HER2 Gene Status Research Articles

Association between HER2 status in gastric cancer and clinicopathological features: a retrospective study using whole-tissue sections.

BackgroundGastric cancer is usually diagnosed in an advanced stage of disease and treatment options are sparse. Trastuzumab was recently approved for metastatic or locally advanced carcinomas arising in the stomach or in the gastroesophageal junction in patients with HER2-positive tumors. However, data on the frequency of HER2-positive cases among Brazilian patients are limited. Our aim was to characterize HER2 protein and gene status in a series of Brazilian patients with gastric cancer and to evaluate its association with clinicopathological data.MethodsHistological slides from 124 primary gastrectomies were reviewed and their pathological reports were retrieved from the files at a Brazilian university hospital. Automated immunohistochemistry for HER2 was performed on whole-tissue sections from each tumor. HER2-equivocal cases by immunohistochemistry were submitted to automated dual in situ hybridization for gene amplification evaluation. HER2 status was confronted with clinicopathological parameters in order to assess statistically significant associations.ResultsImmunohistochemistry analysis revealed that 13/124 cases (10.5 %) were HER2 positive (3+), 10/124 cases (8.1 %) were equivocal (2+) and 101/124 cases (81.4 %) were negative, being 7 cases 1+. None of the equivocal cases showed gene amplification. The overall HER2 positivity rate was 10.5 %. There was an association between HER2 expression and Laurén’s intestinal histological subtype (P = 0.048), well to moderately differentiated tumors (P = 0.004) and presence of lymphovascular invasion (P = 0.031). No association was found between HER2 status and tumor topography.ConclusionsConfronted with data published by other authors, the lower percentage of HER2-positive cases found in our series might be partially explained by the lower frequency of tumors arising at the gastroesophageal junction in comparison with distal gastric carcinomas in Brazilian patients. This could also account for the lack of statistically significant association between HER2 status and tumor topography in our study.

Reproducibility in the Assessment of HER2 DISH in Breast Cancer

OBJECTIVE: HER2 gene status is an important biological marker that determines both the prognosis and response to Trastuzumab in patients with breast cancers. Although FISH assay has long been used to determine the HER2 gene copy number, DISH is gaining popularity since it can be assessed with a conventional microscope. The DISH test relies on an individual inspection however, so inter-observer variation may be an issue. MATERIALS AND METHODS: In the current study, reproducibility in the assessment of HER2 DISH test was evaluated in 69 breast cancer samples with HER2 IHC score 2+ and 3+. Two independent investigators evaluated the digitallycaptured images of DISH, without the knowledge of HER2 IHC status. RESULTS: The HER2/CEP17 ratio obtained from both observers were highly correlated (Rho = 0.883, p < 0.001). Based on the ASCO 2013 guideline, agreement of both investigators for HER2 gene diagnosis was 92.75 % (ĸ = 0.848). The HER2/CEP17 ratio of the discordant cases ranged from 1.6 (non-amplifi ed HER2) to 2.6 (low level of HER2 gene amplifi cation). CONCLUSION: It is concluded that the DISH method produces good reproducibility between independent observers, and the result also implies that the HER2 gene copy number is fairly homogenous across an individual breast cancer sample.

Associations between TOX3 rs3803662 polymorphisms and immunological markers of breast cancer

