide
paper strips, and 4 equal open sections permitted distance views of
0.5 to 3m. The experimental cage had a 5000 lux near light (a 50 watt
desk lamp 12cm from the cage floor), while the outside was
illuminated with ~60 lux, ~140 lux, or typical overhead fluorescent
lighting (~600 lux). The control cage had no near light but equal
outside illumination. One eye, with the lens, viewed the experimental
cage for 1hr. The lens and occluder were then switched, and the
fellow eye viewed the control cage through the lens for 1hr. Chicks
had 2 exposures per condition on one day, and 2 more the following
day over 24hrs.
Results: The control eyes became hyperopic as expected with +3D
lenses. However, the experimental eye’s hyperopic responses were
statistically significantly reduced or reversed in the experimental eye
for all three background light levels (~60, ~140, ~650 lux), based on
a thinner choroid (-69±12 vs. 6±17, -53±12 vs. -4±20, 31±16 vs.
64±16µm), larger vitreous chamber depth (86±18 vs. 2±29, 55±23 vs.
-11±17, -27±19 vs. -99±12µm), and more negative refraction (1.59±0.53 vs. +1.19±0.64, -1.85±0.58 vs. +0.12±0.9D, not tested).
Axial changes were not expected due to the short time period.
Conclusions: A visual scene’s brighter areas seem to affect lens
compensation more than darker areas, even when light levels are
comparable to common environments. Plus lens compensation was
inhibited or even reversed when the brightest areas had hyperopic
defocus. This phenomenon may help explain some of the seemingly
contradictory results between human epidemiological and animal
emmetropization studies.
View from inside cage
Commercial Relationships: Alan G. Busby, None
Support: NIH 1K08EY014245
Effects of a human anti-VEGF antibody (Bevacizumab) on
myopia development and choroidal thickness in the chicken
Frank Schaeffel1, Ute Mathis1, Focke Ziemssen. 1Section
Neurobiology of Eye, Ophthalmic Research Institute, Tuebingen,
Germany; 2Center of Ophthalmology, University of Tuebingen,
Tuebingen, Germany.
Purpose: Intravitreal injection of inhibitors of vascular epithelial
growth factor (VEGF) represent currently the most promising
treatment option for age related macular degeneration and myopic
choroidal neovascularization. Bevacizumab has also been shown to
affect the ERG, choroidal blood vessel leakage and turtuosity in
rabbits. Sheng, Zhu and Wallman (ARVO 2012) found that VEGF
isoform V165 can transiently thin the choroid in chicken in vitro. We
have studied whether Bevacizumab can affect myopia development
and choroidal thickness also in vivo.
Methods: 24 chickens, 10 days old, were studied. After a single
intravitreal injection containing either 625µg Bevacizumab, or
vehicle alone, refractions and ocular dimensions were followed by
infrared photoretinoscopy and A-scan ultrasonography, respectively,
and daily OCT was performed to track choroidal thickness. Three
experiments were done: (1) one eye Bevacizumab, the other vehicle,
both covered with diffusers for 4 days (2) one eye Bevacizumab, the
other vehicle, and both normal vision and (3) both eyes
Bevacizumab, but only one eye covered with a diffuser for 4 days.
Results: (1) A single injection of Bevacizumab reduced the amount
of deprivation myopia (Bevacizumab -3.1+-2.7 D, vehicle -5.6+-2.7
D, p<0.02) and suppressed choroidal thickening that normally occurs
when eyes recover from myopia on both sides (choroidal thickness in
vehicle injected animals on the third day of recovery 925+-90 µm; in
Bevacizumab treated eyes 294+-54 µm, and 377+- 64 µm in vehicle
injected fellow eyes; p<0.001 in both cases) (2) Bevacizumab showed
a trend of reducing choroidal thickness in animals with normal vision
over a period of 8 days, relative to vehicle injected fellow eyes (3)
However, binocular injections of Bevacizumab did NOT reduce
choroidal thickness and myopia in a third experiment, compared to
binocularly vehicle-injected animals.
Conclusions: In the first experiment, a single injection of
Bevacizumab had long-lasting and powerful effects on choroidal
thickening and myopia, even though Bevacizumab is an antibody
raised against the human VEGF receptor. It remains unclear why the
effect was weak or absent in the two other experimental paradigms.
Potential causes might be species-specifity of the antibody or variable
impact of local VEGF concentrations and isoforms, calling for more
detailed studies in the future.
Commercial Relationships: Frank Schaeffel, None; Ute Mathis,
None; Focke Ziemssen, Novartis (C), Bayer Healthcare (C), Alcon
(R)
Anti-Diuretic Hormone Arginine-Vasopressin Promotes an
Increased Myopic Shift in Refractive Compensation to Optical
Defocus in Physiologically Stressful Environmental Light
Conditions
Melanie J. Murphy1, Sheila G. Crewther1, Sarah N. Kiely1, Nina
Riddell1, Loretta Giummarra1, David P. Crewther2. 1School of
Psychological Science, La Trobe University, Melbourne, VIC,
Australia; 2Centre for Human Psychopharmacology, Swinburne
University of Technology, Melbourne, VIC, Australia.
Purpose: Arginine-Vasopressin (AVP) is a naturally occurring antidiuretic hormone that acts to control osmolarity and body fluid
volume. AVP has been localized to the inner retina, reduces IOP and
is associated with retinal AQP-4 expression (ARVO 2009). Thus we
aimed to determine whether AVP’s anti-diuretic action would affect
refraction and ocular growth compensation to optical defocus
especially in the presence of asymmetric flicker, a physiologically
stressful stimulus of environmental origin.
Methods: Chicks were raised from days 5-9 post-hatching in one of
3 lens (+, -10D or No Lens) and in one of 3 light conditions (a
Normal Diurnal 12 hr day/night cycle or either Fast ON/Slow OFF or
Fast OFF/Slow ON sawtooth flicker generated by a light emitting
diode array at 1 Hz) following a single intravitreal injection of 5μl of
either PBS alone or as the carrier solution for Vasopressin (1.84 x 104 mM) or the dual V1/V2 Vasopressin receptor antagonist ([desGly9-β-Mercapto-β, β-cyclopentamethylenepropionyl1, O-Et-Tyr2,
Val4, Arg8]-Vasopressin) (5.53 x 10-4 mM). Retinoscopy and Ascan ultrasonography was performed on Day 9.
Results: AVP-injected, -10D lens-wearing eyes all showed a myopic
shift as expected of an anti-diuretic agent in all light conditions, as
did the +10D eyes reared under Fast ON flicker. In comparison, the
AVP antagonist prevented the myopic shift typically induced by
flicker conditions in the +10D lensed eyes. Changes in refraction
were closely mirrored by changes in posterior eye growth. Significant
interaction effects between drug, light and lens conditions were
observed for refractive state, axial length and vitreous chamber depth,
while more variable growth patterns were observed for anterior
chamber depth.
Conclusions: The finding that the natural anti-diuretic hormone,
AVP, but not its antagonist, increased myopia and axial elongation in
the presence of negative defocus highlights the importance of the
interaction between modulation of fluid dynamics and responses to
light-induced environmental stress in ocular growth and refractive
compensation.
Commercial Relationships: Melanie J. Murphy, None; Sheila G.
Crewther, None; Sarah N. Kiely, None; Nina Riddell, None;
Loretta Giummarra, None; David P. Crewther, None
The expression of vaoactive intestinal peptide receptors and
ZENK protein in form-deprivation myopia
Hikmet Basmak1, Huseyin Gursoy1, Ayse Idil Cakmak1, Nilgun
Yildirim1, Nese Tuncel2, Nilufer Erkasap2, Mete Ozkurt2. 1Dept of
Ophthalmology, Eskisehir Osmangazi Univ Med Sch, Eskisehir,
Turkey; 2Physiology, Eskisehir Osmangazi Univ Med Sch, Eskisehir,
Turkey.
Purpose: Many candidate molecules, including vasoactive intestinal
peptide (VIP), have been proposed to be involved in the myopia
patho-physiology. Our aim was to investigate the expression of
mRNA of VIP receptors and ZENK protein on the retina and choroid
of form-deprived myopic chick eyes and the effects of intravitreal
VIP injection on these receptors and protein.
Methods: Three groups of eight white Leghorn chicks were included.
They wore a unilateral translucent hemispherical plastic diffuser to
induce myopia in their right eyes. Group 1 was the control group that
received nothing. The intravitreal injections of 10 μl of saline and 10
μl of VIP (0.5 ng/μl) were applied in group 2 and 3 respectively
every 24 hours for seven days. On the first and eight day of
experiment the refraction was assessed with retinoscopy.
On the eight day of the experiment the chicks were sacrificed. The
mRNA levels of VIP1 and VIP2 receptors and ZENK protein on the
retina and choroid of the right eyes, in relation to the housekeeping
gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was
determined using real time PCR (RT-PCR) with Taqman prob. Total
RNA was extracted from retinal tissue by the RNA stabilization
reagent and quantified by measuring the absorbance at 260 nm. RTPCR was performed by monitoring in real time the increase in the
amount of Taqman prob using Rotor-Gene 6000 RT-PCR. RT-PCR
data were collected using the Rotor-Gene 6000 detection system.
Cycle threshold (CT) values were determined by automated threshold
analysis.
Results: The mean final refraction (D) detected in the right eyes was
-13.5±2.7, -9.9±3.6 and -1.8±2.8 in group 1, 2 and 3 respectively.
The final myopia obtained was significantly lower in VIP injected
eyes (p<0.05). mRNA levels for VIP1 receptors were undetectable.
The mean delta-delta CT for VIP2 receptors was 1.11±0.16,
1.37±0.20 and 0.36±0.08 in group 1, 2 and 3, respectively (p=0.013
for group 1 vs 3 and p=0.002 for group 2 vs 3). The mean delta-delta
CT for ZENK protein was 2.48±1.58, 3.64±0.65 and undetectable in
group 1, 2 and 3, respectively (p=0.059 for group 1 vs 3 and p<0.001
for group 2 vs 3).
Conclusions: Prevention of experimental myopia by exogenous VIP
seems to be mediated by down-regulating the expression of VIP2
receptors and ZENK protein, indicating that it may be a promising
agent for the treatment of myopia.
Commercial Relationships: Hikmet Basmak, Eskisehir Osmangazi
University (F); Huseyin Gursoy, Eskisehir Osmangazi University
(F); Ayse Idil Cakmak, Eskisehir Osmangazi University (F); Nilgun
Yildirim, None; Nese Tuncel, ESOGÜ (S); Nilufer Erkasap,
ESOGU (S); Mete Ozkurt, ESOGÜ (S)
Support: Eskisehir Osmangazi University 2011.11034 BAP project
Effects of Adenosine A2A Receptor on the Development of FormDeprivation Myopia in Mice
Jianhong An1, Xiangtian Zhou1, Fanjun Shi1, Jiangfan Chen1, 2, Jia
Qu1. 1School of Ophthalmology & Optometry, Wenzhou Medical
College, Wenzhou, Zhejiang, China; 2Department of Neurology,
Boston University School of Medicine, Boston, MA.
Purpose: We have reported that the A2A receptor knockout mice
confer a relatively myopia during the first two months of postnatal
development. The present studies seek to evaluate the role of the
adenosine A2A receptor in form deprivation myopia (FDM) using
A2A KO mice.
Methods: The right eye of A2A KO mice and WT littermates
(postnatal days 23) were subjected to form deprivation to induce
myopia. Ocular parameters were measured at 4 and 6 weeks after
form deprivation. Refraction was measured by eccentric infrared
photorefraction (EIR). Corneal radius of curvature was evaluated by a
keratometer with a + 20.0 D aspherical lens mounted before it.
Ocular dimensions (including anterior chamber depth, lens thickness,
vitreous chamber depth and axial length) were measured by a
custom-designed optical coherence tomography.
