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Abstract
Background Gastric cancer is a leading cause of cancer-related mortality, and current treatment outcomes for advanced disease remain poor. HER2 has been identified as a potential candidate for targeted therapy in gastric cancers displaying HER2 gene amplification and protein overexpression.
Aims To study the prevalence rate of HER2 gene amplification/overexpression in a local population, and determine the concordance rate between the various modalities.
Methods 128 gastric cancer samples were analysed by fluorescence (FISH) and chromogenic (CISH) in situ hybridisation and immunohistochemistry (IHC). The relation between HER2 status and various clinicopathological parameters was also analysed.
Results 11.7% (15/128) and 9.4% (12/128) of gastric cancers displayed HER2 gene amplification and protein overexpression (score 3+), respectively, with a perfect correlation between the FISH and CISH analyses. There was also a significant inverse correlation between overall survival and HER2 protein overexpression in intestinal-type gastric carcinomas (p<0.05).
Conclusion Results, besides corroborating existing reports that discrepancies exist between HER2 ISH and IHC assays, also suggest the need to rigorously evaluate CISH as an independent reference standard for assessment of HER2 amplification in gastric cancers.
- Gastric cancer
- HER2
- fluorescence in situ hybridisation (FISH)
- chromogenic in situ hybridisation
- immunohistochemistry
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- Gastric cancer
- HER2
- fluorescence in situ hybridisation (FISH)
- chromogenic in situ hybridisation
- immunohistochemistry
Introduction
Gastric cancer is one of the most prevalent cancers and is a leading cause of cancer-related mortality.1 To date, treatment outcomes for gastric cancer remain poor, particularly for patients who present with advanced disease.2 There is a need to identify and develop effective therapeutic regimes for such patients.
The advent of targeted therapeutics has led to a paradigm shift in cancer treatment.3 HER2 is the prototype of a target for which an effective targeted therapeutic, in the form of a monoclonal antibody (trastuzumab), has been developed.4 HER2 belongs to the epidermal growth factor receptor family of tyrosine kinase receptors that drive tumourigenic pathways such as cell survival, invasion, metastasis and angiogenesis.5 HER2 gene amplification and protein overexpression were first identified in around 30% of breast cancers,6 and were subsequently similarly demonstrated in various tumour types including gastric cancers.7–9 In some studies, a correlation between HER2 status and survival has been reported in gastric cancers.10
Trastuzumab in combination with standard chemotherapy displays efficacy against HER2 gene-amplified/protein-overexpressing primary11 12 and metastatic breast cancers,13 and recent studies show that this might similarly hold true for gastric cancers.14 In our current study, one objective was to determine the prevalence of HER2 gene amplification by fluorescence in situ hybridisation (FISH) and chromogenic in situ hybridisation (CISH), and HER2 protein expression by immunohistochemistry (IHC) in our local cohort of gastric cancers, and to evaluate the concordance rate between results obtained from these various modalities. Another objective was to study the correlation between HER2 status and various clinicopathological parameters.
Materials and methods
Study population
Clinicopathological data were obtained from 190 patients who had undergone surgical resection for histologically proven gastric cancer at the National University Hospital of Singapore between 1 January 2000 and 31 December 2008, representing the entire population of surgically operable gastric cancer patients at our institution who had given consent for research. The results were statistically analysed against available clinicopathological criteria—gender, age, tumour size, tumour stage, histological grade, vascular invasion, perineural invasion, lymphatic invasion and survival time. Survival was measured from the date of diagnosis until the date from death from gastric cancer, or was censored until the date of the last follow-up for non-gastric cancer related deaths and survivors. The study was carried out in the context of a research project approved by the National University of Singapore Institutional Review Board (NUS-IRB 06-063).
Tissue microarray construction
A total of 190 formalin-fixed, paraffin-embedded samples of gastric cancer with matched normal tissue were obtained from the archives of the Department of Pathology, National University Hospital. Tissue cylinders with a diameter of 1 mm were punched from tumour and normal areas of the donor tissue blocks and deposited into a recipient block using a tissue-arraying instrument (Beecher Instruments, Silver Spring, Maryland, USA).
