Analysis of HER2/neu Gene Amplification in Microdissected Breast

Analysis of HER2/neu Gene Amplification in Microdissected Breast
Cancer Tumour Samples
Rishie Seth 1, Angelika Burger 2, and Harriette J. Kahn 1
1
Department of Anatomic Pathology, Sunnybrook and Women’s College Health Sciences Centre and Department of
Laboratory Medicine and Pathobiology, University of Toronto
2
Department of Molecular and Cellular Biology, Sunnybrook and Women’s College Health Sciences Centre
Abstract:
One in nine women in Canada will develop breast cancer during their lifetime. Generally, breast cancer progresses
sequentially from atypical hyperplasia to ductal carcinoma in situ (DCIS) to an invasive stage, which eventually
metastasizes to distant sites. Our laboratory has been interested in studying the role of HER2/neu amplification and its
correlation with breast cancer progression. The HER2/neu oncogene has been reported to be amplified in >20% of
sporadic breast tumours. However, it is not clearly understood at what stage HER2/neu amplification occurs. In order to
investigate HER2/neu status in different stages of tumour progression, we chose to utilize the Laser Capture
Microdissection (LCM) system to isolate pure cell populations for gene amplification analysis. Five-micron formalin
fixed paraffin embedded tissue sections from an archival collection at the Sunnybrook and Women’s College Health
Sciences Centre were stained with hematoxylin and eosin, to visualize cell morphology. The Pixcell IIe Laser Capture
Microdissection System was used to capture specific tumour cell populations. Desired tissue areas were identified and
captured on a plastic film after pulsing the region with laser light for a duration of six milliseconds, at a power of twentyone to thirty-three milliwatts. Genomic DNA was isolated by a modified proteinase K- phenol extraction method. β-globin
was used as a housekeeping gene, and HER2/neu as the primary oncogene in this study. The control genomic DNA was
obtained from the HT29 tumour cell line. We preformed a serial dilution of control genomic DNA to determine the lower
limit of HER2/neu gene detection. Tissue samples corresponding to normal, ductual carcinoma in situ (DCIS), and
invasive ductal carcinoma (IDC) from nine samples were dissected. PCR analysis showed that we were able to amplify βglobin gene fragments from all the cases. Preliminary data revealed that one of six cases examined had HER2/neu gene
amplification, which is in agreement with reported studies. Our results show that Laser Capture Microdissection is a
powerful technique for isolating pure populations of cells from paraffin embedded tissue sections and that these cells can
be used to prepare DNA, RNA, or protein for molecular applications.
Introduction:
Breast cancer is one of the leading causes of death in women in Canada. Generally, breast tumour growth begins when a
specific cell sustains a somatic mutation that causes it to proliferate uncontrollably. Breast tumour development
progresses in several stages, from atypical hyperplasia, where there is abnormal cell growth to in situ cancer where the
growth is still confined in the ducts, and eventually to invasive cancer where the tumour cells begin to invade underlying
tissue and shed cells into the lymphatics or blood vessels (Figure 1) . Breast tumour tissue consists of a heterogeneous
mixture of cell types ranging from normal cells such as, stroma, blood, and fat to cancer cells at various stages of
progression. The obvious problem with these diverse tumour samples is to isolate pure cell populations for molecular
analysis. Current conventional methods utilize DNA/RNA extraction protocols where whole tumour sections are
processed without any separation. If the ratio of tumour tissue to normal tissue in a given sample is small, the data may
become obscure. For this reason, we chose to utilize laser capture microdissection (LCM) a novel technique for obtaining
pure cell populations (5).
Figure 1: Breast cancer progression – The above figure provides a basic view of breast cancer progression from normal ductal tissue
to ductal carcinoma in situ to invasive ductal carcinoma to metastatic cancer. The first image (a) shows normal breast tissue with
adjacent lymph nodes (4). The next image (b) demonstrates the normal breast ducts (purple) surrounded by stroma (pink). The next
stage is DCIS (c), in which there is the proliferation of abnormal cells confined to the ducts by an intact basement membrane (BM).
