HER2 Expression, Test Deviations, and Their Impact on Survival in Metastatic Gastric Cancer: Results From the Prospective Multicenter VARIANZ Study

PURPOSE Trastuzumab is the only approved targeted drug for first-line treatment of human epidermal growth factor receptor 2–positive (HER2+) metastatic gastric cancer (mGC). However, not all patients respond and most eventually progress. The multicenter VARIANZ study aimed to investigate the background of response and resistance to trastuzumab in mGC. METHODS Patients receiving medical treatment for mGC were prospectively recruited in 35 German sites and followed for up to 48 months. HER2 status was assessed centrally by immunohistochemistry and chromogenic in situ hybridization. In addition, HER2 gene expression was assessed using qPCR. RESULTS Five hundred forty-eight patients were enrolled, and 77 had HER2+ mGC by central assessment (14.1%). A high deviation rate of 22.7% between central and local test results was seen. Patients who received trastuzumab for centrally confirmed HER2+ mGC (central HER2+/local HER2+) lived significantly longer as compared with patients who received trastuzumab for local HER2+ but central HER2− mGC (20.5 months, n = 60 v 10.9 months, n = 65; hazard ratio, 0.42; 95% CI, 8.2 to 14.4; P < .001). In the centrally confirmed cohort, significantly more tumor cells stained HER2+ than in the unconfirmed cohort, and the HER2 amplification ratio was significantly higher. A minimum of 40% HER2+ tumor cells and a HER2 amplification ratio of ≥ 3.0 were calculated as optimized thresholds for predicting benefit from trastuzumab. CONCLUSION Significant discrepancies in HER2 assessment of mGC were found in tumor specimens with intermediate HER2 expression. Borderline HER2 positivity and heterogeneity of HER2 expression should be considered as resistance factors for HER2-targeting treatment of mGC. HER2 thresholds should be reconsidered. Detailed reports with quantification of HER2 expression and amplification levels may improve selection of patients for HER2-directed treatment.

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Profiling of circulating tumor cells isolated from 105 metastatic gastric cancer patients revealed HER2 overexpression/activation for potential use in clinical setting.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.10535 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 10535-10535 ◽

Cited By ~ 1

Author(s):

Saswati Hazra ◽

Jeeyun Lee ◽

Phillip Sangwook Kim ◽

Kyoung-Mee Kim ◽

Limin Liu ◽

...

Keyword(s):

Gastric Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Predictive Marker ◽

Patient Treatment ◽

Her2 Status ◽

Her2 Overexpression ◽

Her2 Expression ◽

Her2 Positive ◽

Over Expression

10535 Background: Gastric cancer (GCA) is the second leading cause of cancer mortality in the world. Survival of patients with advanced GCA treated with chemotherapy remains low. New targeted therapies are urgently needed. There is mounting evidence of the role of HER2 overexpression in patients with GCA, and it has been highly correlated to poor outcomes with more aggressive disease. The ability to accurately determine HER2 status by testing circulating tumor cells (CTCs) may improve patient treatment by allowing ongoing assessment of HER2 status during treatment and/or identifying additional patients who could potentially benefit from HER2- targeted therapy. Methods: The Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) was utilized to determine the expression and activation (phosphorylation) levels of HER2 in CTCs isolated from blood specimens obtained from 105 metastatic GCA patients. Results: Utilizing the CEER platform, the levels of HER2 expression and phosphorylation were determined for CTCs isolated from metastatic GCA patients. Evaluable CTCs were found in 33% (35/105) of enrolled patients. Out of 35 patients, 7 patients (20%) have high HER2 over expression, 6 patients (17%) have moderate HER2 expression and 11 patients (31%) have HER2 activation (phospho positive) with no HER2 over-expression. Conclusions: When CTCs were present, the CEER assay identified varying levels of HER2 involvements in 68% of metastatic GCA patients. HER2 positive CTCs could serve as a prognostic and/or predictive marker in patients with advanced GCA and CTC-HER2 profile shifts can be utilized to monitor the treatment efficacy.