Immunohistochemistry
HER2 IHC was performed on 4 μm thick array sections using an automated slide stainer, BenchMark XT (Ventana Medical Systems, Arizona, USA) and ultraViewTM DAB detection kit (Ventana Medical Systems, Arizona, USA). The primary antibody was anti-HER2/neu rabbit monoclonal antibody (clone 4B5) (Ventana Medical Systems, Arizona, USA). Slides were scored by a pathologist (BP) according to the recommendations by a consensus panel15 as follows: 0, no reactivity, or membranous reactivity in <10% of cells; 1+, faint/barely perceptible membranous reactivity in >10% of cells, cells are reactive only in part of their membrane; 2+, weak to moderate complete or basolateral membranous reactivity in >10% of tumour cells; 3+, moderate to strong complete or basolateral membranous reactivity in >10% of tumour cells.
Fluorescence in situ hybridisation
Array sections (4 μm) were treated according to the PathVysion HER2 DNA Probe Kit and Paraffin Pretreatment Kit II (Vysis, Abbott Molecular, Des Plaines, IL, USA) protocol, being subjected to pretreatment solution at 80°C for 15 min followed by protease solution for 45 min at 37°C. The slides were analysed using an Olympus BX61 microscope equipped with DAPI, Spectrum Orange, Spectrum Green and double-band pass filters for simultaneous visualisation of the Spectrum Green and Spectrum Orange signals. Images were captured using CytoVision version 3.93 software (Applied Imaging, Newcastle upon Tyne, UK). The relative ratio of the orange HER2/neu gene signals to the green CEP-17 signals was evaluated. A ratio above 2.2 and less than 1.8 indicated amplification and non-amplification, respectively. A ratio between 1.8 and 2.2 was interpreted as borderline.
Chromogenic in situ hybridisation
Array sections (4 μm) were treated according to the SPOT-Light HER2 CISH Kit (Invitrogen, Camarillo, CA, USA) protocol with extended enzyme pretreatment time to 20 min. HER2 amplification was defined as the presence of more than five dots, or small clusters, or large clusters per nucleus, in more than 50% of tumour cells. Non-amplification was defined as the presence of less than six dots per nucleus in more than 50% of tumour cells.
Statistical analyses
The Kaplan–Meier method was used for estimating survival rates with the group differences assessed by log-rank test. Statistical analyses were performed using SPSS (V.15.0 for Windows) at the 5% significance level.
Results
A total of 128 of 190 samples, comprising 76 intestinal, 29 diffuse and 23 mixed gastric carcinomas could be evaluated by all three assays. In total, 11.7% (15/128) of gastric cancers displayed HER2 gene amplification (by FISH and CISH) (figure 1). Among these 15 cases, 10 displayed an HER2 IHC score of 3+, two displayed a score of 2+, one displayed a score of 1+, and two displayed a score of 0. The five cases which showed HER2 gene amplification but not HER2 IHC 3+ scores were considered discrepant cases. To ascertain if these discrepancies were genuine, or artifactual as a result of tissue microarray analysis, a repeat evaluation on the full sections of these five cases was performed. Of these five cases, three retained their original scores on repeat IHC, while two cases (originally 0 and 2+) were found to be 3+ on full section IHC. The discrepancy originally seen with the tissue microarray analysis was noted to be due to heterogeneous HER2 expression in the full sections.
Representative examples of HER2 immunohistochemistry (IHC), fluorescence in situ hybridisation (FISH) and chromogenic in situ hybridisation (CISH) in gastric cancer. (A–C) Sample with IHC score 0, HER2 gene non-amplification. (D–F) Sample with IHC score 3+, HER2 gene amplification. Orange and green signals in FISH represent HER2 and CEP-17, respectively; brown signals in CISH represent HER2. Original magnification 400× (A,D), 1000× (B,C,E,F).
Tables 1 and 2 show the final analyses. A perfect correlation between the FISH and CISH analyses was observed (table 3). When analysed by the Lauren's classification, 15.8% (12/76) of intestinal, 13% (3/23) of mixed and 0% (0/29) of diffuse tumours displayed HER2 gene amplification. Among the mixed cancers displaying amplification, all three cases displayed amplification in the intestinal component, while one displayed amplification in the diffuse component.