Results: In WT mice, after form deprivation for 4 and 6 weeks, the
treated (T) eyes showed significant myopic shift compared with their
fellow (F) eyes (4 w: T: 2.23±1.43D, F: 9.13±1.22D, P<0.001; 6 w:
T: 3.06±1.92D, F: 8.28±1.51D, P<0.001) and the myopic shift was
correlated with the increase in axial length (4 w: P<0.001; 6 w:
P<0.05) and increased vitreous chamber depth (4 w: P<0.01; 6 w:
P<0.01), and no significant difference was found in corneal radius of
curvature. In A2A R KO mice, after form deprivation for 4 and 6
weeks, the treated (T) eyes showed significant myopic shift compared
with their fellow (F) eyes (4 w: T: -0.39±1.63D, F: 5.77±1.73D,
P<0.001; 6 w: T: -1.69±1.48D, F: 7.29±1.43D, P<0.001). However,
there was significant difference in corneal radius of curvature after
form deprivation for 4 and 6 weeks (4 w: T: 1.432±0.023mm, F:
1.440±0.030mm, P<0.05; 6w:T:1.465±0.027mm, F:1.475±0.026mm,
P<0.001) and no significant difference in ocular axial length, vitreous
chamber depth, anterior chamber depth, and lens thickness between T
eyes and F eyes in all groups was found.
Conclusions: Genetic inactivation of the A2A receptor can induce
myopia accompanied with corneal radius of curvature changes
compared with WT mice by form deprivation, suggesting that
adenosine A2A receptor have a role in development form deprivation
myopia in mice.
Commercial Relationships: Jianhong An, None; Xiangtian Zhou,
None; Fanjun Shi, None; Jiangfan Chen, None; Jia Qu, None
Support: Wenzhou Science and Technology Projects Y20100206,
NSFC 81000386
521 Ocular and Orbital Tumors
Thursday, May 09, 2013 8:30 AM-10:15 AM
Program #/Board # Range: 5874-5899/D0237-D0262
Malignant Cancer After Trauma or Presumed Trauma in Cats
Richard R. Dubielzig. Pathobiol Sciences, Univ of WisconsinMadison, Madison, WI.
Purpose: To use the archives of a large collection of comparative
ocular pathology to catalogue the predisposing factors, presenting
characteristics, and morphologic variants of feline ocular posttraumatic sarcoma (FOPTS).
Methods: The database of the Comparative Ocular Pathology
Laboratory of Wisconsin (COPLOW) were searched and records of
FOPTS were examined. The signalment, age at enucleation, age at
trauma, type of trauma, and the morphologic variant of tumor were
recorded and compared.
Results: 325 cases of FOPTS were identified and studied. The
spindle cell variant made up (67%) of the cases, followed by the
round-cell variant (21%), and the osteosarcoma / chondrosarcoma
variant (10%). Males accounted for 67% of cases. 81 cases (25%)
listed trauma as the original disease process and all cases (100%) had
a history of chronic eye disease. The average time between trauma
and enucleation was 6.2 years.
Conclusions: There are three morphologic variants of FOPTS:
spindle-cell, round-cell, and osteosarcoma/chondrosarcoma. All of
the variants present after a history of chronic eye disease. A historic
traumatic event was often specified in the distant past but most cases
had a history consistent with this scenario even if the event was not
specified. Males are over represented in the series
Commercial Relationships: Richard R. Dubielzig, OSOD, LLC
(I), Allergan (C)
Canine Primary Ocular Osteosarcomas and Chondrosarcomas
Felicia D. Duke, Richard R. Dubielzig. Pathobiological Sciences,
University of Wisconsin, Madison, WI.
Purpose: To describe a case series of primary ocular osteosarcomas
and chondrosarcomas in dogs.
Methods: The COPLOW database was mined for cases of primary
osteosarcomas and chondrosarcomas. Cases were reviewed and
described, and additional clinical and follow-up data were collected
from submitting veterinarians where available.
Results: There are 4 cases of canine primary osteosarcoma and 5
chondrosarcomas in the COPLOW collection. The average age at
enucleation is 10.6 years old (range 6-15), and 8 of the 9 cases are in
male dogs. There are 2 Golden retrievers, 2 Rottweilers, and 1 each
Blue heeler, Collie, Staffordshire bull terrier, Maltese, and Cocker
spaniel. One case is bilateral. Increased intraocular pressure,
hyphema, and uveitis are the most common presenting complaints.
Histologically the tumors are comprised of variable amounts of
spindle cells that carpet the ciliary body epithelium, and variable
amounts of extracellular matrix consistent with either osteoid or
cartilaginous matrix. The carpeting effect is variably extensive
among the cases but the pattern of lining the ciliary body epithelium
is consistent. Nine of the 10 eyes have thick preiridal fibrovascular
membranes. None of the dogs have a known history of skeletal
lesion, or a clinical history suggesting one. Four dogs died or were
euthanized with suspected or confirmed metastatic disease, a fifth
died of “old age.” No follow-up data were available for the remaining
cases. Normal canine globes contain no bone or cartilage, thus the
cell of origin in these cases is unclear.
Conclusions: We propose that these cases represent primary ocular
neoplasms. Canine primary osteosarcomas and chondrosarcomas
frequently present with hyphema, uveitis, and increased intraocular
pressure in older dogs. There is a possible male predilection.
Commercial Relationships: Felicia D. Duke, None; Richard R.
Dubielzig, OSOD, LLC (I), Allergan (C)
AJCC TNM COLLABORATIVE INTERNET STAGING
VALIDATION FOR OPHTHALMIC TUMORS
Priya Selvakumar. 1The Ophthalmic Oncology Task Force, American
Joint Committee on Cancer, Chicago, IL; 2Health informatics
research, Princess Margaret Cancer Centre, Toronto, ON, Canada.
Purpose: The current 7th edition of American Joint Committee on
Cancer - International Union Against Cancer (AJCC-UICC) tumor,
node, metastasis (TNM) staging for ophthalmic tumors is largely
based on the consensus of 46 eye cancer specialists from 11
countries, since there is a paucity of evidence-based clinical trials in
ocular oncology. In order to prepare for the 8th edition, we developed
a collaborative internet survey evaluating the prognostic accuracy for
the 7th edition.
Methods: Data fields were designed by multicenter clinical
collaboration. Web-based algorithms were constructed to calculate
cancer-specific TNM stage, based on ocular and systemic extent of
disease. The accuracy of the algorithms was authenticated by the
participating clinican-scientists through a demo online survey.
Research features not yet established in treatment were included.
Ongoing, after each participating center has obtained institutional
ethics approval, they enter data anonymized data on all relevant
patients treated in their institution over a 10 year period. Statistical
analysis is applied to the collective multicenter data.
Results: The algorithms include all data points required to calculate
the TNM stage, including tumor site and dimensions at initial
presentation, lymph node involvement, pathological grading,
biomarkers, systemic metastasis, and research biomarkers and broad
treatment categories and outcome at last follow-up. In the ongoing
study of uveal melanoma, participating centres have entered more
than 2529 uveal melanoma patients. The demo online surveys for
retinoblastoma, conjunctival melanoma and conjunctival carcinoma
are under construction.
Conclusions: The multicenter collaboration using the internet and
TNM staging algorithms is collecting accurate data and outcomes
with efficiency, ready for analysis. International collaboration
provides large numbers of patients. The assessment of the 7th edition
TNM staging to accurately predict outcomes will be used to improve
the prognostic capability of the 8th edition.
Commercial Relationships: Priya Selvakumar, None
Support: The Eye Cancer Foundation, Inc and a Bioinformatics
Grant from John and Myrna Daniels
Prognostic Factors Associated with Histopathology in Intraocular
and Ocular Adnexal Lymphoma: A Twenty Years Review for the
University of Wisconsin Madison
Pimkwan Jaru-ampornpan1, Amir Azari1, Mozhgan Rezaei Kanavi1, 2,
Heather Potter1, Daniel M. Albert1. 1Ophthalmology and Visual
Sciences, University of Wisconsin, Madison, Madison, WI;
2
Ophthalmic Research Center, Shahid Beheshti University of Medical
Sciences, Tehran, Islamic Republic of Iran.
Purpose: To investigate the prognostic factors in ocular and ocular
adnexal lymphomas that correlates with histological findings.
Methods: After obtaining approval from institutional review board
(IRB) committee, a retrospective review of clinical and histologic
findings for patients with diagnosis of ocular and/or ocular adnexal
lymphoma was conducted at the University of Wisconsin School of
Medicine and Public Health between 1992 and 2012. Data examined
included patient’s age, gender, presenting signs and symptoms, tumor
location, tumor laterality, clinical diagnosis, histological diagnosis
and length of survival.
Results: Pertinent clinical and histopathological data was collected
on 67 eyes of 60 patients. The average age at diagnosis was 64 ± 16
years (range 14 - 94 years) with a female: male ratio of 1.2. Overall,
the total five-year mortality for the ocular lymphomas regardless of
location or histological diagnosis was 36% in our study. In regards to
tumor location, the intraocular lymphomas carried the worst
prognosis with a five-year mortality rate of 100 % in our series
(N=8), while the orbital lymphomas carried the best prognosis with a
mortality rate of 12% (N=17). In our case series patients with
histological diagnosis of large cell lymphoma had a five-year
mortality rate of 83% while those with small cell lymphoma
(mortality rate of 25%), follicular lymphoma (0% mortality), and
mucosa-associated-lymphoid-tissue (MALT) lymphoma (0%
mortality) carried a far better prognosis.
Conclusions: After reviewing the experience of a single institution
serving a large geographic region of the Midwest, we discovered that
the location and the type of lymphoma were similar to those
previously reported in the literatures. Patient's prognosis and
mortality were influenced by tumor location and tumor histology. In
addition, there has not been any significant change in patient’s
survival in the recent years with current treatment regimens.
Commercial Relationships: Pimkwan Jaru-ampornpan, None;
Amir Azari, None; Mozhgan Rezaei Kanavi, None; Heather
Potter, None; Daniel M. Albert, None
Uveal Lymphoma: Comparison of Diagnostic Imaging Studies
Mary E. Aronow1, Craig Portell2, John Sweetenham2, Arun D.
Singh1. 1Cole Eye Institute, Cleveland Clinic, Shaker Heights, OH;
2
Hematologic Oncology and Blood Disorders, Cleveland Clinic,
Taussig Cancer Institute, Cleveland, OH.
Purpose: To compare imaging results from fluorescein angiography
(FA), indocyanine green angiography (ICG), B-scan ultrasonography,
computed tomography (CT), and magnetic resonance imaging (MRI)
in patients with uveal lymphoma.
Methods: Data were collected regarding patient characteristics,
clinical features on ophthalmic examination, and ancillary imaging
studies in 16 patients (25 affected eyes) diagnosed with uveal
lymphoma between 1997 and 2012.
Results: 12 patients were male (75.0%) and the median age at
diagnosis was 67.5 years. Choroidal involvement was present in 15
(93.7%) and ciliochoroidal disease was present in 1 (6.3%) case.
Lymphoma of overlapping ocular adnexal structures was observed in
8 cases (50.0%) including: the conjunctiva in 4 (25.0%), the orbit in 3
(17.7%), and conjunctiva and orbit in 1 (6.3%) case. Bilateral disease
was present in 9 cases (56.3%). The most characteristic clinical
examination finding was yellow-white choroidal infiltrates, observed
in 25 (100.0%) eyes. Infiltrates were located anterior to the arcades
(84.0%), most commonly in a diffuse (32.0%) or superotemporal
(32.0%) distribution. FA yielded a variety of patterns including early
hyperfluorescence (84.6%), hypofluorescent lesions (15.4%),
choroidal folds (38.5%), and normal angiographic findings (7.7%). In
contrast, ICG demonstrated hypofluorescent lesions corresponding to
clinically observed infiltrates in 100% of cases. B-scan
ultrasonography detected extrascleral extension (ESE) in 70% of
eyes. In such cases, CT orbit detected 25.0% and MRI orbit detected
75.0% of eyes with ESE.