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Clinicopathologic significance of HER2 expression in gastric cancer: A retrospective study in Indian population.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e15143 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e15143-e15143

Author(s):

Sudheer Reddy Kataru ◽

Venkata Sampath Vankamamidi ◽

Amit Kumar Chowhan ◽

Meenakshisundaram Manickavasagam ◽

Khajjayam Radhakrishna ◽

...

Keyword(s):

Gastric Cancer ◽

Retrospective Study ◽

Indian Population ◽

Smoking Status ◽

Rank Test ◽

Her2 Status ◽

Published Data ◽

Her2 Expression ◽

Her2 Gene ◽

Kaplan Meier

e15143 Background: Gastric cancer is the second most common cause of cancer related death worldwide. HER2 expression is an important biomarker for identifying patients who could respond to targeted therapy. The purpose of this study was to evaluate the frequency of HER2 expression and its clinicopathological significance in Indian population with gastric cancer. Methods: In this retrospective study, clinicopathological details and formalin fixed embedded tissue from patients with gastric and gastro-esophageal junction Adenocarcinoma of all stages from August 2009 to January 2012 were collected. HER2 status in the tissue was evaluated by the validated methods. IHC (Immunohistochemistry) scores of 3+ were considered positive, 2+ equivocal, 1+ and 0 negative for HER2. Equivocal staining was further evaluated with FISH (Fluorescence In-situ Hybridization) testing. The significance of HER2 expression was correlated with epidemiological, clinical, pathological and biochemical parameters using Pearson’s test. Progression free survivals (PFS) were compared using Log Rank test (Mantel-Cox). Kaplan-Meier curves were determined for PFS. SPSS version 17.0 was used for statistical analysis. Results: We studied 135 patients, of which IHC for HER2 was 3+ in 8.8%, 2+ in 10.4%, 1+ in 8.9% and zero in 71.9%. Among HER2 equivocal samples (n=14), FISH showed HER2 gene amplification in 14.2%. HER2 status was negative in 89.6% and positive in 10.4%.HER2 status significantly correlated with stage (p=0.03), smoking status (p=0.022) and not correlated with Age, Gender, Site, Lauren’s classification, Albumin, Hemoglobin. HER2 negative patients have better PFS (13.1 versus 7.1 months; p=0.005) and the levels of HER2 expression significantly influenced the PFS (p=0.006).Median PFS in patients with 3+, 2+, 1+ and zero IHC scores were 8.2, 8.8, 9.3, and 14.2 months respectively. Conclusions: The frequency of HER2 expression in gastric cancer in Indian population is comparable to the published data. HER2 in Gastric cancer serves as a prognostic marker.

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Central validation of HER2 status to determine heterogeneity of marker expression in HER2-positive gastric cancer (GC).

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.4_suppl.12 ◽

2017 ◽

Vol 35 (4_suppl) ◽

pp. 12-12

Author(s):

Ivonne Haffner ◽

Birgit Luber ◽

Dieter Maier ◽

Albrecht Kretzschmar ◽

Ludwig Fischer von Weikersthal ◽

...

Keyword(s):