TOX3 gene was considered to be breast cancer susceptibility gene in the European population and East Asian populations. This study was aimed to investigate the relevance of TOX3 gene rs3803662 single nucleotide polymorphisms and sporadic breast cancer susceptibility among the Han Nationality in Henan Province. A case-control study was performed among 253 patients with sporadic breast cancer and 343 control subjects in Henan Province. The SNP rs3803662 in TOX3 was genotyped by imLDR technique. Association analysis based on unconditional logistic regression was carried out to determine the odds ratio (OR) and 95% confidence interval (95% CI) for the SNP between different alleles and breast cancer. There was no statistically significant difference in the distribution of TOX3 rs3803662 allele in different Ki-67 value and HER2 gene status in the case group. The distribution of TOX3 rs3803662 allele between breast cancer and the control group were different. Compared with allele AA and GA, allele GG increased the risk of breast cancer in codominant inheritance (OR=2.19, 95% CI:1.19-4.02) and recessive genetic models (OR=2.06, 95% CI: 1.15-3.70). Further stratifying analysis was conducted based on estrogen receptor status. The SNP rs3803662 showed significant associations with ER status, and was associated with positive ER status in the recessive (OR=1.92; 95% CI:1.00-3.67; P=0.05) and codominant models (OR=2.07; 95% CI:1.05--.08; P=0.036). And this SNP was associated with negative ER status breast cancers in both recessive (OR=2.38; 95% CI:1.10-5.15; P=0.028) and codominant models (OR=2.43; 95% CI:1.08-5.48; P=0.032). But there was no statistically significant difference in each subgroup stratified by ER status. This was a verification study in a Han population. In codominant and recessive genetic models, allele GG increased breast cancer risk and was associated with the pathogenesis of different ER status breast cancer. But there was no obvious correlation between this SNP and Ki-67 or HER2 gene. This is the first breast cancer susceptibility loci that is confirmed in Henan population. Our study only analyzes the correlation between the SNP and ER status in breast cancer. More studies and analyses about the association between SNPs and different characteristic of breast cancer should be performed.

Alterations of biomarker profiles after neoadjuvant chemotherapy in breast cancer: tumor heterogeneity should be taken into consideration.

Tumor biomarkers including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67 are routinely tested in breast cancer patients and their status guides clinical management and predicts prognosis. A few retrospective studies have suggested that neoadjuvant chemotherapy (NAC) in breast cancer may change the status of biomarker expression, which in turn will affect further management of these patients. In this study we take advantage of a relatively large cohort and aim to study the effect of NAC on biomarker expression and explore the impact of tumor size and lymph node involvement on biomarker status changes. We collected 107 patients with invasive breast cancer who received at least three cycles of NAC. We retrospectively performed and scored the immunohistochemistry (IHC) of ER, PR, HER2 and Ki-67 using both the diagnostic core biopsies before NAC and excisional specimens following NAC. HER2 gene status was assessed by fluorescence in situ hybridization for cases with IHC result of 2+. We demonstrated that there was a significant decrease in expression of PR (P = 0.013) and Ki-67 (P = 0.000) in post-NAC specimens compared to pre-NAC core biopsies. In addition, cases with large tumor size (≥ 2 cm) and cases with lymph node metastasis were more frequently to have biomarker changes. Finally we studied cases with HER2 status changes after NAC treatments in detail and emphasized the nature of tumor heterogeneity.

Protecting Quantum Dot Fluorescence from Quenching to Achieve a Reliable Automated Multiplex Fluorescence In Situ Hybridization Assay.

Quantum dots (QD) are novel inorganic fluorochromes that are ultra-bright, photo-stable, and available in multiple, highly-resolvable colors. QDs represent an ideal detection material for in situ hybridization (ISH) because they may provide unprecedented resolution and strong signal intensities that are not attainable with traditional fluorophores. Unfortunately, lack of reliability has been an impediment to widespread adoption of QD-based fluorescence in situ hybridization (QD FISH) technology. By optimizing QD-to-target accessibility, we have developed a QD FISH staining procedure that dramatically improves the reliability of an automated ERG/PTEN QD FISH assay (91% 1st pass rate). Here, we report improvements to the assay that protects QD fluorescence from quenching due to trace amounts of heavy metals and minimizes QD background signals. When using this method, highly-consistent staining was observed with the ERG/PTEN QD FISH assay in prostate tissue. Successful staining of several other clinically-relevant genetic markers was also possible. We further demonstrated improved reliability for determining HER2 gene status in breast cancer, identifying anaplastic lymphoma kinase (ALK) gene break-apart in non-small cell lung cancer, and detecting human papillomavirus 16 (HPV16) in cervical intraepithelial neoplasia. The enhanced QD FISH assay allows for examining complicated genetic aberrances without use of enzymatic amplification. Our optimized methods now demonstrate reliability sufficient for QD FISH technology to be a diagnostic tool in a clinical setting.