The next stage (d) is invasive ductal carcinoma (IDC) NOS*, containing nests of tumour cells with no interfering stroma. In this stage
the tumour extends beyond the BM into surrounding tissue. Subsequently, tumour cells are shed into the regional lymph nodes. It is
possible for the tumour cells to metastasize to distant sites in the body.
*(not otherwise stated)
The laser lifts desired tissue onto a plastic film attached to a Capsure Macro LCM cap (5). The tissue captured by the laser
can than be used for a variety of applications, including genomic DNA extraction and RNA/protein purification. The laser
is set at a specific wavelength to ensure that the tissue sections remain undamaged. The point and shoot method allows for
accuracy beyond any other current microdissection techniques. The process consists of several sequential steps that
eventually produce high quality microdissected tissue, which is suitable for gene analysis (Figure 2).
Figure 2: Laser capture microdissection system technology –In the top panel (a) the Capsure Macro LCM cap is shown just above
the cells of interest. The ‘cap’ is swung into the position of interest by the cap arm, which securely holds the cap above the tissue. The
cap arm is designed so that the cap never actually comes in direct contact with the tissue. The centre figure (b) shows the laser pulse
activating the film; this is the actual process by which the tissue becomes attached to the cap. This technique is of high precision and
accuracy in that the tissue is undamaged and attached to the film. The bottom panel (c) shows the area of interest after the cells have
been lifted and attached to the cap. The cells have been physically lifted from their previous position and are now securely fastened to
the cap. (6)
We utilized this technique to microdissect non-invasive and invasive breast carcinomas to determine the status of
oncogenes during breast cancer progression. Proto-oncogenes play major roles in converting normal cells to tumourgenic
cells and can be activated through translocation, promoter insertion, or gene amplification. One such proto-oncogene is
the tyrosine kinase receptor, HER2/neu that is overexpressed in breast cancer as a result of gene amplification (3).
HER2/neu is the only oncogene for which there is a specific molecular therapy available (1). The monoclonal antibody,
Herceptin is a therapy developed to treat women who have tumourous tissue in the metastatic phase (1). This therapy
functions to treat patients with an overexpression in HER2/neu by attaching itself to the HER2 protein, which restricts the
epidermal growth factor ligand from reaching the breast cancer cells (1). Ultimately, this blocks the ability of cells to
divide and grow. There are several methods to determine the amplification or overexpression of HER2/neu, which include
fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH), or polymerase chain reaction (PCR)
amplification for detection of amplification or immunohistochemistry to detect overexpression (3). Our study utilized the
PCR amplification method to assess data on HER2/neu amplification.
Objectives:
•
•
To Microdissect different stages of breast cancer using the laser capture microdissection system
To analyze gene amplification in pure cell populations
Materials and Methods:
Tissue samples: Paraffin embedded breast biopsies were obtained from the Department of Anatomic Pathology at
Sunnybrook and Women’s College Health Sciences Centre database. Tissues were previously fixed in formalin for 16 to
24 hours. Five micron sections were cut from the paraffin blocks and mounted on glass slides. Sections were prepared
with a standard protocol for hematoxylin and eosin (H and E) staining. Areas of normal ductal tissue, ductal carcinoma in
situ (DCIS), and invasive ductal carcinoma (IDC) were identified from coverslipped slides, and matched against H and E
stained serially sectioned slides, that were not coverslipped.
Laser capture microdissection: The Pixcell IIe Laser Capture Microdissection system was used for microdissection. A
low-power infrared laser was used to melt 30 µm pulse sizes of special thermoplastic film over the cells of interest. The
optimal settings for power and duration for capturing cells ranged between 21-33 mW and 6-9 milliseconds, respectively.
In denser tissue, the cells were harder to lift so the same area was dissected multiple times to ensure the removal of all the
cells of interest, and the duration setting was increased to over 10 milliseconds. After microdissection, the cap containing
the captured cells was placed on the special Capsure clean up pad to remove any cells that were removed from the tissue
inadvertently. The cap was then fit into a 0.5mL GeneAmp PCR reaction tube with an Arcturus cap insertion tool.