Gastric Cancer ◽

Growth Factor Receptor ◽

Stage Iv ◽

Her2 Status ◽

Her2 Gene ◽

Her2 Positive ◽

Secondary Resistance ◽

Deviation Rate ◽

Epidermal Growth ◽

Egfr Family

12 Background: 10-20% of GC overexpress HER2, a membrane-bound receptor tyrosine kinase (RTK) which belongs to the epidermal growth factor receptor (EGFR) family. Drugs directed against HER2 have shown mixed success in the treatment of advanced GC. While trastuzumab, a monoclonal antibody addressing HER2 has been approved for 1st-line treatment of stage IV HER2+ GC, trastuzumab-emtansine failed to improve outcomes in 2nd-line and lapatinib, a small molecular RTK inhibitor of HER2 and EGFR was not effective in 1st- and 2nd-line. Until now, primary and secondary resistance against HER2-directed treatment of GC is not well understood. The VARIANZ study aims to assess mechanisms influencing efficacy of trastuzumab in HER2+ GC. Methods: In this multicenter study, patients who receive medical treatment for advanced GC are recruited in 31 sites. The HER2 status is verified centrally by two dedicated GI pathologists using immunohistochemistry (IHC, DCS, HI608C0I) and chromogenic-in-situ hybridization (CISH, Zytomed Systems, C-3022-40). Results: From May 2014 to August 2016, we have enrolled 316 patients in this ongoing project (72% male, median age 64 years). At present, 281 samples were fully characterized for the HER2 status. According to criteria from the Trastuzumab for Gastric Cancer (ToGA) study, 53 of 281 samples were characterized HER2+ by central testing. In 38 samples that were diagnosed as HER2+ by local pathologists the HER2 status could not be verified centrally. 7 HER2- probes in local testing were characterized as HER2+ by central testing. The overall deviation rate between local and central testing is 27%. HER2 gene amplification in HER2+ tumors with deviating local report (mean HER2/CEP17: 2.8 ± 0.9, range between 1.9 and 4.5) is lower compared to HER2+ tumors and confirmed local report (mean HER2/CEP17: 5.5 ± 2.6; range between 2.2 and 11.0; p = 0.014). Conclusions: HER2-expression in GC is heterogeneous and still not easy to assess. Variability between local and central HER2 assessment is significant. Robust biomarkers predicting response or resistance to HER2 and other target therapies are needed. Clinical trial information: NCT02305043.

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Detection of HER2 expression and its structural alterations in gastric cancer tissues infected with cagA+ H. pylori

10.1101/2020.03.05.979963 ◽

2020 ◽

Author(s):

Akhileshwar Kumar Srivastava ◽

Divya Singh

Keyword(s):

Gastric Cancer ◽

Growth Factor Receptor ◽

Pcr Amplification ◽

Receptor Expression ◽

Her2 Status ◽

Her2 Overexpression ◽

Her2 Expression ◽

Gastric Cancer Cells ◽

H Pylori ◽

Cancer Tissues

AbstractBackgroundHelicobacter pylori (HP) cagA is the causing agent for development of gastric cancer (GC). H. pylori also involves to trigger the EGFR (epidermal growth factor receptor) expression in gastric cancer cells. However, the prognostic relation of cagA with HER2 status in GC was not well understood.ObjectiveThe main aim of this study was to investigate the link of HER2 expression with CagA+ H. pylori in GC tissues.Materials and MethodsThe study was performed on 85 GC tissues of GC patients. The specific primers of 16S rDNA and cagA for PCR amplification were used. For investigation of HER2 status in GC tissues, immunohistochemistry and PCR amplification were performed. In silico study was performed for the investigation of interactive potential of HER2 with CagA protein.ResultsPCR amplified the 54 (63.52 %) of 85 GC tissues for HP that showed 34 (62.96 %) cagA+ HP. Immunohistochemistry of tissues revealed 57 (67.05 %) diffuse and 28 (32.94 %) intestinal type cancer. Of 85 cases, 21 GC tissues scored (2 + or 3 +) for positive HER2 expression and score (0 or 1 +) of 64 (75.29 %) showed negative. Of 21 HER2 + GC tissue, 15 biopsies had cagA+ HP and 2 were negative. PCR amplified single amplicon in 17 (20 %) CagA+ tissues and 3 (5.55 %) in CagA - HP. The molecular interactions of CagA was also showed its efficiency for HER2 expression.ConclusionThe study concluded that CagA+ HP may induce HER2 overexpression in GC tissues.

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Detection and HER2 expression of circulating tumor cells in advanced gastric cancer patients.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.10541 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 10541-10541

Author(s):

Satoshi Matsusaka ◽

Keisho Chin ◽

Mariko Ogura ◽

Mitsukuni Suenaga ◽

Eiji Shinozaki ◽

...