HER2 gene and protein expression status of breast carcinoma can be reliably tested on a single slide.

Human epidermal growth factor receptor 2 (HER2) status in breast carcinomas serves as a predictor of benefit from anti-HER2 therapy. In providing clinicians with the information necessary to decide whether or not to treat with targeted therapy, it might be necessary to choose between methods assessing HER2 protein overexpression or gene amplification. A new diagnostic approach could be a combination of both tests on the same slide. If accurate and reproducible, this approach might optimize patient stratification for therapy. In this study, formalin-fixed paraffin-embedded tumor samples from 77 breast cancer patients were examined for HER2 by immunohistochemistry (IHC) and silver in situ hybridization (SISH) using HER2 IHC (clone 4B5), HER2/CEN17SISH, and combined IHC and SISH assay, called gene protein (GP). Cases were selected to ensure a sufficient number of borderline cases on the basis of IHC scores (0, 1+, 2+, 3+), obtained during diagnostic histopathological workup. The concordance between the HER2 IHC score obtained during diagnostic histopathological workup and GP was 93 %. Discordances had no influence on therapy decisions. The concordance between ISH results using dual ISH (DISH) and GP was 96 %. Of the 77 cases studied by GP, three cases with a ratio close to 2 would have been called amplified by DISH. The use of GP reduced the time for slide reading for a trained pathologist by up to 25 %, relative to sequential reading of IHC followed by SISH. For cases with an IHC score of 2+, the final result was obtained in 1 day, while the sequential technique would have required retesting by ISH on a second day. In conclusion, assessment of HER2 status by GP is an improvement for pathologists and facilitates clinical decision-making for breast cancer management.

Evaluation of HER2 Gene Status in Breast Cancer Samples with Indeterminate Fluorescence in Situ Hybridization by Quantitative Real-Time PCR

Administration of drugs targeting HER2 (official symbol ERBB2) is an important component of therapy for breast cancer patients with HER2 amplification/overexpression as determined by in situ hybridization (ISH) and immunohistochemistry (IHC). In approximately 5% of breast cancers, ISH assays fail. In these cases, HER2 protein expression is evaluated by IHC alone that may yield false negatives/positives for poor-quality samples. Therefore, we developed a method that was based on quantitative real-time PCR applicable for DNA from formalin-fixed, paraffin-embedded tissue samples. Its limit of detection was determined with breast cancer cell lines and validated with 223 breast cancer patient samples. On the basis of comparisons with fluorescent ISH (FISH) and IHC data, the sensitivity of the new method was 94.2% and 95.1%, its specificity was 100% and 99.1%, and overall concordance between results obtained with the quantitative real-time PCR method and FISH/IHC was 97.6% for both methods. The quantitative real-time PCR method was then used to evaluate the HER2 status of 198 of 3696 breast cancer tissues that yielded indeterminate FISH results. The HER2 copy number was successfully determined in 69.2% of these indeterminate samples. Thus, the DNA-based technique appears to be a specific, sensitive method for determining HER2 copy numbers when the FISH assay fails, which may complement IHC tests.

P269 Silver in situ hybridization and FISH for determination of HER2 gene status in breast carcinoma

P269 Silver in situ hybridization and FISH for determination of HER2 gene status in breast carcinoma

The HER2 Gene and HER2 Protein Status and Chromosome 17 Polysomy in Gastric Cancer Cells in Own Material