DNA extraction: Each GeneAmp reaction tube was filled with 100 µL of DNA digestion buffer (10mM Tris–HCl (pH
8.0), 1mM EDTA (pH 8.0), and 1% Tween 20) and 10 µL of proteinase-K (0.05%). Once the cap was inserted into the
reaction tube, the tube was then inverted to allow contact between the buffer and the cells on the cap. The reaction tubes
were then incubated at 65 °C for 16 to 24 hours and centrifuged for one minute at 1,000 x g. The cap was removed from
the tube and the solution was heated to 95 °C for 10 min to inactivate proteinase-K. To ensure the cleanliness of the
purified DNA, phenol extraction followed by ethanol precipitation was carried out.
Genomic DNA Analysis: The first pair of HER2/neu primers had the following sequence: forward - ata tcc agg agg tgc
agg g, reverse: ctt cga agc tgc agc tcc c. The second pair of primers (nested) had the following sequence: forward - ctc aca
acc aag tga ggc ag, reverse - cag ggg tgg tat tct tca. The annealing temperatures were calculated based on the lowest
melting temperature for the pair of primers. The purified genomic DNA was amplified by PCR with the following
conditions: initial denaturing at 96 °C for ten minutes, 35 cycles of denaturing at 96 °C, annealing at 58 °C, and
elongation at 72 °C. The PCR product was then analyzed by gel electrophoresis. We also utilized a nested PCR technique
to increase the yield of amplified DNA, so it can be visualized by gel electrophoresis. The first pair of primers amplified
exon 3 for an expected size of 205 bp, while the nested primers were expected to produce a size of 126 bp. From the first
PCR amplification, 4 µL of the product was used as template for the second PCR reaction. During the first round of PCR,
the annealing temperature was adjusted to 53 °C and then to 58 °C for the second PCR.
Figure 3: Pathological data on cases for microdissection
KEY
*Invasive Ductal Carcinoma
** Ductal Carcinoma In Situ
*** Fluorescence In Situ Hybridization
+ Not Done
++ 3/3 Nuclear Grade
Case
Tumour
Number Type
Tumour In Situ
Size
Component
Histological Estrogen
Grade
Receptor
(modified
Bloom and
Richardson)
1
IDC*
3 cm
2/3
2
IDC/DCIS** 4 cm
3
DCIS
4
IDC
Progesterone Overexpression of
Expression
Receptor
HER2/neu – By
of
Immunohistochemistry HER2/neu
– By PCR
<Borderline Positive
Positive
Negative
Negative>
Present (40% 3/3
DCIS)
Negative
Negative
Negative
Negative
3.2 cm
Extensive
DCIS
3/3
Positive
Positive
ND+
Negative
2.5 cm
Present
3/3
Negative
Negative
Positive by FISH***
Positive
5
Invasive
1.2 cm
Lobular
Carcinoma
Present
2/3
Positive
Positive
ND
Negative
6
IDC/DCIS
1.6 cm
DCIS
3/3
Negative
Negative
Positive
Positive
7
DCIS
Less
than
1.0
mm
High
DCIS
++
N/A
N/A
ND
Negative
8
IDC
2.3 cm
Present
2/3
Positive
Positive
Negative
ND
9
IDC
1.5 cm
Lobular/DCIS 2/3
Positive
Positive
Negative
ND
Absent
Grade
Figure 3: Pathological data on cases for microdissection – The following data was obtained on the breast cancer cases: tumour type,
tumour size, histological grade, estrogen and progesterone receptors, and HER2/neu status. Also, the HER2/neu status was recorded
to compare HER2/neu amplification by standard PCR.