Keyword(s):

Gastric Cancer ◽

Advanced Gastric Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Her2 Status ◽

Her2 Expression ◽

Her2 Positive ◽

Cellsearch System ◽

Primary Tumors ◽

Gastric Cancer Patients

10541 Background: This study was aimed at detecting and HER2-expressiong circulating tumor cells (CTC) in peripheral blood of chemo-naïve or chemo-resistant patients with advanced gastric cancer. Methods: All patients were enrolled using institutional review board-approved protocols at the Cancer Institute Hospital in the Japanese Foundation for Cancer Research and provided informed consent. The study population consisted of patients of age 18 years or older with histologically proven advanced gastric cancer. A total of 140 patients with advanced gastric cancer were enrolled into a prospective study. We used CellSearch system for detection and HER2 expression for CTC. Results: We detected ≥1 CTC/7.5ml in 80 of 140 patients (57.1%). HER2-positive CTC were observed in 19 of 80 CTC-positive patients (23.8%), including 6 patients with HSR2-negative primary tumors. 21 patients with HER2-positive primary tumors administered trastuzumab in combination with chemotherapy (Xeloda+cisplatin; 15, weekly Paclitaxel; 5). We detected ≥1 CTC/7.5ml at the baseline in 11 of 20 patients with trastuzumab treatment. HER2-positive CTCs were observed in 6 of 11 CTC-positive patients (54.5%). Patients with HER2-positive CTC at the baseline were shorter PFS than those with HER2-negative CTC at the baseline. Conclusions: HER2 expression on CTC was associated with chemo-resistance. Information on the HER2 status of CTC might be helpful for stratification of HER2-directed therapies.

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Assessment of the clinical utility of chip-based digital PCR for HER2 assessment in formalin fixed paraffin-embedded breast carcinoma tissue.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e23120 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e23120-e23120

Author(s):

Parth Shah ◽

Shiva Murarka ◽

Jacob Sands ◽

Bhavna Mehta ◽

Anupam Joshi ◽

...

Keyword(s):

Clinical Utility ◽

Growth Factor Receptor ◽

Digital Pcr ◽

Her2 Status ◽

Her2 Amplification ◽

Tissue Samples ◽

Amplification Ratio ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

e23120 Background: 15%-25% of breast cancer neoplasms exhibit Human epidermal growth factor receptor-2(HER2) amplification, as the driver mutation.Techniques to identify HER2 amplification include Immunohistochemistry (IHC) and Fluorescence in situ Hybridization (FISH). Digital PCR (dPCR) has been increasingly explored in determining HER2 status in cases of indeterminate results on IHC, mainly in archived samples. In this study, we aim to demonstrate the clinical utility of the Quantstudio 3D Digital PCR system to evaluate HER2 levels from Formalin Fixed Paraffin Embedded (FFPE) tissue with RNaseP as a control target. Methods: 61 tissue samples were analyzed by IHC and dPCR in parallel in a double blinded manner. IHC equivocal samples were reflexed to FISH and compared to the results obtained from dPCR. Samples suboptimal for IHC or FISH were satisfactorily processed by dPCR. dPCR results were analyzed on the Thermofisher Cloud platform. The general turnaround time(TAT) was about 2 and 3 days for IHC and FISH respectively with that of dPCR being 24 hours. Results: All 9 IHC positive and 35 negative samples had similar results on dPCR using an amplification ratio threshold for a positive result of 1.8. Of 17 IHC-equivocal samples, 5 resulted as positive, 10 negative and 2 as equivocal by dPCR. There was 100% concordance between the dPCR and FISH results. Two IHC equivocal samples that were unanalyzable by FISH were negative on dPCR Conclusions: Our results demonstrate that the chip based dPCR was non-inferior for HER2 detection in FFPE samples in a clinical setting. Superior TAT's and objective results were obtained compared to more subjective techniques like FISH and IHC even with low sample input. dPCR requires controls but no standards for calibration as it gives absolute copy numbers. Further study is needed to understand dPCR interpretation in cases of chromosomal aneuploidy. [Table: see text]