The aims of the study were to assess the expression of HER2 protein, the amplification of the HER2 gene, and the occurrence of chromosome 17 polysomy in gastric cancer cells and to analyze the relation between the results of such a determination and the selected clinicopathologic parameters in patients treated for gastric cancer. Tissue samples of primary tumor from 83 consecutive patients who underwent gastric cancer resection were analyzed by immunohistochemical (IHC) analysis and fluorescence in situ hybridization (FISH). A positive result of the IHC test, with a minimum score +, was obtained among 22.8% patients. The FISH test was carried out successfully among 58 patients, including 10.3% cases with a positive result, whereas the presence of chromosome 17 polysomy was confirmed among 13.8% patients. A statistically significant dependence was found between the presence of HER2 overexpression and: the lower stage of tumor infiltration, the higher grade of cancer differentiation, no mucinous component, and the intestinal type according to the Lauren classification. Statistically significant relation was found between chromosome 17 polysomy and the tumor location in the proximal part of the stomach, the performance of the palliative procedure, the presence of distant metastases, and a higher frequency of postoperative complications. There is no complete coincidence in gastric cancer between the occurrence of the HER2 gene amplification and the HER2 receptor expression. The impact of the HER2 gene status and HER2 protein on prognosis in gastric cancer remains unclear. Chromosome 17 polysomy may be an important negative prognostic factor in gastric cancer.

Evaluation of HER2 gene amplification status in invasive breast cancer patients by Fluorescence in Situ Hybridization analysis and its correlation with clinical features

Precise assessment of HER2 gene status as an important biomarker plays a significant role in identifying the eligible patients for Trastuzumab therapy and determining their clinical outcomes. In this study, the researchers assigned HER2 amplification status in invasive breast cancer specimens by Fluorescence in Situ Hybridization (FISH) and determined its association with other clinical features. Formalin-fixed paraffin embedded tumor tissue specimens of 46 patients with invasive breast cancer were collected from November 2011 till May 2012. HER2status was evaluated by FISH. The Zytolight SPEC HER2/CEN17 dual color probe kit was applied for assessment of HER2status. HER2 gene amplification was defined as HER2/CEP17 ratio>2.2.The association between HER2status and clinical features like tumor grade, tumor type, tumor size, axillary lymph node involvement and age of patient was done using Chi squared test at the 0.05 level of significance (p value). Amplification of HER2 gene was detected in twelve cases (26%). On statistical analysis HER2status showed correlation with tumor grade (p=0.02).There was no correlation between HER2status and tumor type, tumor size, lymph node status and age of patients. The results of this study are consonant with the findings of other studies about the presence of HER2 gene amplification in invasive breast cancer. Statistical analysis showed patients with HER2 amplified gene have tumors with higher grade. In these patients the probability of increased proliferation and metastasis is high therefore evaluation of HER2 gene amplification status in breast cancer patients specially in high grade tumor with an accurate method such as FISH is essential

Gene status in HER2 equivocal breast carcinomas: impact of distinct recommendations and contribution of a polymerase chain reaction-based method.

The primary objectives of this study on carcinomas with equivocal HER2 expression were to assess the impact of distinct recommendations with regard to identifying patients eligible for anti-HER2 agents by fluorescence in situ hybridization (FISH) and to elucidate whether multiplex ligation-dependent probe amplification (MLPA) may be of support in assessing HER2 gene status. A cohort of 957 immunohistochemistry-evaluated HER2-equivocal cases was analyzed by dual-color FISH. The results were assessed according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2007 and 2013 guidelines for dual- and single-signal in situ hybridization (ISH) assays. A subgroup of 112 cases was subjected to MLPA. HER2 amplification varied from 15% (ASCO/CAP 2007 HER2/CEP17 ratio) to 29.5% (FDA/EMA HER2 copy number). According to the ASCO/CAP 2013 interpretation of the dual-signal HER2 assay, ISH-positive carcinomas accounted for 19.7%. In contrast with the ASCO/CAP 2007 ratio, this approach labeled as positive all 32 cases (3.34%) with a HER2/CEP17 ratio <2 and an average HER2 copy number ≥6.0 signals per cell. In contrast, only one case showing a HER2 copy number <4 but a ratio ≥2 was diagnosed as positive. MLPA data correlated poorly with FISH results because of the presence of heterogeneous HER2 amplification in 33.9% of all amplified carcinomas; however, MLPA ruled out HER2 amplification in 75% of ISH-evaluated HER2-equivocal carcinomas. The ASCO/CAP 2013 guidelines seem to improve the identification of HER2-positive carcinomas. Polymerase chain reaction-based methods such as MLPA can be of help, provided that heterogeneous amplification has been ruled out by ISH.