Results:
Our first aim was to microdissect pure cell populations from different stages of breast cancer using the laser capture
microdissection system. We obtained tissue samples from the Sunnybrook and Women’s College Health Sciences
Centre’s archival paraffin embedded tissue bank. The following information was obtained, tumour type, tumour size,
histological grade, estrogen and progesterone receptors, and HER2/neu status (Figure 3 and 4). We obtained serially
sectioned slides from nine cases: 2 cases were pure DCIS, 6 cases were invasive ductal carcinoma, and of the six cases
that were IDC, 2 also contained DCIS. Depending on the tissue quality, a selected area was pulsed three times for six
milliseconds each time (Figure 5 and 6). We noticed that when the tissue was completely dry, the cells of interest were
easily dissected, and little or no contamination from neighbouring normal cells was found. On the other hand, poor quality
tissue was pulsed more than three times for a longer duration to ensure complete lifting of tissue. Although, with breast
tissue, it is difficult to obtain good quality dissections because of tissue heterogeneity, we were able to capture all the
nuclei from different stages of breast cancer by pulsing a specific area several times (Figure 5 and 6). Our second aim
was to analyze gene amplification in pure cell populations from various stages of breast cancer. We chose to examine
HER2/neu because previous reports indicate that it is amplified in >20% of breast cancer (3). DNA was isolated from pure
cell populations of normal tissue, and DCIS and invasive breast carcinomas (materials and methods). To investigate the
lower limit of DNA that could be observed by our PCR protocol as described in the materials and methods; we amplified
the HER2/neu gene using serially diluted cancer cell line DNA.
Figure 4: Detection of HER2/neu overexpression by immunohistochemistry – This case demonstrates strongly positive membranous
staining for HER2/neu using the antibody CB11, indicating overexpression of HER2/neu.
Figure 5: Ductal carcinoma in situ (DCIS) microdissected tissue samples – The figure demonstrates a high grade DCIS section of a
tumour magnified at 20X. a) The DCIS section of the tumour is composed of a nest of tumour cells confined to the ducts with adjacent
fat, stroma, and lymphocytes. b) The area of interest was lifted by the LCM technique, but the necrosis was removed inadvertently by
the cap. However, the capsure cleanup pad was used to remove cells that were not directly lifted by the laser pulse.
Figure 6: Invasive ductal carcinoma-microdissected – Both images, magnified at 20X, show H and E stained sections of an invasive
ductal carcinoma NOS: a) The tumour is composed of nests of tumour cells surrounded by stroma. The aim for this specific dissection
was to avoid the stroma tissue, and focus on lifting the purest tumour tissue as possible. b) Pure areas of the tumour were lifted by the
LCM technique.
PCR amplification was examined by agarose gel electrophoresis. From this it was evident that 1.725ng was sufficient to
visualize HER2/neu bands (Figure 7, Lane # 7). We estimated that each Capsure Macro LCM cap had DNA
corresponding to 150 cells. From this data, we estimated that 1700pg of DNA was present on the cap before dilution into
the digestion buffer (1 cell =~ 10pg of DNA) (2). The HER2/neu gene was amplified by standard PCR using forward and
reverse primers (materials and methods). Figure 8 shows faint HER2/neu specific bands in lanes 3 (IDC), 4 (normal), and
5 (DCIS) (Figure 8). Since we were unable to see clear bands, we performed a second round of PCR using nested primers
to re-amplify the HER2/neu fragment. By performing two rounds of PCR we were successful in producing single
HER2/neu specific bands. The nested PCR technique involves amplifying a segment by standard PCR, and then reamplifying the PCR product using internal (nested) primers (Figure 9). >From the nine cases that we microdissected, two
of them showed overexpression of HER2/neu by PCR (Figure 10). One of the HER2/neu bands was purified and
confirmed by DNA sequence analysis (Figure 11). When HER2/neu gene is overexpressed it corresponds to the 34.5ng
band in the serial dilution experiment, and when HER2/neu is normal we see very faint bands, even by nested PCR. In
addition, we observed HER2/neu amplification in one case in the IDC/DCIS stage of the tumour tissue, and in one case
that was purely IDC, which corresponds to 22% of the cases showing HER2/neu amplification. In every case, normal
tissue was negative for HER2/neu amplification. Of the two cases that exhibited DCIS and IDC carcinomas, one was
HER2/neu positive for amplification in both stages, and one was negative in both stages. Overall, our study is in
agreement with previous studies that investigated HER2/neu expression by FISH or immunohistochemistry.