Conclusions: Ancillary imaging is essential for evaluating the full
extent of ocular involvement and for treatment planning in
individuals with uveal lymphoma. ICG is a superior imaging study
than FA to assess choroidal lymphoma. B-scan ultrasonography is
more sensitive in detecting ESE than either CT or MRI of the orbit.
Commercial Relationships: Mary E. Aronow, None; Craig
Portell, None; John Sweetenham, None; Arun D. Singh, None
Support: Research to Prevent Blindness Challenge Grant,
Department of Ophthalmology, Cleveland Clinic Lerner College of
Medicine
Clinical and epidemiologyc features of lymphoproliferative
lesions in the orbital and adnexal region at the Asociación Para
Evitar la Ceguera en México
Linda Guakil1, Guillermo Salcedo1, Abelardo A. Rodríguez-Reyes2,
Rosa Isela Rubio1. 1ORBIT AND OCULOPLASTICS,
ASOCIACION PARA EVITAR LA CEGUERA EN MEXICO,
MEXICO, Mexico; 2PATHOLOGY, ASOCIACION PARA EVITAR
LA CEGUERA EN MEXICO, MEXICO, Mexico.
Purpose: Lymphatic neoplasms are tumours derived from lymphatic
ganglia or extraganglionar tissues that arise from lymphocytes and its
precursor cells expressing B or T phenotype. Lymphomas are
classified as Hodgkin and non- Hodgkin and then subdivided. Ocular
adnexal lymphomas develop as primary or secondary tumours in the
conjunctiva, lid, orbit, lacrimal gland, or lacrimal sac. Most patients
presents with localized disease. Being an ophthalmology reference
center, it is important to know the epidemiology of this disease, so
the purpose of this study is to evaluate lymphoproliferative lesions of
the orbital and adnexal region diagnosed at Asociación Para Evitar La
Ceguera en México in a period of 10 years, describe its epidemiology
and relationship with the literature, characterize its
clinicopathological features and to present clinical cases to exemplify
it.
Methods: Patients diagnosed with lymphoproliferative lesions in the
ocular adnexa in the years 2002 to 2012 were included. We made a
retrospective, observational, descriptive analysis, using the SPSS
statistical Package
Results: 74 cases were identified. 45 Female and 29 Male patients
with median age of 60 years. Most common presenting symptoms
were increase of volume (64.38%) and proptosis (10.96 %). Only
27.12 % presented pain. Pathologic and immunohistochemistry
diagnosis coincided in 96.3% of cases.
All specimens represented B cell non-Hodgkin’s lymphomas:
extranodal marginal zone lymphoma (EMZL) and MALT (n=39),
diffuse large B cell lymphoma (n=10), mantle cell lymphoma (n=5),
follicle centre lymphoma (n=4), others (n=5). Lymphoid hyperplasia
in 11 cases. The orbit (49.32 %) and conjunctiva (35.62%) were the
most commonly affected sites. Bilateral involvement in 16.44% of
cases. The mayority of patients (76.47%) were sent to a cancerology
institute for follow up. The main form of treatment was radiotherapy.
Conclusions: In our experience lymphoproliferative lesions in the
orbital and adnexal region are most commonly found in elderly
women coinciding with the literature. Orbit and conjunctiva are the
most frequently involved sites, this shows some variation in the
literature, weret the most common sites are the orbit and eyelid. We
found a mayority of low grade b cel lymphoma wich also
corresponds with the literature. We found an increase of incidence
over time.
Commercial Relationships: Linda Guakil, None; Guillermo
Salcedo, None; Abelardo A. Rodríguez-Reyes, None; Rosa Isela
Rubio, None
The Usefulness of Cell Block Technique in Cytological diagnosis
of Primary Intraocular Lymphoma
Takako Ito1, HIroshi Yoshikawa1, Atsunobu Takeda1, Takeru
Yoshimura1, Sayaka Hirakawa1, Koh-hei Sonoda2, Takaomi Koga3,
Tatsuro Ishibashi1. 1Ophthalmology, Kyushu University, Fukuoka,
Japan; 2Ophthalmology, Yamaguchi University, Yamaguchi, Japan;
3
Pathology, Kyushu University, Fukuoka, Japan.
Purpose: The diagnosis of primary intraocular lymphoma (PIOL) is
difficult in many cases with vitreous smear preparation. We here in
report the usefulness of cell block technique in patients with PIOL.
Methods: Retrospective review was conducted of 23 PIOL patients
(13 women, 10 men ; 25 eyes, average 63.9 years old) who had
vitreous biopsy in Kyushu University Hospital from January 2001 to
December 2011. Undiluted vitreous specimens were obtained at the
time of vitrectomy, centrifuged, then supernatants and pellets were
subjected to ELISA (interleukin (IL) -6 and IL-10) and
Giemsa/Papanicolaou stains, respectively. Irrigation fluid was
collected from cassette/tubing/drain bag at the end of vitrectomy,
centrifuged, fixed, and then embedded in paraffin for cell block. Thin
sections were stained with hematoxylin-eosin and by
immunochemistry. Final diagnosis of PIOL was made when atypical
lymphoid cells were detected from either smear preparation or cell
block, concurrent with clinical findings and IL-6/IL-10 ratio.
Results: Atypical lymphoid cells were detected in 44% (11 eyes in
25 eyes) with smear preparation, while 76% (19 eyes in 25 eyes) with
cell block.
Conclusions: Cell block technique is useful for the diagnosis of
PIOL.
Commercial Relationships: Takako Ito, None; HIroshi
Yoshikawa, None; Atsunobu Takeda, None; Takeru Yoshimura,
None; Sayaka Hirakawa, None; Koh-hei Sonoda, None; Takaomi
Koga, None; Tatsuro Ishibashi, None
Distinct Profiles of Vitreous micro RNA in Primary Vitreoretinal
Lymphoma and Uveitis
Jingsheng Tuo, DeFen Shen, Chi-Chao Chan. Laboratory of
Immunology, National Eye Institute/NIH, Rockville, MD.
Purpose: Primary vitreoretinal lymphoma (PVRL), a subset of
primary CNS lymphoma, affects the retina, vitreous, and optic nerve
head, with the most common symptoms of blurred or decreased
vision. Because the clinical appearances overlap heavily with
inflammatory and infectious uveitis, PVRL is often masqueraded as
uveitis and treated initially with corticosteroids, which may further
mystify the physicians. Recently, studies have unveiled the high
stability of miRNA in biofluids and the correlation of miRNA
expression profiles with diseases and disease states, which have
positioned extracellular miRNA quantification in biofluids as a
promising tool for diagnostic applications.
Methods: The study included 11 B-cell PVRL and 13 uveitis
patients. Vitreous specimens were obtained through a standard three
port pars plana vitrectomy. The diluted and undiluted vitreous
supernatant was pooled and subjected to total RNA extraction using a
commercial column-based system (Qiagen miRNeasy® Mini Kit).
cDNA was synthesized by using mature miRNA as templates with
miRCURY LNA™ Universal RT cDNA Synthesis Kit (Exiqon A/S).
Six selected samples (PVRL n=3, uveitis n=3) were arrayed by a RTPCR-based microRNA panel that detects 168 human mature
miRNAs. The markers promising in distinct levels between uveitis
and lymphoma were further tested by individual RT-PCR analysis.
Results: Of 168 human mature miRNAs assayed by RT-PCR array,
29.8% (50 miRs) were detectable in vitreous samples. The miR-484,
miR-197, and miR-132 were consistently higher in the PVRL
vitreous, while miR-155, miR-200c, and miR-22* consistently higher
in the uveitis vitreous. The results were normalized by different
combination of 7 control miRs (miR-103, miR-191, miR-42-5p, miR-
16, miR-425, miR-93 and miR-451) as recommended by the
manufacture. After optimization, the normalization against miR-16
showed equally reliable as the average of 7 control miRs. Individual
assay for the selected miRs did not succeed in other 18 samples but
the results support the pattern yielded from the array analysis.
Conclusions: Mature miRs are detectable in vitreous samples. B-cell
PVRL and uveitis might be characterized with distinct miR
signatures. Assay of selected miR might be helpful in the differential
diagnosis of these two diseases. Further investigation on disease miR
may provide new insight into ocular lymphoma and uveitis.
Commercial Relationships: Jingsheng Tuo, None; DeFen Shen,
None; Chi-Chao Chan, None
Support: NEI intramural research program
Using a Murine Xenograft Model for Von Hippel-Lindau
Disease-Associated Retinal Hemangioblastomas to Test Novel
Therapies
Stanley Park1, 2, Yujuan Wang1, Kristin Huntoon3, DeFen Shen1,
Zhengping Zhuang3, Chi-Chao Chan1. 1Immunopathology Section,
Laboratory of Immunology, NEI, NIH, Bethesda, MD; 2Howard
Hughes Medical Institute, Chevy Chase, MD; 3Surgical Branch,
NINDS, NIH, Bethesda, MD.
Purpose: Von Hippel-Lindau (VHL) disease is an autosomal
dominant multisystemic disorder that gives rise to cystic and/or
highly vascularized tumors. VHL is mutated or deleted and makes
non-functional VHL protein leading to constitutive expression of
hypoxia-inducible factor (HIF). This causes high levels of vascular
endothelial growth factor (VEGF), platelet-derived growth factor
(PDGF) and erythropoietin (EPO) to be expressed in the tumor cell.
Patients commonly present with retinal hemangioblastomas (RH)
which can cause severe visual morbidity. VHL-null animal models do
not fully recapitulate common features of human VHL disease and
leave us with a limited understanding on targeted therapies for RH.
Here we designed a murine xenograft model to evaluate new
treatments for VHL disease-associated RH.
Methods: We used severe combined immunodeficiency (SCID) mice
and intravitreally inoculated them with UMRC6 cells, a human VHLnull renal cell carcinoma cell line. The mice were either given an
EPO receptor antagonist or a dual VEGF and PDGF kinase receptor
antagonist. Treatment efficacy was evaluated by clinical fundoscopy.
Eyes showing progressive obscuration of the retinal vasculature and
optic nerve head from baseline were given +1 score. Those that
showed clinical improvement were given -1, those that had no change
were given 0.
Results: At least two experiments were performed with two different
agents and the data were repeatable. By clinical scoring of the fundus
photographs, mice treated with the dual receptor inhibitor (n= 13)
showed no significant improvements when compared to those given
vehicle solution (n= 13). No treated mice demonstrated clinical
improvement while 53.8% (7/13) worsened. On the other hand, mice
treated with the EPO receptor antagonist (n= 16) demonstrated
modest clinical improvement when compared to mice given PBS (n=
17, p<0.01). 31.3% (5/16) improved while only 12.5% (2/16)
worsened. On histology, layers of UMRC6 cells with cysts and fine
vascular-like structures formed behind the posterior capsule of the
lens. Scattered inflammatory cells were also admixed with UMRC6
cells in some areas. There was no significant improvement in any
treated mice on histology.
Conclusions: This model may be a useful tool for testing therapeutic
agents. EPO receptor antagonism appears to be a promising
therapeutic option for treating this model.
Commercial Relationships: Stanley Park, None; Yujuan Wang,
None; Kristin Huntoon, None; DeFen Shen, None; Zhengping
Zhuang, None; Chi-Chao Chan, None
Support: The NEI Intramural research program
Transvitreal endoresection of retinal hemangioblastoma after
ligating feeder vessels
Gibran S. Khurshid. Ophthal & Visual Sci, Univ of Texas Medical
Branch, Galveston, TX.
Purpose: To evaluate the clinical outcome of transvitreal
endoresection of retinal hemangioblastoma (RH) after feeder vessel
ligature.
Methods: Prospective interventional case series of three consecutive
patients aged 21 to 24 years with unilateral solitary RCH. Tumors 3.4
to 5.2 mm in diameter were endoresected transvitreally, after ligation
of feeder vessels with a 20 guage bimanual technique.