234P - Circulating Tumor Cells As Biomarkers During Neoadjuvant Chemotherapy in Bladder Cancer

ABSTRACT Aim: Cisplatin based neoadjuvant chemotherapy (NAC) improves survival in some patients with resectable muscle-invasive bladder cancer but carries significant toxicity. Development of predictive biomarkers to direct NAC is an urgent clinical need, Circulating tumor cells (CTC) are prognostic for survival in several metastatic epithelial cancers and for recurrence in patient undergoing cystectomy without NAC in muscle-invasive bladder cancer. Current CTC assays have poor sensitivity in non-metastatic disease. We hypothesized that CTC numbers before and after 1 cycle of chemotherapy could serve as potential biomarkers and investigated a novel microfluidic immunomagnetic CTC assay IsoFlux to detect CTCs in patients receiving NAC. Methods: An IRB approved prospective pilot study at the University of Michigan Comprehensive Cancer Center to establish the feasibility of detecting CTCs in patients with locally advanced resectable bladder cancer (cT2-T4aNxM0) before and during cisplatin based neoadjuvant chemotherapy has enrolled 14 patients. Peripheral blood (7.5 mL) was drawn in EDTA tubes prior to NAC (baseline) and again after one cycle (3-4 weeks). We enumerated CTCs by the CellSearch and IsoFlux assays in parallel and by IsoFlux in duplicate samples in a subset of patients. We correlated CTC numbers with pathological stage at cystectomy. We also explored the feasibility of assessing EGFR and HER2 expression by RT-PCR in CTCs in 5 patients and assessed their baseline tumor HER2 gene status by Ion Torrent sequencing. Results: Median age of patients (n = 14) was 63.5 yrs with 83% male. Clinical stage was 79% cT2Nx, 7% cT3Nx and 14% cT4aNx. NAC was cisplatin/gemcitabine in 6 patients and MVAC in 8 patients. The IsoFlux assay detected more CTCs per sample than CellSearch (median =10 vs. 0) at baseline (n = 5) and after 1 cycle (5.5 vs. 0, n = 3). Exact concordance was 40% by IsoFlux assay in tubes drawn in parallel; a paired t-test showed no difference (p = 0.9). After 1 cycle, CTCs by IsoFlux declined from median of 10.5 to 3.75. Patients with Conclusions: CTC detection in patients undergoing NAC for bladder cancer is feasible, potentially more sensitive with the novel assay and a promising predictive biomarker. Disclosure: C. Ionescu: Employee of Fluxion Biosciences Inc, CA; M. Schwartz: Employee of Fluxion Biosciences Inc, CA. All other authors have declared no conflicts of interest.

Concomitant detection of HER2 protein and gene alterations by immunohistochemistry (IHC) and silver enhanced in situ hybridization (SISH) identifies HER2 positive breast cancer with and without gene amplification.

IntroductionHER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number.MethodsTissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis.ResultsOverall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival (p = 0.088, reaching nearly statistical significance) compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification.ConclusionsThe novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.

Correlation of HER2 gene status assessment by fluorescence in situ hybridization between histological sections and cytological specimens of breast cancer.

While HER2 gene detection in cytological specimens using fluorescence in situ hybridization (FISH) has been reported, the appropriate criteria for such specimens remain controversial. Fine needle aspiration (FNA) samples collected from surgically resected breast cancer specimens were rinsed in a cytopreservative solution containing fixative. Then, slides of the FNA samples were prepared by liquid-based cytology (LBC) (ThinPrep system, Hologic) according to the manufacturer's instructions, and a PathVision HER2 DNA probe kit (Abbott) was used for FISH staining. The results were evaluated using an automated MetaCyte imaging system (MetaSystems, Altlussheim, Germany). HER2 gene amplification was scored using the HER2/chromosome enumeration probe 17 (CEP17) signal count ratio as follows: amplified, >2.2; equivocal, 1.8-2.2; and unamplified, <1.8. The cytology results were compared with the histology results from concordant cases. Successful results were obtained in 98 of 100 cases, and results from the FNA specimens were in agreement with those from the histological sections in 97 of these 98 cases (accuracy rate, 99%; kappa, 0.962). FISH-based assessment of the HER2 gene status is consistent between histological sections and cytological specimens of breast cancer.