Concordance between fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) results
Concordance between chromogenic in situ hybridisation (CISH) and immunohistochemistry (IHC) results
Concordance between fluorescence in situ hybridisation (FISH) and chromogenic in situ hybridisation (CISH) results
In our analyses of various clinicopathological parameters with HER2 status, we found a significant inverse correlation between HER2 protein overexpression (3+) status and overall survival in intestinal-type gastric cancers (p<0.05).
Discussion
Our findings with regard to the prevalence of HER2 gene amplification and protein overexpression are similar to figures reported in the literature (generally around 10–20% and 10–30% for gene amplification and protein overexpression, respectively15). Our data are also consistent with the observation that HER2 amplification is more prevalent in intestinal-type carcinomas than diffuse-type carcinomas,10 16–18 and that HER2 gene overexpression status correlates inversely with overall survival in a population of gastric cancers.10
Our study is important for several reasons. First, it highlights that, although generally correlated, discrepancies exist between HER2 gene amplification and protein expression levels in gastric cancer, which is in line with the existing literature.15 Such discrepancies are well documented in breast cancer, and are related to a variety of factors, both biological and technical. In breast cancer, HER2 gene amplification correlates better with responsiveness to trastuzumab or lapatinib treatment than HER2 protein overexpression.19 At present, it is not known whether this similarly holds true with regard to gastric cancer.
Second, we observed homogeneous HER2 gene amplification but heterogeneous HER2 protein expression in certain samples subjected to repeat full section analyses (as detailed above). These observations are consistent with the literature,20 21 and have important clinical implications if HER2 IHC is employed as a triaging or predictive assay for trastuzumab therapy, as false negatives might arise particularly when insufficient material is examined, such as in gastric biopsy specimens.
Last, we observed a perfect correlation between the FISH and CISH results. To the best of our knowledge, there is at present no study that has reported a systematic analysis to determine the concordance rate between HER2 FISH and CISH assays in gastric cancer. We feel that this is an important area for further investigation.
Conventionally, detecting HER2 gene amplification by FISH is considered the reference standard in breast cancer.22 However, FISH suffers from the following drawbacks: the analysis is labour intensive and time consuming, requiring a fluorescence microscope and specific observer training; the photolability of the probes mandates storage of data in the form of archival micrographs, which necessitate the incorporation of a high-resolution digital camera; and the correlation with tumour morphological features, such as tumour heterogeneity, is difficult with FISH. In contrast, the analysis of CISH is less time consuming because it can be performed with a bright field microscope, yields a more long-lasting record than FISH, and allows for correlation of amplification with morphological features.23
One proposed drawback of CISH is that it does not allow for distinction between polysomy and low level amplification due to the lack of a comparison centromere probe. In breast cancers however, several studies have reported that true polysomy of chromosome 17 is either a very rare or non-existent event, and that multiple chromosome centromere 17 (CEP17) signals are due to amplification of the centromeric region as well.24 25 Since the reliance on CEP17 signals for determination of amplification might be misleading, the theoretical concern that CISH does not distinguish between polysomy and amplification is of little significance in practice, at least in breast cancers. Similar studies will be required to determine the prevalence and therapeutic implications of chromosome 17 polysomy in gastric cancers.
In summary, based on the various factors discussed above and the results of our study, it appears that CISH has many merits as a potential diagnostic assay for determination of HER2 gene amplification in gastric cancers and should be rigorously evaluated as a reference standard independent of FISH.
Take-home messages
HER2 amplification or protein expression status may predict responsiveness to trastuzumab in gastric cancer, and although the amplification and protein expression statuses are generally correlated, discrepancies exist.
The strong concordance rate between fluorescence in situ hybridisation and chromogenic in situ hybridisation results suggest the need to rigorously evaluate CISH as an independent reference standard for determination of HER2 amplification in gastric cancers.
References
Footnotes
BY and YEX contributed equally to this work.
Funding MS-T receives funding support from SCS grants MN-05, MN-05R and MN-77, awarded by the Singapore Cancer Syndicate, Agency for Science, Technology and Research, Singapore, Cancer Science Institute of Singapore and the Singapore Gastric Cancer Consortium.
Competing interests None.
Ethics approval This study was conducted with the approval of the National University of Singapore Institutional Review Board (NUS-IRB 06-063).
Provenance and peer review Not commissioned; externally peer reviewed.