Results: Reduction of exudative retinal detachment and resolution of
macular exudative changes were observed with parallel improvement
in vision. No tumor recurrence was noted, and visual acuity remained
stable at the last follow-up.
Conclusions: Considering the natural history of RH, this study may
offer endoresection as a treatment option for large solitary RH in
advanced stages that are refractory to conventional treatment
modalities.
Commercial Relationships: Gibran S. Khurshid, None
Prognostic Factors after Treatment of Patients with Retinal
Capillary Hemangioma(RCH)
Hyesun Kim, Jeong Ho Yi, Hee Jung Kwon, Christopher S. Lee,
SungChul lee. Department of Ophthalmology, Yonsei University
Severance Hospital, Seoul, Republic of Korea.
Purpose: To report observations regarding the regression and visual
outcome after treatment of eyes with retinal capillary hemangioma
(RCH)
Methods: Retrospective consecutive case series. A total of 37 eyes of
32 patients with RCH were included in this study. Data were
analyzed for basic characteristics, regression of RCH and visual
outcome after treatment.
Results: The mean age at diagnosis in cases with RCH was 30.6 year
(range, 5~56 years).The RCH was bilateral in 6 cases (19%), and von
Hippel-Lindau disease was positive in 15 cases(47%). Seven of
tumors (19%) touched optic disc and were classified as juxtapapillary
RCH, and the remaining 30 (81%) were extrapapillary in location.
Twenty (54%) cases were single tumor and 17(46%) were multiple
(mean: 2.4, range:1~21). Nine (24%) RCH were 1.5mm or smaller
and 3 (8%) were larger than 6mm in size. Initially accompanying
exudation, serous fluid and retinal detachement were 27 (73%), 11
(30%) and 7 (19%).Twenty four (75%) of 32(87%) treated RCH were
regressed. The mean number of treatment was 1.87 (range, 1~9) and
cases experienced complication after treatment was 17 (53%). RCH
was treated in 29 of 30(97%) extrapapillary cases vs 4 of 7(57%)
juxtapapillary cases (p<0.001). Visual improvement after the
treatment was significantly related to the regression of RCH after the
treatment (p=0.024) and the number of treatment(1.26 vs 2.50,
p=0.02). Multivariate logistic regression identified the regression of
RCH after the treatment(p=0.028,odds ratio=13.76;confidence
interval=1.3,142) as predictive factor for visual improvement. Better
final vision (>20/50) was significantly related to initial better vision
(>20/50) (p=0.0002), regression of RCH (p=0.0014), absence of
retinal detachement (RD) (p=0.012), absence of complication after
treatment (p=0.015), initially no need for treatment (p=0.036).
Conclusions: This study suggests that the prognostic factors for
better visual outcome after treatment of RCH are initial better vision,
regression of RCH, absence of retinal detachement (RD), absence of
complication after treatment, initially no need for treatment.
Commercial Relationships: Hyesun Kim, None; Jeong Ho Yi,
None; Hee Jung Kwon, None; Christopher S. Lee, None;
SungChul lee, None
Safety and Efficacy of Anti-VEGF Therapy for Choroidal
Neovascularization Overlying Choroidal Osteoma
Mohammed A. Khan1, Francis C. DeCroos2, Philip P. Storey2, Jerry
Shields3, Sunir J. Garg2, Carol L. Shields3. 1Ophthalmology, Wills
Eye Institute, Philadelphia, PA; 2Retina Service, Wills Eye Institute,
Philadelphia, PA; 3Oncology Service, Wills Eye Institute,
Philadelphia, PA.
Purpose: To investigate the safety and efficacy of intravitreal
bevacizumab or ranibizumab therapy for treatment of choroidal
neovascularization (CNV) associated with choroidal osteoma.
Methods: Retrospective, observational, consecutive case series of
patients diagnosed with choroidal osteoma and CNV who were
treated with intravitreal bevacizumab and/or ranibizumab, on the
Retina or Oncology Services of the Wills Eye Hospital, Philadelphia,
PA.
Results: Six tumors in six eyes were treated with intravitreal antiVEGF therapy. Mean tumor basal diameter was 8.9 mm (range 4.5 14 mm), with mean tumor thickness 1.2 mm (range 1.0 - 1.6 mm). A
mean of 10 intravitreal injections were completed (median 9, range 3
- 19 injections), with regression of CNV and resolution of fluid on
optical coherence tomography achieved in 5 of 6 (83%) cases. Two
tumors required 1 session of photodynamic therapy (PDT) for control
in addition to anti-VEGF therapy. Improvement or stability of visual
acuity was observed in 5 of 6 eyes (83%, range 0 to 6 Snellen line
improvement). Patients were followed over a mean period of 21
months (median 18, range 5 to 56 months), with one complication
(traumatic cataract) observed.
Conclusions: Therapy with intravitreal bevacizumab and/or
ranibizumab can be effective for CNV secondary to choroidal
osteoma, with resolution of CNV and preservation of visual acuity.
Commercial Relationships: Mohammed A. Khan, None; Francis
C. DeCroos, None; Philip P. Storey, None; Jerry Shields, None;
Sunir J. Garg, Lux (F), EyeGate (F), Regeneron (F), Genentech (F),
Allergan (C); Carol L. Shields, None
Effect of menopausal status on the progression of orbital
cavernous hemangiomas
Anupam Jayaram, Gary Lissner. Ophthalmology, Northwestern
University, Chicago, IL.
Purpose: Cavernous hemangiomas are among the benign neoplasms
of the orbit that are often found incidentally and present
asymptomatically. It has been shown that orbital cavernous
hemangiomas stain positive immunohistochemically for progesterone
receptor antibody, suggesting that they are a mesenchymal lesion
with possible response to sex steroid (Di Tomassa et al). It has also
been described that cavernous hemangiomas of other locations such
as the liver and the uterus regress post-menopausally. It can therefore
be hypothesized that as circulating levels of estrogen and
progresterone decrease in post menopausal women, these lesions in
the orbit may also cease progression or possibly even regress. The
purpose of this study is to document the likelihood of stabilization or
regression of orbital cavernous hemangiomas in post-menopausal
women and thus be able to better reassure this patient population and
substatiate management by observation.
Methods: This study is a retrospective chart review of all patients
who had lesions defined as "radiologically consistent with cavernous
hemangioma of the orbit" seen through Northwestern Memorial
Hospital. All imaging studies were reviewed noting specific
dimensions of lesions as well as time interval between studies, and
each patient's lesion was defined as progressed, stable, or regressed.
Progression and regression were defined as a statistically significant
change in size of the lesion. Lesions in men and pre-menopausal
women were used as controls to which the lesions in post menopausal
women were compared.
Results: A total of 28 patients seen by the oculoplastics department
at Northwestern Hospital were identified with imaging studies
consistent with cavernous hemangioma of the orbit. Of these patients,
the percentage of post menopausal women who demonstrated stable
or regressing lesions was greater than the total population of men and
women with orbital cavernous hemangiomas who demonstrated
stable or regressing lesions.
Conclusions: While cavernous hemangiomas of the orbit frequently
remain stable over time, because of their response to hormone levels,
these lesions in post-menopausal women seem to remain stable or
even regress more often than in men or pre-menopausal women.
Commercial Relationships: Anupam Jayaram, None; Gary
Lissner, None
The effect of Transforming growth factor-beta-induced protein
(TGFBIp) in lymphangiogenesis
Yong-Sun Maeng, Tae-im Kim, Hyung Keun Lee, Eung Kweon Kim.
Ophthalmology, Yonsei University, Seoul, Republic of Korea.
Purpose: Purpose: Transforming growth factor-beta-induced protein
(TGFBIp, also known as βig-H3 and keratoepithelin) is an
extracellular matrix protein that plays a role in a wide range of
physiological and pathological conditions including diabetes, corneal
dystrophy and tumorigenesis. However, the role of TGFBIp signaling
in lymphangiogenesis, the growth of lymphatic vessels, is poorly
understood. The purpose of this study was therefore to analyze the
lymphangiogenic response to TGFBIp and determine if these
responses are important for the pathogenesis of lymphangiogenesisdependent diseases.
Methods: Methods: We used transwell migration, matrigel tube
formation and extracellular matrix adhesion models in order to study
the effect of TGFBIp in in vitro lymphangiogenesis. Cytodex threedimensional bead sprouting assay, 3D spheroid sprouting assay and
thoracic duct sprouting assay were utilized to analyzed the function
of TGFBIp in in vivo lymphangiogenesis
Results: Results: In this study, we report a novel role of TGFBIp as a
potent lymphatic endothelial activator, promoting
lymphangiogenesis. TGFBIp increased adhesion, migration, and
morphologic differentiation of human lymphatic endothelial cells
(LECs), consistently with increased the LEC sprouting in threedimensional bead sprouting assay, 3D spheroid sprouting assay and
lymphatic ring assay. TGFBIp also increased phosphorylation of
intracellular signaling molecules FAK, ERK, AKT and JNK.
Furthermore, TGFBIp-induced LEC adhesion, migration and
morphologic differentiation were specifically blocked by neutralizing
antibody of beta 3 integrin. In addition, TGFBIp-induced LEC
sprouting in 3D spheroid sprouting assay was inhibited by
neutralizing antibody of beta 3 integrin and pharmacologic inhibitors
of FAK, ERK, AKT or JNK. These data demonstrate that TGFBIp
promotes lymphangiogenesis by stimulating lymphatic endothelial
cells via the beta 3 integrin-FAK-ERK-AKT-JNK signaling pathway.
Conclusions: Conclusions: These findings open new perspectives for
the role of TGFBIp in the pathogenesis of lymphangiogenesisdependent diseases.
Commercial Relationships: Yong-Sun Maeng, None; Tae-im Kim,
None; Hyung Keun Lee, None; Eung Kweon Kim, None
Support: This research was supported by the Converging Research
Center Program funded by the Ministry of Education, Science and
Technology (2012K001354)
RAS Mutations Coexist with PIK3CA and MET Mutations in
Lacrimal Gland Epithelial Neoplasms
Bita Esmaeli1, Vivian T. Yin1, Khalida Wani2, Kenneth Aldape2,
Diana Bell2. 1Head & Neck Surgery/Ophthal, UT MD Anderson
Cancer Center, Houston, TX; 2Pathology, UT MD Anderson Cancer
Center, Houston, TX.
Purpose: Oncogenic mutations for PIK3CA, RAS (KRAS, NRAS),
BRAF and MET have been identified in various malignancies, and
activate the PI3K/ AKT/mTOR and RAS/RAF/MEK pathways
respectively. Both pathways are critical drivers of tumorigenesis.
Methods: Tumor tissue from 21 patients with lacrimal gland
epithelial neoplasms (3 benign pleomorphic adenomas and 18
malignant: 14 adenoid cystic carcinomas, 2 low grade myoepithelial
carcinoma-ex pleomorphic adenomas, one high grade salivary ductlike carcinoma-ex pleomorphic adenoma, one squamous carcinoma)
treated at M. D. Anderson Cancer Center were analyzed for PIK3CA,
RAS (KRAS, NRAS), BRAF and MET using polymerase chainreaction based DNA sequencing.
Results: KRAS mutations were found in 10 (48 %) of 21 patients
tested; NRAS in 2 (10 %); MET in 3 (14%) of 21 patients tested;
PIK3CA in 1 (5 %) of 21 patients tested; BRAF in 0 of 21 patients
tested.
Half of oncogenic mutations were found in adenoid cystic carcinoma
(8/14, 57%). KRAS mutations were most frequent in adenoid cystic
carcinoma (5/10, 50%), as well as MET mutations (2/3, 67%), NRAS
mutations (1/2, 50%) and none for PIK3CA/ BRAF.
In one benign mixed tumor KRAS mutations coexisted with NRAS
and PIK3CA; in another benign mixed tumor both KRAS and NRAS
mutations were found. One high grade carcinoma was found to have
concurrent KRAS and MET mutations.
Conclusions: RAS (KRAS, NRAS), PIK3CA and MET mutations
are frequent in diverse epithelial neoplasms of lacrimal gland with the
highest proportion of mutations found in adenoid cystic carcinoma.