A novel gene-protein assay for evaluating HER2 status in gastric cancer: simultaneous analyses of HER2 protein overexpression and gene amplification reveal intratumoral heterogeneity.

Human epidermal growth factor receptor 2 (HER2) protein overexpression and gene amplification are important biomarkers for trastuzumab treatment in breast and gastric cancer patients. Gastric cancer presents high rates of tumor heterogeneity, which may influence the results of HER2 testing. A novel gene-protein assay (GPA) can allow the simultaneous analysis of HER2 protein and gene status on a single slide. Using the tissue microarray technique, the HER2 status of each of 875 gastric cancer cases was evaluated by immunohistochemistry (IHC), brightfield dual-color in situ hybridization (DISH), and GPA. Intratumoral phenotypic and genotypic heterogeneity were evaluated by comparing the HER2 statuses of two tissue cores from each case. There was excellent concordance between GPA and IHC (99.2%), as well as between GPA and DISH results (99.3%). HER2 positivity obtained by GPA was almost identical (99.8%) to the results obtained by IHC and DISH assays. Intratumoral phenotypic heterogeneity was more frequently observed in IHC 2+ cases (63.5%) compared with IHC 3+ cases (28.3%). Phenotypic heterogeneity (48.8%) was more frequently observed than genotypic heterogeneity (26.8%). Tumor heterogeneity was consistently observed from early to advanced stages. HER2-positive gastric cancers presented different levels of HER2 protein expression and gene amplification statuses within the same lesion in almost half the cases examined. Evaluating both phenotypic and genotypic heterogeneity may contribute to a deeper understanding and improved prediction of clinical outcome in gastric cancer patients treated with trastuzumab. This newly established GPA technology may also be useful for developing biomarkers for other molecularly targeted therapies.

Characterisation of HER heterodimers in breast cancer using in situ proximity ligation assay

HER2 plays an important role in breast cancer progression and provides predictive and prognostic information. HER2 receptor family members function through dimerisation, which can lead to impact on cell function, growth and differentiation; however, their value in breast cancer development remains to be defined. This study aims to examine the relationships of HER2 heterodimers to breast cancer characteristics in trastuzumab naïve and treated cases. HER2 protein (IHC), HER2 gene (chromogenic ISH) and HER2 heterodimerisation status [chromogenic in situ proximity ligation assay (PLA)] were assessed in two breast cancer series prepared in tissue microarray (TMA) format. A range of signals/cell for each HER2 heterodimer was detected (0-34.6 signals/cell). The vast majority of cases with HER2 heterodimers showed HER2 gene amplification and/or protein expression. There was an association between HER2 dimerisation with HER3 and HER4 and their protein expression level but no such association was found in with HER1 (EGFR). Of the HER2+ cases, 74, 66, and 58 % showed heterodimers with EGFR, HER3 and HER4, respectively. 51 % of HER2+ tumours expressed all three heterodimers whereas 23 % of the cases did not show expression of any of the three heterodimers. There was an inverse association between the presence and levels of HER2 heterodimers and hormone receptor expression in HER2+ tumours. Tumours exhibiting high levels of HER2 heterodimers demonstrated aggressive clinicopathological features and poor outcome. In the HER2+ cases, dimerisation with EGFR and HER3 but not with HER4 showed an association with aggressive features. There was no association between HER2 heterodimers with patient breast cancer-specific survival or recurrence in HER2+ breast cancer in those patients receiving trastuzumab or not. Our results demonstrate that HER2 dimerisation is a complex process that may underlie the biological heterogeneity of HER2 positive tumours and may identify patients suitable for a specific targeted therapy but does not predict patient outcome for those receiving trastuzumab. PLA proved to be a useful tool for detecting, visualising and quantifying the frequency of protein-protein interactions in archival formalin-fixed paraffin-embedded tissue samples.