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Changing HER2-status of the primary gastric tumor after neoadjuvant trastuzumab-based therapy.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e16529 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e16529-e16529

Author(s):

Rashida Orlova ◽

Natalia P. Beliak ◽

Svetlana Kutukova ◽

Natalia V. Zhukova ◽

Inna Avramenko ◽

...

Keyword(s):

Gastric Cancer ◽

Tumor Cells ◽

Tumor Regression ◽

Locally Advanced ◽

Neoadjuvant Treatment ◽

Disease Free Survival ◽

Residual Tumor ◽

Her2 Status ◽

Her2 Expression ◽

Her2 Positive

e16529 Background: Aproximetly 16 % of patients with metastatic gastric cancer (GC) have HER2+ tumors. With localized stages, the detection rate of HER2+ is known to be lower. Addition of trastuzumab (T) to chemotherapy (CT) improved survival in metastatic, HER2+ GC. Unlike the metastatic disease, Her-2 did not represent an independent prognostic biomarker for early stages during exploratory analysis of MAGIC trial. The aim of the study was to compare human epidermal growth factor receptor 2 (HER2) expression before and after trastuzumab-based chemotherapy in patients with locally advanced HER2-positive gastric cancer, to evaluate the contribution of trastuzumab to the effectiveness of neoadjuvant treatment in this rare patient population. Methods: We assessed HER2 expression using immunohistochemistry in pre-treatment biopsied specimens and post-treatment resected specimens obtained from 10 patients with locally advanced HER2-positive (3+) gastric cancer receiving trastuzumab-based neoadjuvant chemotherapy: 8 men (80%) and 2 women (20%), from 30 to 80 years old, the median age was 63,5 years. Included 7 patients with resectable adenocarcinoma of the stomach and 3 patients with adenocarcinoma of the esophageal-gastric junction. All patients received neoadjuvant therapy with trastuzumab (100%) + chemotherapy: FLOT (2 patients), FOLFOX (7 patients), XELOX (1 patient). All patients underwent R0 gastrectomy. Tumor regression grading (TRG) after treatment were determined according to Mandard system. Results: Two patients showed a complete pathomorphological response of the tumor (20%,TRG1), therefore, the determination of postoperative HER2 status was not possible. Two patients (20%) maintained the HER2-positive status and six patients (60%) had a change in HER2 expression from positive to negative. Three patients had a (y)pT3 and 5 had a (y)pN+ tumor. Basing on histological changes the tumor regression TRG3 ( < 10% residual tumor cells) was observed in 5 patients (50%). TRG 4-5(preponderance of tumor cells/tumors without changes of regression) observed in 3 patients (30%). The follow-up period is too short for disease-free survival or overall survival to be assessed. Conclusions: HER2 expression can change after trastuzumab-based chemotherapy in patients with locally advanced HER2-positive gastric cancer. Continuous monitoring of HER2 expression after neoadjuvant treatments may be utilized to determine whether the continued use of trastuzumab is advisable.

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HER2 Heterogeneity in Gastric Cancer: A Comparative Study, Using Two Commercial Antibodies

Journal of Oncology ◽

10.1155/2020/8860174 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Catalin Bogdan Satala ◽

Ioan Jung ◽

Raluca Ioana Stefan-van Staden ◽

Zsolt Kovacs ◽

Calin Molnar ◽

...