PIK3CA and MET mutations can coexist with RAS mutations.
Commercial Relationships: Bita Esmaeli, None; Vivian T. Yin,
None; Khalida Wani, None; Kenneth Aldape, None; Diana Bell,
None
Screening of Optic Pathway Gliomas in Children with
Neurofibromatosis Type-1
Raffaele Parrozzani1, Maurizio Clementi2, Gloria Orlando3,
Giacomo Miglionico3, Eva Trevisson2, Edoardo Midena3, 1. 1GB
Bietti Eye Foundation, IRCCS, Rome, Italy; 2Department of
Pediatrics, Clinical Genetics, University of Padova, Padova, Italy;
3
Department of Ophthalmology, University of Padova, Padova, Italy.
Purpose: The aim of this study was to compare visual function
assessment (VA), optic disc evaluation by indirect ophthalmoscopy
and retinal nerve fibres layer (RNFL) analysis by optical coherence
tomography (OCT) as screening tools for optic pathway gliomas
(OPGs) in pediatric patients (2-15 years) affected by
neurofibromatosis type-1 (NF1).
Methods: Fifty-seven consecutive pediatric patients affected by NF1
with recent (<6 moths) orbital/brain MRI were included. Patients
underwent VA (Hyvarinen symbols chart, HOTV or Snellen charts in
patients aged 2-4, 4-6 and 6-15 years respectively) and optic disc
evaluation by indirect ophthalmoscopy by experienced, masked
paediatric ophthalmologist. Spectral domain-OCT (SD-OCT;
Spectralis™, Heidelberg, Germany) was performed by a single
masked operator to assess RNFL (16 to 100 averaged images by
Automatic Real Time-ART).
Results: Fifteen of fifty-seven enrolled patients were affected by
MRI-proven ONGs. VA was judged feasible and reliable in ten
(66%) of fifteen OPGs patients (40%, 60% and 100% in patients aged
<5, 5-10 and 10-15 years respectively) versus thirty-for of forty-two
(81%) no-OPGs patients (57%, 86% and 100% respectively). VA
was clinically judged to be suspected of OPGs in six of ten (60%)
OPGs examined patients versus five of thirty-four (15%) no-OPGs
examined patients (p=0.01). Optic disc evaluation was judged
clinically feasible in fourteen of fifteen (93%) OPGs patients (100%,
80% and 100% respectively) versus forty of forty-two (95%) noOPGs patients (93%, 93% and 100% respectively). Optic disc aspect
was clinically judged to be suspected of OPGs in four of fourteen
(40%) OPGs examined patients versus four of forty (10%) no-OPGs
examined patients (p>0.05). SD-OCT analysis was clinically feasible
in twelve (80%) of fifteen OPGs patients (60%, 80% and 100%
respectively) versus thirty-nine (93%) no-OPGs patients (79%, 93%
and 100% respectively). SD-OCT analysis documented RNFL loss in
eleven of twelve patients affected by OPGs (92%) and in no-one of
thirty-nine no-OPGs patients (p<0.001).
Conclusions: RNFL assessment using SD-OCT is superior to visual
function assessment and optic disc evaluation as a clinical screening
tool of OPGs in NF1pediatric patients. The use of SD-OCT to detect
RNFL loss under the age of 5 years may be questionable due to the
low rate of children compliance, although more feasible and reliable
than standard visual function assessment.
Commercial Relationships: Raffaele Parrozzani, None; Maurizio
Clementi, None; Gloria Orlando, None; Giacomo Miglionico,
None; Eva Trevisson, None; Edoardo Midena, None
Association of AJCC pT category with outcome in sebaceous
carcinoma of the eyelid
Valerie A. White1, 2, Kaustubh Mulay3, Sneha Agrawal4, Santosh
Honavar4. 1Department of Pathology and Laboratory Medicine,
University of British Columbia, Vancouver, BC, Canada;
2
Ophthalmology and Visual Science, University of British Columbia,
Vancouver, BC, Canada; 3Ophthalmic Pathology, LV Prasad Eye
Institute, Hyderabad, India; 4Oculoplastic and Orbital Surgery, LV
Prasad Eye Institute, Hyderabad, India.
Purpose: The 7th edition of the AJCC classification of ophthalmic
tumors is in the process of being validated. Only one previous study
has validated the updated classification for sebaceous carcinoma of
the eyelid with outcome (Esmaeli B et al. Ophthalmology 2012;
119:1078-82) We wanted to perform a validation study from another
global region.
Methods: The medical records and pathology slides of all patients
treated in the Oculoplastic and Oncology Department of LV Prasad
Eye Institute with a diagnosis of sebaceous carcinoma of the eyelid
from 2001 to 2011 were retrieved. The slides were reviewed for
diagnosis, type of specimen, size and spread of tumor, and margins of
excision. The following clinical information was extracted: age, sex,
previous occurrence prior to presentation at LVPEI, treatment,
recurrences, lymph node and/or distant metastases.
Results: 242 pathology specimens from 197 patients were examined.
Only 54 patients had adequate follow-up for analysis. There were 35
women and 19 men with a mean age of 56 years. The following table
shows the number of patients in each pT category and the
relationship of pT category to local recurrence and metastases.
Conclusions: Follow-up was difficult to obtain in this setting.
However, as most patients were treated by surgery and have a
pathologic specimen, a pT category could be applied and predicted
increasing incidence of local recurrence, and local and distant
metastases. A source of uncertainty was whether to classify those
patients with predominantly in-situ disease that had been treated by
exenteration as Tis or T3b.
Correlation of pT category with outcome in sebaceous carcinoma of
the eyelid
Commercial Relationships: Valerie A. White, None; Kaustubh
Mulay, None; Sneha Agrawal, None; Santosh Honavar, None
Stratifin- A novel biomarker in the prognosis of Ocular Surface
Squamous Neoplasia patients
Sheetal Chauhan1, Seema Sen1, Anjana Sharma2, Seema Kashyap1,
Shyam S. Chauhan3, Radhika Tandon4, Neelam Pushker4, Murugesan
Vanathi4, Rajvardhan Azad4. 1Ocular Pathology,R.P.Centre, All India
Institute of Medical Sciences, New Delhi, India; 2Ocular
Microbiology, All India Institute of Medical Sciences, New Delhi,
India; 3Biochemistry, All India Institute of Medical Sciences, New
Delhi, India; 4Ophthalmology, All India Institute of Medical
Sciences, New Delhi, India.
Purpose: Stratifin (14-3-3σ)/HEM (human epithelial marker) is a
p53 mediated inhibitor of cell cycle progression; it has been shown to
be a target of epigenetic deregulation in many carcinomas. In the
present study, the association of Stratifin expression with it’s
promoter methylation status and mutant p53 expression was
undertaken. The prognostic significance of Stratifin in ocular surface
squamous neoplasia (OSSN) patients was also evaluated.
Methods: Sixty-four histopathologically confirmed OSSN cases (44
squamous cell carcinomas and 20 conjunctival intraepithelial
neoplasias) were included in this study. AJCC TNM staging was
done and patients were followed up for 32 months.
Immunohistochemical expression of Stratifin (clone-ab14123) and
mutant p53 (clone-DO7) protein was evaluated; methylation status of
Stratifin was determined by methylation specific PCR using specific
primers. Kaplan-Meier survival and Cox regression analysis was
done to assess the prognostic significance of Stratifin.
Results: Loss of Stratifin immunoexpression was observed in 75%
(48/64) and its promoter hypermethylation in 63% (40/64) OSSN
cases. Loss of Stratifin expression significantly correlated with it’s
promoter hypermethylation (P=<0.0001).
Expression of mutant p53 protein was seen in 48% (31/64) cases and
this inversely correlated with Stratifin immunoexpression (P=0.009).
Both loss of Stratifin immunoexpression and it’s promoter
hypermethylation, were significantly associated with tumor size 2cm,
T3 and T4 category and reduced disease free survival (P 0.05). Cox
analysis showed Stratifin to be independent prognostic marker for
OSSN (p =0.03).
Conclusions: Our results indicate that loss of Stratifin expression
occurs in OSSN and is caused by aberrant DNA methylation.
Relationship between mutant p53 and Stratifin suggests that
abnormalities in p53-stratifin pathway could play an important role in
the pathogenesis of OSSN. Loss of Stratifin immunoexpression could
prove to be a useful biomarker to identify high risk OSSN patients.
Commercial Relationships: Sheetal Chauhan, None; Seema Sen,
None; Anjana Sharma, None; Seema Kashyap, None; Shyam S.
Chauhan, None; Radhika Tandon, None; Neelam Pushker, None;
Murugesan Vanathi, None; Rajvardhan Azad, None
Incidence and immunostaining pattern of oncocytomas of the
caruncle - a single center 12 year analysis
Sandra C. Bajorat1, Mara Joana von Schoenfels1, Ivo Leuschner2,
Johann Roider1, Stefan O. Koinzer1. 1Ophthalmology, University of
Kiel, University Medical Center, Kiel, Germany; 2Pathology,
University of Kiel, University Medical Center, Kiel, Germany.
Purpose: Oncocytomas are benign tumors that have been reported to
account for 3-8% of excised caruncle lesions. These cystic lesions
grow slowly, are usually asymptomatic and occur predominantly in
elderly women. Oncocytomas may be found in other glandular organs
as well. Therapy is by excision. Malignancy has only been described
in single cases, but not at the caruncle. It is unclear whether the
tumour is of glandular or myoepithelial origin. We examined
histologically all caruncular oncocytomas that have been diagnosed
in our clinic between 2000 and 2012.
Methods: Retrospective chart review of all conjunctival probe
excisions performed in a single center over 12 years. Routine HEhistological analysis of all oncocytomas of the caruncle, and, if
sufficient material was available, immunohistochemical analysis for
pancytokeratin (pan-CK), smooth muscle antigen (SMA), lysozyme,
estrogen (ER) - and progesterone receptors (PR).
Results: In the observed period, we excised 641 conjunctival probes
where one oncocytotic adenocarcinoma was diagnosed. 51 / 641 (8%)
probes originated from the caruncle with 53 different pathologies.
The most frequent caruncular diagnoses were nevi (22 / 53 [43%]),
cysts and oncocytomas (6 / 53 each [12%]), sebaceous gland
hyperplasia (5 / 53 [10%]), papillomas (4 / 53 [8%]), and others. The
gender ratio of oncocytoma patients was female : male = 5 : 1. The
mean age was 79 years. The diagnosis could be established by routine
HE histology in all cases. Material for immunostaining was available
for 3 / 6 cases. All 3 immunohistochemical patterns showed positivity
for pan-CK and SMA but negativity for lysozyme, ER and PR.
Conclusions: In our series, oncocytomas occurred more frequently
(12%) than in other retrospective analyses (3-8%) and were among
the second most frequent diagnoses of caruncle tumors. As reported
by others, the majority originated from elderly women, but the gender
preference could not be explained by positive ER or PR expression in
the 3 cases eligible for immunostaining. The findings favor
myoepithelial origin over glandular origin. 1 oncocytotic
adenocarcinoma in 641 cases occurred distant from the caruncle, and
no malignant transformation of the tumor was found in the caruncular
localization.
Commercial Relationships: Sandra C. Bajorat, None; Mara
Joana von Schoenfels, None; Ivo Leuschner, None; Johann
Roider, Novartis (F), Bayer (F); Stefan O. Koinzer, None
Classification Schemes for Conjunctival Melanocytic Neoplasia:
the Effect of Immunohistochemistry on C-MIN Scoring
Emilia H. DeMarchis1, Peter Egbert2, Jinah Kim3, 4. 1School of
Medicine, Stanford University, Palo Alto, CA; 2Department of
Ophthalmology, Stanford University, Stanford, CA; 3Department of
Pathology, Stanford University, Stanford, CA; 4Department of
Dermatology, Stanford University, Stanford, CA.