Reproducibility in the assessment of HER2 DISH in breast cancer

HER2 gene status is an important biological marker that determines both the prognosis and response to Trastuzumab in patients with breast cancers. Although FISH assay has long been used to determine HER2 gene copy number, DISH is gaining popularity since it can be assessed with a conventional microscope. Because the DISH test relies on an individual inspection, inter-observer variation might be an issue. In the current study, reproducibility in the assessment of HER2 DISH test was evaluated in 69 breast cancer samples with HER2 IHC score 2+ and 3+. Two independent investigators evaluated the digitally-captured images of DISH (10–20 images per case), taken at x600 magnification, without the knowledge of HER2 IHC status. The HER2/CEP17 ratio obtained from both observers were highly correlated (R = 0.883, p

Abstract P1-02-03: Clinical utility of combined brightfield HER2 gene-protein assay in IHC equivocal cases

Abstract The combined gene protein assay (GPA) for HER2 (Ventana Medical Systems Inc, Tucson AZ) is a newly developed technique where both HER2 gene and protein can be simultaneously assessed on a single slide using routine bright field microscopy. This is made possible by combining HER2 immunohistochemistry (using clone 4B5 from Ventana) with the HER2 Dual in-situ hybridization (DISH) DNA probe cocktail. The main goal of the study was to compare this new GPA methodology with standard fluorescence in situ hybridization (FISH) on the most challenging group of breast cancers, i.e. HER2 immunohistochemical (IHC) 2+ cases. This validation study included whole tissue sections of 36 invasive breast cancer cases with equivocal HER2 IHC results (IHC score 2+) on clinical testing. FISH and GPA were performed on the same tumor blocks. The GPA assay staining results were enumerated by counting at least 20 nuclei by a pathologist blinded to FISH results. Similar to FISH, areas with stronger IHC staining were counted in heterogeneous cases. For both FISH and GPA, HER2 amplification was defined as HER2/CEP17 ratio ≥ 2.0. Cases with HER2/CEP17 ratio &amp;lt;2.0 were considered as non-amplified. In addition, 25 known HER2 IHC 3+ invasive carcinoma cases (represented on a tissue microarray) and 22 known IHC negative (scores 0 and 1+ also represented on tissue microarray) were also evaluated by GPA. GPA was successful in all cases in the first attempt. All previously tested IHC 2+ cases were again scored as 2+ on GPA. The agreement for IHC 2+ cases between FISH and GPA with respect to HER2 gene status was 94.4% (see table). The 2 discordant cases were amplified by GPA and not amplified by FISH. The HER2 to CEP17 ratio on these 2 cases by GPA was 2.0 and 2.34 with average HER2 copies/cell of 5.7 and 5.5 respectively. For the 36 IHC 2+ cases, the mean HER2 copies per cell for FISH were 3.09 (std dev: 1.8; range: 1.9 to 11.07) versus 3.52 (std dev: 1.8; range: 1.8 to 9.95) for GPA. The mean CEP17 signals per cell were 2.24 (std dev: 0.6; range: 1.65 to 4.2) for FISH versus 2.3 (std dev: 0.6; range: 1.6 to 3.5) for GPA. The mean for HER2:CEP17 ratio was 1.34 (std dev: 0.6; range: 1.03 to 4.32) by FISH and 1.50 (std dev: 0.7; range: 1.02 to 4.72) by GPA. GPA versus FISH for IHC 2+ cases GPA amplifiedGPA not amplifiedTotalFISH amplified202FISH not amplified23234Total43236FISH: Fluorescence in-situ hybridization; GPA: Gene protien assay All 25 (100%) of IHC 3+ cases showed clusters of HER2 gene consistent with HER2 amplification by GPA. These 25 cases also showed strong membranous (“chicken wire”) reactivity consistent with 3+ IHC score. Similarly, the 22 known IHC negative cases on the TMA, were again IHC negative, and lacked HER2 gene amplification by GPA. The HER2 GPA shows excellent concordance with both HER2 IHC assay and FISH assay. It performs accurately not only for unequivocally positive and negative cases but also on the most challenging (IHC 2+) breast cancer cases. This new assay combines the advantages of both IHC and FISH, i.e. accuracy similar to FISH for IHC equivocal cases, faster turn around time (less than 24 hours) like IHC and evaluation under bright-field microscope where the tissue morphology is well preserved and heterogeneity is better appreciated. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-03.