Figure 7: DNA dilution scheme on HT29 cancer cell line – We used the HER2/neu primer to test the lower limit of DNA that could
be detected by our DNA purification method. In the second lane, the New England Biolabs 100 bp ladder was used as a marker to
measure the size of the PCR products. The third to the seventh lanes display a decreasing range of DNA from the cell line, beginning
with 34.5ng of DNA as the highest level and 1.725ng of DNA as the lowest level. It is evident that the intensity of the bands decreases,
as less DNA is present. Since the band from the seventh well is fairly strong, we hypothesize that less than 1.725ng of DNA can be
detected by the performed method of DNA purification. Lane 8 displays a negative control, which ensures there is no primer
contamination.
Figure 8: Analysis of microdissected tissue by standard PCR – PCR amplified products were analyzed by 2% agarose gel
electrophoresis and ethidium bromide staining. The second lane contains the 100 bp New England BioLabs ladder, and shows
markers at 100 and 200 bp. The next four lanes, 3 (IDC), 4 (normal tissue), 5 (DCIS), and 6 (IDC) displays microdissected tissue at
various stages with the top band being the HER2/neu amplified fragment. The bottom band in these four lanes seems to be a nonspecific signal. The seventh lane contains the positive control DNA obtained from a cancer cell line. This strong band is estimated to
have a size of 126 bp. Lane 8 contains microdissected DNA from a case of pure DCIS. This lane contains two full caps of
microdissected tissue, as compared to lane five, which has the same tissue, but only one full cap. The last lane contains the negative
control, ensuring no primer contamination.
Figure 9: Schematic diagram of nested PCR – The first pair of primers amplifies the HER2/neu target DNA by standard PCR (first
amplicon). The second pair of primers (nested primers) binds within the first PCR product to produce a fragment shorter in length
(second amplicon). The result is numerous copies of amplified fragments of HER2/neu target DNA.
Figure 10: Detection of HER2/neu overexpression by nested PCR – This figure demonstrates the overexpression of HER2/neu in one
case vs. the normal expression of HER2/neu in another case. This data is the result of a nested PCR using the HER2/neu genes from
exon 3. The first lane depicts the New England Biolabs 100 bp ladder. The next lane (2) is the positive control from the cancer cell
line, HT29. The next three lanes (3,4,5) contains the PCR product of invasive microdissected genomic DNA, which is overexpressed.
The next two lanes (6,7) show the microdissected tissue of a tumour with no HER2/neu amplification. In these lanes there are no
prominent bands that are comparative to the previous three lanes, indicating no overexpression. Lane 8 shows the negative control
and verifies no primer contamination.
Figure 11: Electropherogram obtained from ABI 3100 Genetic Analyzer – A HER2/neu DNA band was extracted from lane 10 in
figure 10 and purified using a gel extraction kit and sequenced using the ABI DNA sequencing kit. Samples were loaded into the ABI
3100 Genetic Analyzer and the results are shown in panel A. We carried out a blast analysis of the DNA sequence in the National
Center for Biotechnology Information’s (NCBI) Genbank and found that the sequence corresponds to the HER2/neu gene fragment on
exon 3, as shown in panel B.
Conclusion:
Our study illustrates that the LCM technique can be successfully used in providing ample DNA for gene analysis by PCR.
We were able to show amplification of HER2/neu gene with DNA isolated from pure cell populations by LCM. Our
results indicated that 22% of the cases studied were positive for HER2/neu amplification, which corresponds to the
literature regarding HER2/neu amplification/overexpression. In addition, HER2/neu amplification could be detected not
only in the invasive stage, but as early as the DCIS stage. Moreover, LCM can be also used for different tissue types
including, kidney, lung, and prostate. Total RNA and genomic DNA isolated from these pure cell populations can be used
for a variety of studies including, microarray assessments and LOH analysis.
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