Purpose: To evaluate the use of immunohistochemistry staining in
scoring and prognostic classification of conjunctival melanocytic
intra-epithelial neoplasia (C-MIN), and analyze how the diagnosis of
primary acquired melanosis at Stanford University correlates with CMIN scoring.
Methods: A histologic review was performed of conjunctival
melanocytic lesions at Stanford University Medical Center from 1990
to present with immunohistochemistry staining for Ki67/MelanA and
MiTF. Lesions were evaluated using the C-MIN scoring system by
hematoxylin and eosin staining alone, versus in conjunction with the
aforementioned special staining.
Results: Immunohistochemical staining enhanced the identification
of pagetoid spread (5/18 cases), vertical spread (8/18 cases), and
permitted objective measurement of mitotic index. In seven cases
(38.9%), the addition of these studies altered the C-MIN score and in
one case, the change was clinically significant. In addition, the CMIN scores showed statistically significant correlation with the
mitotic index.
Conclusions: The use of immunohistochemical staining is helpful,
and arguable necessary, for proper evaluation of conjunctival
melanocytic lesions using the C-MIN scoring system, and can have
clinical consequences on the management of lesions. MelanA and
MiTF staining allow for more accurate identification of melanocytes,
especially in terms of pagetoid spread, and Ki67 provides insight into
the virulence of lesions. The incorporation of mitotic index may aid
classification of these pigmented lesions.
Commercial Relationships: Emilia H. DeMarchis, None; Peter
Egbert, None; Jinah Kim, None
Immunohistochemical Analysis with a Novel Red Chromagen of
Benign and Malignant Melanocytic Lesions of the Conjunctiva
Seymour Brownstein1, 2, Kailun Jiang1, 2, Kay Lam1, 2, Bruce F.
Burns2, James Farmer2. 1Ophthalmology, Ottawa Hospital, Ottawa,
ON, Canada; 2Pathology, Ottawa Hospital, Ottawa, ON, Canada.
Purpose: In North America, immunohistochemical analysis of
heavily pigmented melanocytic lesions of the conjunctiva and uvea
rely almost exclusively on the 3, 3 diaminobenzidine (DAB) as a
colored substrate. DAB use may require bleaching of samples to
remove the confounding melanin pigment, but bleaching can affect
cellular antigenicity and may be incomplete. We evaluated an
alternative substrate, Vector Red (VR), whose vibrant red color is
distinct from the melanin pigment and renders bleaching
unnecessary. Using VR, we identified an immunological marker that
is sensitive for all melanocytes and another marker that is sensitive
and specific for activated/atypical melanocytic lesions.
Methods: Retrospective and prospective case series. 8 specimens
each of normal conjunctiva and choroid, choroidal melanoma,
conjunctival nevus, PAM with mild or no atypia, PAM with moderate
or severe atypia, and conjunctival melanoma were obtained from our
laboratory from 2005 to 2012. We compared the
immunohistochemical profile of these specimens. Each specimen was
immunolabeled with dPANMEL, S100, HMB45, Melan A, and Ki67
using the VR substrate. The HMB45 immunolabeled specimens were
additionally developed with the DAB substrate with or without
overnight 4% H2O2 bleaching. The immunoreactivity data were then
analyzed using a 2-tailed Mann-Whitney test (p<0.05 is significant).
Results: The degree of immunoreactivity in specimens developed
with VR was similar to those treated with 4% H202 and developed
with DAB. Atypical melanocytes were most specifically labeled with
HMB45 (93% specific and 88.3% sensitive). The percentage of
HMB45 and Ki67 positive cells increased significantly with
worsening atypia. dPANMEL and Melan A labeled all benign and
malignant melanocytic lesions indiscriminately. 40% of normal
conjunctival melanocytes and 58% of PAM specimens stained
negative for S100.
Conclusions: We recommend using VR as a standard substrate for
the immunohistochemical analysis of melanocytic lesions as the
recent VR products with their vivid red colour are relatively
inexpensive and do not require bleaching of the samples. We found
Melan A and HMB45 to be the 2 minimal markers necessary to fully
characterize a given melanocytic lesion. Melan A has superior
sensitivity for both benign and malignant melanocytes compared to
S100, and HMB45. HMB45 is able to identify atypical melanocytes
most specifically.
Commercial Relationships: Seymour Brownstein, None; Kailun
Jiang, None; Kay Lam, None; Bruce F. Burns, None; James
Farmer, None
Conjunctival Nevus: Clinicopathological Review of 724 cases
Stephanie De la O-Pérez1, Héctor A. Rodríguez-Martínez2, Dolores
Ríos y Valles-Valles1, Ofelia Pérez-Olvera2, Alfredo Gómez-Leal1,
Armando Medina-Cruz2, María del Mar Y. Namba-Bando1, Luis H.
De La O-Cerna3, Abelardo A. Rodríguez-Reyes1. 1Ophthalmic
Pathology Service, Hospital "Dr. Luis Sánchez Bulnes", Asociación
Para Evitar la Ceguera en México,, Mexico city, Mexico;
2
Anatomopathological Research Laboratory "Roberto Ruiz Obregón”
Experimental Medicine Department, Medical Faculty, National
Autonomous University of Mexico, General Hospital of Mexico,
Mexico City, Mexico; 3Eye Bank, University Hospital, Autonomous
University of Coahuila, Torreón, Mexico.
Purpose: Present clinicopathological characteristics of Conjuntival
Nevus (CN) in a Mexican population.
Methods: All surgically excised cases with histopathological
diagnosis of CN were collected form the Ophthalmic Pathology
Service archives at a referral center in Mexico City over a period of
54 years (1957-2011). Clinical and demographical data were obtained
from clinical charts. All slides were reviewed and reclassified.
Results: From a total of 777 cases, 724 were confirmed as CN.
Prevalence in women was 54% compared to 46% in men, with a
mean age of 23 (SD 18) years at the time of surgical excision (range,
6 months - 85 years). In 52% of the cases, the left eye (OS) was
affected, compared to 48% in the right eye (OD). The vast majority
were located on bulbar conjunctiva (67%). The average size was 4
mm (SD 2, range, 0.5mm - 15mm). Most of the CN were observed
clinically as flat lesions (91%). Histopathologically, 87% were
compound, 12% subepithelial and 1% intraepithelial nevus. Inclusion
cysts were present in 73% of the cases. Melanin was observed in all
cases, 95% in nevus cells and 93% in melanophages. Accompanying
inflammatory infiltrate was observed 62% of the time, 32% presented
eosinophils. Melanosis (26%) and hypervascularity (27%) were
associated features. Five cases corresponded to balloon cell type
nevus.
Conclusions: CN in Mexican population occur in young individuals,
slightly more in women than in men, both eyes affected equally.
Mostly apparent as a flat pigmented lesion in the bulbar conjunctiva.
Compound nevus is the most frequent type, very often associated
with inclusion cysts; an inflammatory component is present in more
than half the cases. The balloon cell nevus is rare in this location. To
the best of our knowledge, this is the largest series of CN until now
presented.
Classical clinical appearance of a nevus: flat pigmented lesion in
bulbar conjunctiva.
Compound nevus.
Commercial Relationships: Stephanie De la O-Pérez, None;
Héctor A. Rodríguez-Martínez, None; Dolores Ríos y VallesValles, None; Ofelia Pérez-Olvera, None; Alfredo Gómez-Leal,
None; Armando Medina-Cruz, None; María del Mar Y. NambaBando, None; Luis H. De La O-Cerna, None; Abelardo A.
Rodríguez-Reyes, None
The Growth of Small Choroidal Melanocytic Lesion after
Intravitreal Bevacizumab Injection
Hee Jung Kwon, Hae Min Kang, Hyesun Kim, SungChul lee,
Christopher S. Lee. Ophthalmology, Institute of Vision Research,
Yonsei University College of Medicine, Seoul, Republic of Korea.
Purpose: To describe the clinical effect of intravitreal bevacizumab
injection (IVB) for small choroidal melanocytic lesions with
subretinal fluid and to determine features that are predictive of
growth into choroidal melanoma.
Methods: The medical records of 34 patients who diagnosed with
small choroidal melanocytic lesion between December 2004 and
October 2011 were retrospectively reviewed. Univariate logistic
regression was performed to evaluate the degree of relationship of
initial clinical findings (tumor size, symptom, surface features
[orange pigment, drusen and RPE alteration], subretinal fluid [SRF])
to tumor growth. We also describe the clinical course of small
choroidal melanocytic lesion treated with intravitreal injection of
bevacizumab (IVB) for the purpose of decreasing SRF.
Results: Six patients of small choroidal melanocytic lesions (17.6%)
demonstrated growth. Lesions that demonstrated growth had
significantly higher rate of thicker tumor (>2.0mm, 83.3% vs. 28.6%,
p=0.018) and orange pigment (83.3% vs. 21.4%, p=0.018). Ten
patients were treated with IVB, but IVB had minimal effect in
reducing SRF and six (60%) of them showed tumor growth. We
could calculate tumor growth rate in 3 patients, and tumors growth
rate accelerated after cessation of serial IVB. Histopathologic finding
of enucleated eye with enlarged tumor after IVB confirmed choroidal
melanoma and showed severe necrosis coinciding necrotic type
choroidal melanoma.
Conclusions: Intravitreal injection of bevacizumab seems to be
inefficacious in resolving subretinal fluid accompanied with small
choroidal melanocytic lesion. Moreover, IVB seems to stimulate
tumor growth by disrupting homeostasis of vascular endothelium
growth factor and other mediators. Therefore, the use of bevacizumab
for treatment of SRF in choroidal melanoma should be considered
carefully because of the possible adverse effects of IVB.
Commercial Relationships: Hee Jung Kwon, None; Hae Min
Kang, None; Hyesun Kim, None; SungChul lee, None;
Christopher S. Lee, None
Clinical utility of SD-OCT -Enhanced Depth Imaging in
Evaluation of Retinal and Choroidal Tumors of the Human Eye
Peter G. Hovland. Colorado Retina Associates, Denver, CO.
Purpose: To present illustrative examples of clinical cases in which
the use of the Heidelberg Spectralis instrument created uniquely
useful images which allow for the precise determination of diagnosis,
anatomic localization and dimension, and local tissue effects of
intraocular tumors.
Methods: A non-randomized series of patients presenting to an
ocular oncology clinic were selected for testing with the Heidelberg
Spectralis instrument on the EDI-OCT setting. Correlation with
clinical history was applied to the interpretation of the images. EDIOCT images were obtained of CHRPE, choroidal nevus, treatment
naive choroidal melanoma, treated choroidal melanoma (status-post
brachytherapy, diode laser, intravitreal bevacizumab and/or argon
laser), and metastatic choroidal and retinal lesions.
Results: In an analysis of over 50 patients, the EDI-OCT could
precisely determine the depth of the lesions when the maximal depth
did not exceed 2 mm. For choroidal lesions, overlying tissue changes
of the retina could be described in terms of presence or absence of
subretinal fluid, photoreceptor changes, retinal edema or
degeneration, and RPE abnormalities. Changes in these tissue
elements can be followed over time as a means of surveillance to
monitor for evidence of neoplasm, or response to treatment.
Conclusions: The EDI-OCT provides an extremely useful adjunct to
standard techniques in the work-up and surveillance of ocular
oncology patients, and especially so for the monitoring of smaller
suspicious lesions.
Commercial Relationships: Peter G. Hovland, None
Waardenburg Syndrome: Iris and Choroidal Hypopigmentation
findings on Anterior and Posterior Segment Imaging
Stephanie Nickerson, Carol L. Shields, SAAD AL-DAHMASH, Jerry
Shields. Ocular Oncology Service, Wills Eye Institute, Philadelphia,
PA.