Correlation between ER, PR, HER-2, Bcl-2, p53, proliferative and apoptotic indexes with HER-2 gene amplification and TOP2A gene amplification and deletion in four molecular subtypes of breast cancer

The aim of our study was to investigate HER-2 and TOP2A gene status and their correlation with Bcl-2, p53, Ki67, ssDNA, and clinicopathological parameters in four molecular subtypes of breast cancer. Seventy-four paraffin-embedded samples are immunohistochemically studied for the expression of estrogen receptor (ER), progesterone receptor (PR), HER-2, p53, Bcl-2, ssDNA, and Ki67, while HER-2 and TOP2A gene status by fluorescence in situ hybridization was investigated in 60 samples. Luminal A and B subtypes were characterized with small tumor size, intermediate histological grade, negative lymph node, and metastatic status, while triple negative and HER-2 positive subtypes were associated with larger tumor size, poorly differentiated tumors, and positive lymph node status. p53, Ki67, and ssDNA expression was higher in triple negative and HER-2 positive than in luminal subtypes, while ER, PR, and Bcl-2 dominated in luminal subtypes. HER-2 gene status was higher in luminal B and HER-2 positive than in luminal A and triple negative subtypes, while TOP2A gene status was similar. HER-2 gene status positively correlated with TOP2A gene status, HER-2 receptor, and histological grade, while negative correlation characterized relationship between HER-2 gene status and ER, PR, and Bcl-2. The shortened overall survival period characterized patients from triple negative breast cancer subtype (18.7months). HER-2 and TOP2A gene amplification showed a tendency to be associated with larger tumor size, positive lymph node status, high level of apoptotic and proliferative indexes, and low level of p53 and Bcl-2 expression, which all together indicate group of patients with similar outcome during the progression of the disease.

Differing deregulation of HER2 in primary gastric cancer and synchronous related metastatic lymph nodes.

BackgroundThe aim of this study was to investigate how differences in expression of HER2 between primary gastric cancers (PGCs) and their corresponding metastatic lymph nodes (LMNs) might affect its potential as a prognostic indicator in treatments including anti-HER2 agents.MethodsThe analysis was conducted in 102 patients who underwent surgical resection for primary gastric cancers (PGCs; adenocarcinoma, intestinal type) with synchronous LNMs. HER2 gene status and protein expression were investigated by immunohistochemistry (IHC) in all patients; fluorescence in situ hybridization (FISH) was performed in 22 patients. The correlation between HER2 gene status in PGCs and their LNMs was evaluated.ResultsPositive HER2 expression as detected by IHC + FISH was observed in 27/102 PGC samples (26.5%) and 29/102 LNM samples (28.4%). HER2 amplification status in 102 paired PGC and LNM samples as evaluated by FISH + IHC was concordant in 92 patients (90.2%), 69 (67.6%) were unamplified and 23/102 (22.5%) were amplified at both sites, and discordant in 10 patients (9.8%), 4 (3.9%) were positive for PGC and negative for LNM, while 6 (5.9%) were positive for LNM and negative for PGC. The results of FISH + IHC showed very strong concordance in HER2 status between the PGC and LNM groups (k = 0.754).ConclusionThe high concordance between HER2 results for PGCs and their LNMs indicates that assessment of HER2 status in the primary cancer alone is a reliable basis for deciding treatment with anti-HER2 agents in patients with LNMs from gastric adenocarcinoma.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9365749431029643.