Purpose: To demonstrate iris and choroidal hypopigmentation in
Waardenburg syndrome (WS) with high resolution ocular imaging
Methods: Retrospective review of 7 patients from 6 families
Results: There were 3 males and 4 females who referred for
evaluation of presumed ocular melanocytosis. In all cases the
diagnosis of WS was established. The non-ocular features included
white forelock in 4/7 (57%), tubular nose in 5/6 (83%), and small
nasal ala in 5/6 (83%). In 2 cases hearing deficit was documented on
audiology testing. Family history of Waardenburg syndrome was
elicited in 5/7 cases (71%). The ocular features (n=7 patients)
included telecanthus in 5 (71%), synophrys in 2 (29%), iris
hypopigmentation in 5 (71%), and choroidal hypopigmentation in 5
(71%) patients. No patient had muscle contractures or Hirschsprung
disease. Visual acuity was 20/20-20/50 in all cases. The iris
hypopigmentation (n=8 eyes) was sector in 6 (75%) or diffuse
(complete) in 2 (25%). The choroidal hypopigmentation (n=9 eyes,
100%) was sector in 6 (67%) or diffuse in 3 (33%). Iris
hypopigmentation was not symmetric in any patient whereas
choroidal hypopigmentation was symmetric in 4 of 5 (80%) affected
patients. Iris hypopigmentation showed minimal correlation with
choroidal hypopigmentation. Imaging with anterior segment optical
coherence tomography (OCT) revealed the hypopigmented iris to be
thinner and with shallower crypts than normal iris. Posterior segment
imaging with OCT revealed normal retina in all cases, but the
subfoveal choroid in the hypopigmented region was slightly thinner
(mean 197 microns) compared to opposite normal choroid (243
microns). Fundus autofluorescence demonstrated mild homogeneous
hyperautofluorescence (scleral unmasking) in hypopigmented
choroid and no lipofuscin abnormality. No patient showed features of
ocular melanocytosis, albinism, vitiligo, autoimmune disease,
previous inflammation, melanoma, or other pigmentary conditions.
Conclusions: Waardenburg syndrome manifests hypopigmentation
of the iris and choroid with imaging features of slight reduction in
thickness of affected tissue. Any patient with iris or choroidal
hypopigmentation should be evaluated for this syndrome.
Commercial Relationships: Stephanie Nickerson, None; Carol L.
Shields, None; SAAD AL-DAHMASH, None; Jerry Shields, None
Does beta-ray emitting therapy of ciliary body tumors decrease
central corneal endothelial cell density?
Eva Suranyi1, Andras Berta2, Laszlo Modis3, Eszter Szalai4, Judit
Damjanovich5. 1Ophthalmology, University of Debrecen, Debrecen,
Hungary; 2Ophthalmology, University of Debrecen, Debrecen,
Hungary.
Purpose: To evaluate the effect of plaque radiation therapy, using
beta radioactive isotope for anterior segment tumors on the density
and morphology of endothelial cells.
Methods: Endothelial cell density (ECD) and morphometry in 15
eyes of 15 patients with ciliary body tumor was examined prior to
and 6 weeks after radiation therapy. After irradiation, central ECD
values were also compared with peripheral (2.0 mm from limbus)
ECD values measured around the plaque. ECD, average cell area,
coefficient of variation of cell area and pachymetry measurements
were conducted with contact specular microscopy.
Results: The mean corrected ECD values prior to irradiation was
2147 ± 128 cells/mm2 [95% confidence interval (CI): 2076 - 2218
cells/mm2] and 2050 ± 108 cells/mm2 after the radiation therapy
(95% CI: 1990 - 2109 cell/mm2). After irradiation the mean
peripheral ECD values were 2056 ± 101 cell/mm2 (95% CI: 1997 2114 cell/ mm2). A significant decrease in ECD values was observed
after radiation (P = .0067). Peripheral ECD values measured around
the plaque showed no significant difference (P = .8552) as compared
to central ECD values.
Conclusions: According to our measurements, plaque therapy for
tumors in the anterior segment decreases endothelial cell density
significantly, but not highly, even in case of plaques containing beta
radiation isotope and the plaques are not placed direct on the cornea
surface. The decreased ECD causes no changes in corneal thickness
or transparency, but it may have an influence on a subsequent
cataract surgery, which generates further endothelial loss.
Commercial Relationships: Eva Suranyi, None; Andras Berta,
None; Laszlo Modis, None; Eszter Szalai, None; Judit
Damjanovich, None
526 Anatomy Challenges for the Future
Thursday, May 09, 2013 10:30 AM-12:15 PM
608 Paper Session
Anatomy of CD31+ blood vessels and LYVE1+ macrophages in
the adult human sclera
Simona L. Schlereth1, Falk Schroedl2, Rafael Grajewski1, Claus
Cursiefen1, Ludwig M. Heindl1. 1Ophthalmology, University of
Cologne, Cologne, Germany; 2Paracelsus Medical University,
Salzburg, Austria.
Purpose: The human sclera is a dense connective tissue that covers
the eye. Physiologically it shows only diminished vascularity. The
existence of lymph vessels has been discussed contradictorily. In this
study we investigate the allocation of blood and lymph vessels in
human sclera in the anterior, equatorial, and posterior region of all
four quadrants.
Methods: Human sclera of ten tissue donors (age: 64-79 years; in
compliance with the Declaration of Helsinki) was analyzed for blood
(CD31) and lymph vessel (LYVE1 and podoplanin) formation, using
fluorescence, confocal and transmission electron microscopy.
Different locations were analyzed for their vessel spreading at
anterior, equatorial, and posterior part of the sclera in all four
quadrants, taking into consideration the differences between the three
scleral layers: episclera, stroma proper and lamina fusca.
Results: Human sclera displayed specific locations of blood vessels
within the eye. Whereas the stroma proper and the lamina fusca were
devoid of blood and lymph vessels, the superficial layers of the sclera
revealed a network of small-sized blood vessels (less then 20 μm in
size) and LYVE1+ macrophages, but no LYVE1+/ podoplanin+
lymphatic vessels. Compared to anterior and equatorial regions
LYVE1+macrophages were less frequent in the posterior part,
whereas no difference in the amount of blood vessels was detectable.
Conclusions: Blood vessels, but no lymphatic vessels are detectable
in the healthy human sclera. Additionally, LYVE1+ macrophages are
present in the superficial layers only. These cells may contribute to
inflammatory neovascularization in pathological circumstances e.g.
scleritis.
Commercial Relationships: Simona L. Schlereth, None; Falk
Schroedl, None; Rafael Grajewski, None; Claus Cursiefen, Gene
Signal (C), Alcon (R), Allergan (R), Bayer (R); Ludwig M. Heindl,
None
Support: German research foundation (SFB643B10)
Optic Nerve Parameters in Preterm and Full Term Infants Using
Spectral Domain Optical Coherence Tomography (SDOCT)
Amy Y. Tong1, Ramiro S. Maldonado2, Du Tran-Viet2, Adam L.
Rothman1, Eric L. Yuan2, Adam M. Dubis2, Sandra Stinnett2, Mays A.
El-Dairi2, Sharon F. Freedman2, Cynthia A. Toth2. 1Duke University
School of Medicine, Durham, NC; 2Duke Eye Center, Durham, NC.
Purpose: To evaluate the effect of prematurity on optic nerve
development by comparing optic nerve parameters and morphology
in premature and term infants at 37-42 weeks postmenstrual age
(PMA) using SDOCT.
Methods: Images were obtained in 60 preterm infants and 60 term
infants at 37-42 weeks PMA using a portable SDOCT system
(Bioptigen Inc., Research Triangle Park, NC). For each subject, the
highest quality image from either eye was selected for quantitative
analysis based on a subjective assessment of image clarity, ability to
identify lateral edges of the retinal pigmented epithelium and lamina
cribrosa, centering of the optic nerve, and lack of tilt. Quantitative
analysis included measurement of vertical cup diameter (vCuD), disc
diameter (vDD), cup-to-disc ratio (vC:D), and cup depth, using a
defined protocol. Images were masked, and measurements made
using a custom MATLAB (MathWorks, Natick, MA) program.
Statistical analyses compared measures by term status, race, and
gender.
Results: vCuD (µm), vDD (µm), vC:D, and cup depth (µm) were
measured in 44/60 preterm and 52/60 term infants. Sixteen preterm
and 8 term subjects were excluded due to lack of adequate optic
nerve images. Preterm infants had a mean gestational age (GA) of 26
weeks, birth weight of 872g, and included 18 subjects who were
treated for ROP or had Stage 3 disease; term infants had a mean GA
of 39 weeks and birth weight of 3345g. In comparing preterm vs.
term infants, preterms had significantly larger vCuD (µm) and vC:D
(871 vs. 687, p<0.001; 0.66 vs. 0.52, p<0.001, resp.); no significant
difference was found for vDD and cup depth (also after adjusting for
gender and race). In comparing non-Hispanic Whites vs. African
Americans, the latter had significantly larger vCuD (µm), vDD (µm),
and cup depth (µm), but not vC:D, after adjusting for term status (716
vs. 829, p=0.032; 1267 vs. 1332, p=0.046; 377 vs. 489, p=0.01; 0.57
vs. 0.62, p=0.18, resp.). In comparing genders, no significant
differences in optic nerve parameters were found after adjusting for
term status.
Conclusions: This is the first study using SDOCT to show a
significantly larger vC:D in preterm compared to term infants at 3742 weeks PMA, in contrast to prior papers that have reported similar
findings in school-aged children with a history of low birth weight.
The data show that prematurity impacts optic nerve development at a
very early stage.
Commercial Relationships: Amy Y. Tong, None; Ramiro S.
Maldonado, None; Du Tran-Viet, None; Adam L. Rothman,
None; Eric L. Yuan, None; Adam M. Dubis, None; Sandra
Stinnett, None; Mays A. El-Dairi, Prana pharmaceuticals (C);
Sharon F. Freedman, Pfizer, Inc. (C); Cynthia A. Toth, Genentech
(F), Bioptigen (F), Physical Sciences Inc. (F), Unlicensed (P)
Support: The Hartwell Foundation
Expression of the Lymphangiogenic Marker Podoplanin (D2-40)
in Human Fetal Eyes
Martina C. Herwig1, Kathrin Münstermann2, Ute Klarmann3, Karin
U. Loeffler1, Annette M. Müller2. 1Dept. of Ophthalmology,
University of Bonn, Bonn, Germany; 2Dept. of Pediatric Pathology,
University of Bonn, Bonn, Germany; 3Institute of Medical Biometry,
University of Bonn, Bonn, Germany.
Purpose: To characterize the expression of the lymphangiogenic
marker podoplanin (clone D2-40) in human fetal eyes with regard to
a maturation-dependent localization and expression and the
nondistinctive finding of lymphatic structures in the choroid.
Methods: 40 formalin-fixed paraffin-embedded eyes from 40 human
fetuses (age range: 10 to 32 weeks of gestation (WoG)) were
investigated. The cohort consisted of 22 male and 14 female fetuses
(gender was unknown in 4 cases). Immunohistochemical stains were
performed for D2-40 and - as control for endothelial reactivity - for
CD34. A semiquantitative analysis of immunoreactivity in different
segments of the eye was performed by light microscopy. The
intensity of staining was graded with a score ranging from 0 to 3.
Statistical analysis was performed with SPSS (IBM SPSS Statistics
20; Armonk, NY). The probability for the alpha error was set to p <
0.05.
Results: Podoplanin was expressed in the human fetal eye of 10 - 32
WoG. Immunoreactivity was found with a varying intensity in 39 of
40 eyes (97.5%). It was seen in vascular structures of the conjunctiva
as well as in conjunctival epithelium, chamber angle, and optic nerve
sheaths. In addition, labelling of the corneal endothelium was
detected in four eyes (10, 12, 17, 18 WoG). In some specimens (n =
14) a slight, equivocal staining reaction was noted in the choroid.
However, no definite intraocular lymphatic vessels resp. their
progenitors were found. There was no correlation of antigen
immunoreactivity and the gestational age except for the endothelial
reactivity that was found in fetuses younger than 19 WoG (p =
0.002).
Conclusions: Podoplanin (D2-40) is, even at an early gestational age,
expressed in several structures of the human fetal eye and the ocular
adnexae that are not necessarily associated with future lymphatic
vessels. Thus, it may play a role in the fetal ocular development. In
particular, D2-40 reactive structures were found in the choroid and
the chamber angle but no lymphatic vessels or their progenitors could
not be unequivocally identified at these sites.
Commercial Relationships: Martina C. Herwig, None; Kathrin
Münstermann, None; Ute Klarmann, None; Karin U. Loeffler,
None; Annette M. Müller, None
Canonical Wnt signaling is required for lacrimal gland
formation, eyelid closure and to suppress corneal cell fate in
mouse conjunctival and eyelid epithelium
Jie Huang1, Ying Liu1, David C. Beebe1, 2. 1Ophthalmology,
Washington University, Brentwood, MO; 2Cell Biology and
Physiology, Washington University, St. Louis, MO.
Purpose: Wnt signaling must be suppressed for the corneal
epithelium to differentiate. Failure to inhibit Wnt signaling in Dkk2
knockout mice caused the corneal epithelium became epidermis
(Mukhopadhyay, et al. Development 2006). Wnt signaling has also
been suggested to inhibit lacrimal gland formation (Dean, et al. Dev
Biol 2005). To better understand the function of Wnt signaling in
specifying ocular surface ectodermal derivatives, we examined the
cell fate of the corneal, conjunctival and eyelid margin epithelia in
mice deficient in canonical Wnt signaling.
Methods: Transgenic mice expressing Cre recombinase in the ocular
surface epithelia (LeCre) were mated to mice carrying floxed alleles
of the Wnt co-receptors, Lrp5 and Lrp6. Antibodies to Sox9 and
Aquaporin1 and antisense probes for Otx1, Barx2, Keratin12
andKeratin4 were used for immunofluorescence staining and in situ
hybridization.
Results: Lrp5/6 conditional knockout mice lacked lacrimal glands
and had open eyelids at birth. Sox9 expression, which we found was
required for lacrimal gland formation (2013 ARVO abstract by Chen,
Huang, Liu and Beebe), was decreased at E14.5 in Lrp5/6 conditional
knockout mice. The conjunctival and eyelid margin epithelia of
Lrp5/6 conditional knockout mice showed ectopic patches of
Keratin12 expression, a marker of corneal epithelial differentiation.
Aquaporin1, an early marker of the corneal stroma, was present
beneath the corneal epithelium, but not beneath the ectopic, K12positive cells.
Conclusions: Canonical Wnt signaling is required for lacrimal gland
formation and eyelid closure. Loss of Wnt signaling caused the
transdifferentiation of conjunctival and eyelid epithelium to corneal
epithelium. Suppressing Wnt signaling may assist in producing
corneal epithelium from stem cells.
Commercial Relationships: Jie Huang, None; Ying Liu, None;
David C. Beebe, FivePrime (C), Panoptica (C), Vistakon (Johnson
and Johnson) (C)
Support: Research was supported by an unrestricted grant from
Research to Prevent Blindness, US NIH grant EY04853, EY022643
and EY021505, and core grant EY02687.
Sox9 regulates the formation and branching morphogenesis of
mouse ocular glands
Ziyan Chen2, 1, Jie Huang1, Ying Liu1, David C. Beebe1, 3.
1
Ophthalmology and Visual Sciences, Washington University in St.
Louis, St. Louis, MO; 2Zhongshan Ophthalmic Center, State Key
Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou,
China; 3Cell Biology and Physiology, Washington University in St.
Louis, St. Louis, MO.
Purpose: Lacrimal, Harderian and Meibomian glands develop from
the prospective conjunctival and eyelid epithelia by branching
morphogenesis and produce secretions that lubricate and protect the
ocular surface. To better understand the mechanisms responsible for
their formation, we identified genes expressed selectively in the
prospective conjunctiva and tested the role of one of them, Sox9, in
the formation of the mouse ocular glands.
Methods: Microarray analysis was used to identify transcripts that
were differentially expressed in the prospective cornea, conjunctiva
and eyelid margin at E12.5. Laser microdissected tissues were
isolated, total RNA extracted, reverse transcribed and amplified using
the NuGEN Pico kit and hybridized to Illumina Mouse6 whole
genome bead arrays. Antibodies to Sox9 were used to map its
expression from E10.5 through birth. Transgenic mice expressing Cre
recombinase in the ocular surface epithelia (LeCre) were mated to
mice carrying floxed alleles of Sox9, Fgfr2 or Smad4. Embryos and
postnatal mice were fixed and processed for standard histology,
immunohistochemistry staining, in situ hybridization.
Results: Sox9 transcripts and protein were initially expressed
throughout the ocular surface epithelium at E10.5, but by E12.5 were
preferentially localized to the prospective conjunctival epithelium.
Sox9 expression decreased after E15.5, becoming undetectable in the
conjunctiva at birth. However, Sox9 expression remained in the
epithelium of the lacrimal/Harderian glands. Sox9 conditional
knockout mice lacked lacrimal/Harderian glands and had reduced
numbers of Meibomian glands in the lower eyelid. Mice
heterozygous for Sox9 had variable degrees of defective lacrimal
gland morphogenesis, including absence of the extraorbital (lacrimal)
lobe (62.5%) and small or ectopic branches (9.4%). Although the
epithelial component of the lacrimal/Harderian glands was absent in
Sox9-/- mice, Fgf10, which is required for lacrimal gland formation,
was still expressed in the adjacent mesenchyme. Sox9 expression was
decreased at E14.5 in Fgfr2 and Smad4 conditional knockout mice, in
which the lacrimal/Harderian glands do not form.
Conclusions: Sox9 is required for the formation of
lacrimal/Harderian glands and contributes to Meibomian gland
formation. Sox9 expression in the prospective conjunctiva appears to
be regulated by FGF and BMP signaling. Loss of one Sox9 allele
reduced branching of the lacrimal gland.
Commercial Relationships: Ziyan Chen, None; Jie Huang, None;
Ying Liu, None; David C. Beebe, FivePrime (C), Panoptica (C),
Vistakon (Johnson and Johnson) (C)
Support: EY022643, EY04853, unrestricted grant from RPB, Core
grant EY02687, China State‐Sponsored Postgraduate Study Abroad
Program
Identification of Signaling Pathways involved in Retina-Lens
Tissue Interactions using Embryos Over-Expressing Pairedless
Pax6 (Pax6ΔPD) Gene
Vijay K. Kalaskar1, James D. Lauderdale2. 1Neuroscience Division of
Biomedical & Health Sciences Institute, University of Georgia,
Athens, GA; 2Cellular Biology, University of Georgia, Athens, GA.
Purpose: To identify and evaluate the role of signaling pathways
involved in lens cell survival and retinal development using mouse
embryos over-expressing Pax6ΔPD in a whole embryo culture
system.
Methods: Mouse embryos were obtained from our breeding colony
with noon on the day of plug discovery designated as embryonic day
0.5 (E0.5). Wild-type (WT) embryos were CD1-C57BL/6J. Embryos
overexpressing Pax6ΔPD were obtained as described by Kim and
Lauderdale (2008). Embryos were evaluated for differential
expression for genes involved in different signaling pathways by
qRT-PCR and in situ hybridization (ISH). Of the genes altered, we
first tested the BMP4 signaling by implanting affi-gel agarose beads
treated either with recombinant BMP4 or Noggin proteins in the eye
region of embryos at E10.5 cultured in a standardized serum free
medium. Contralateral eyes implanted with BSA protein treated
beads were used as control. Eye tissues from these embryos cultured
for 18 - 20 hours were analyzed for development and differential
gene expression using immunohistochemistry, western blot, qRTPCR and ISH.
Results: Mouse embryos overexpressing Pax6ΔPD exhibited
apoptotic degeneration of lens cells and analysis of this ocular tissue
revealed up-regulation of BMP4 target genes indicating a possible
role for BMP4 signaling in lens cell survival. When tested in a mouse
embryo culture by implanting BMP4 treated beads in the ocular
region in WT embryos, it did not result in lens cell apoptosis.
Similarly, Noggin treated bead implantation in Pax6ΔPD
overexpressing mouse embryos did not prevent the apoptotic
degeneration of the lens tissue indicating that BMP4 signaling may
not be involved in lens cell survival. However, BMP4 beads in these
embryos inhibited the pigmentation in the retinal pigmented
epithelium (RPE) when implanted at ~30 somite stage (ss) and
decreased pigmentation at ~35ss and as well affected the dorsal-
ventral (D-V) patterning in the retina. Preliminary analysis revealed a
5-fold increase in SOX2 expression apart from other BMP4 target
genes in the BMP4 treated eyes compared to the BSA treated eyes.
Conclusions: BMP4 signaling may not have a role in lens cell
survival. However, it affects the pigmentation in the RPE in a stagedependent manner and has a role in D-V patterning of the retina
indicating a potential role for BMP4 in RPE and retinal development.
Commercial Relationships: Vijay K. Kalaskar, None; James D.
Lauderdale, None
Support: 1) Children's Glaucoma Foundation, 2) Sharon Stewart
Aniridia Research Trust
Topical small molecule translational bypass therapy rescues Pax6
aniridia mutant phenotype
Cheryl Y. Gregory-Evans, Xia Wang, Andrew Metcalfe, Xianghong
Shan, Kevin Gregory-Evans. Ophthalmology, University of British
Columbia, Vancouver, BC, Canada.
Purpose: Congenital aniridia caused by haploinsufficiency of PAX6,
is characterized at birth by absence of the iris, cataracts and foveal
hypoplasia, with later development of glaucoma and corneal
keratopathy. The commonest type of mutation in PAX6 is nonsense
mutations leading to a premature stop codon, present in 40% of
aniridia cases. In this study we tested the efficacy of the small
molecule drug therapy aimed at bypassing stop mutations to
ameliorate the phenotype of the heterozygous Pax6Sey mouse model.
Methods: Pax6Sey heterozygous pregnant mice were treated daily
from E12.5 with subcutaneous injections of either gentamicin or
Ataluren and then the offspring received the same treatment until
P21. In an alternate postnatal only paradigm, Pax6Sey heterozygous
mice received daily drug therapy by either subcutaneous injection or
topical eyedrops from P4-P60. Drug efficacy was tested by histology,
electrophysiology, optokinetic tracking, and quantitative PCR.
Results: Treatment of Pax6Sey pregnant mice with either gentamicin
or Ataluren rescued the proliferative defect in the retina and
normalized the lens, iris and cornea defects in the Pax6Sey mutant
offspring. Early postnatal treatment of Pax6Sey mice either by
systemic or eyedrop delivery similarly rescued the histological
phenotype. Untreated Pax6Sey mice do not exhibit ERG responses
however, the treated mice showed characteristic dark-adapted rod
responses, dark oscillatory potentials, photopic cone responses and a
30Hz flicker that were 70-90% of wildtype responses. In treated
mice, optokinetic tracking demonstrated a spatial threshold frequency
of 0.41 cycles/degree, equivalent to normal mice. In untreated mice,
gene expression changes in Foxc1, Tgfβ2 and Bmp4 in the developing
Pax6Sey iris were normalized after drug treatment.
Conclusions: Translational bypass therapy used in a prenatal or
postnatal paradigm rescued the histological, molecular and functional
defects observed in the Pax6Sey mutant eye. Using a targeted drug
therapy to overcome the underlying Pax6 genetic defect by a
postnatal strategy is the first evidence that it is possible to correct
developmental abnormalities in congenital conditions such as
aniridia. Since Ataluren is an orally bioavailable drug that does not
have the ototoxic and nephrotoxic side effects associated with
gentamicin, it may provide a treatment option for aniridia patients.
Commercial Relationships: Cheryl Y. Gregory-Evans, None; Xia
Wang, None; Andrew Metcalfe, None; Xianghong Shan, None;
Kevin Gregory-Evans, None
Support: Sharon Stewart Research Award

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group

Transcription

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