Keyword(s):

Gastric Cancer ◽

Growth Factor Receptor ◽

Fish Analysis ◽

Her2 Amplification ◽

Her2 Gene ◽

Gene Encoding ◽

Gold Standard Method ◽

Negative Prognostic Factor ◽

Significant Difference ◽

Her2 Heterogeneity

Background. Although amplification of the gene encoding human epidermal growth factor receptor 2 (HER2) is used as an indicator for response to trastuzumab, the reported response rate is low, and few patients with gastric cancer (GC) benefit from this individualized therapy. The aim of this study was to examine the expression of c-erbB-2 oncoprotein (HER2), in GC samples, using two commercial immunohistochemical (IHC) antibodies, and to validate the results by checking HER2 gene amplification by fluorescence in situ hybridization (FISH). Methods. We assessed the IHC expression of HER2 using the polyclonal antibody from Dako and CB11 clone from Leica, in 93 consecutive cases of GC samples. In all of the cases, FISH analysis was also performed using the BOND-MAX platform. Results. No significant difference was observed between the two HER2 antibodies. Of the 93 cases, 22.58% demonstrated at least focal and 1+ HER2 positivity. Seven cases (7.53%) exhibited 3+ expression, and another 7 carcinomas (7.53%) were equivocal (2+). HER2 amplification was seen in 11 cases (11.83%), 10 of which were differentiated adenocarcinomas. In 5 of the cases, 2–5 sections were examined, which proved the extremely high intratumorally/intraglandular heterogeneity. FISH heterogeneity was higher in cases with only 2+ positivity on IHC assessment, compared with those showing at least one small focus of 3+ overexpression. HER2 amplification proved to be an independent negative prognostic factor. Conclusions. Due to the highly heterogeneous aspect of GC, at least 3-4 slides should be assessed by IHC, before considering a tumor to be HER2-negative. In cases with small 3+ foci representing less than 5% of tumor and in equivocal (2+) cases, FISH analysis remains the gold standard method.

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Detection of HER2 Amplification in Breast Carcinomas: Comparison of Multiplex Ligation-Dependent Probe Amplification (MLPA) and Fluorescence In Situ Hybridization (FISH) combined with Automated Spot Counting

Analytical Cellular Pathology ◽

10.1155/2006/741586 ◽

2006 ◽

Vol 28 (4) ◽

pp. 151-159

Author(s):

Elna Moerland ◽

Rens L. H. P. M. van Hezik ◽

Toine C. J. M. van der Aa ◽

Mike W. P. M. van Beek ◽

Adriaan J. C. van den Brule

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Gene Amplification ◽

Her2 Status ◽

Her2 Amplification ◽

Breast Carcinomas ◽

Her2 Gene ◽

Her2 Gene Amplification ◽

Dependent Probe

In this study the detection of HER2 gene amplification was evaluated using Fluorescence In Situ Hybridization (FISH; PathVysion) in comparison with Multiplex Ligation-dependent Probe Amplification (MLPA), a PCR based technique. These two methods were evaluated on a series of 46 formalin fixed paraffin embedded breast carcinomas, previously tested for protein overexpression by HercepTest (grouped into Hercep 1+, 2+ and 3+). HER2 gene amplification (ratio ≥ 2.0) by FISH was found in 9/10, 10/30 and 0/6 in IHC 3+, 2+ and 1+/0 cases, respectively. Digitalized automated spot counting performed with recently developed CW4000 CytoFISH software was 100% concordant with manual FISH scoring. Using MLPA 18/46 samples showed a clear HER2 amplification. Comparing MLPA and IHC showed the same results as for FISH and IHC. All but one FISH positive cases (18/19) were confirmed by MLPA for the presence of the gene amplification. The overall concordance of detection of Her2 gene amplification by FISH and MLPA was 98% (45/46). Furthermore, both the level of amplification and equivocal results correlated well between both methods. In conclusion, MLPA is a reliable and reproducible technique and can be used as an either alternative or additional test to determine HER2 status in breast carcinomas.

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HER2 Amplification Status in Feline Mammary Carcinoma: A Tissue Microarray–Fluorescence In Situ Hydridization–Based Study

Veterinary Pathology ◽

10.1177/0300985818808531 ◽

2018 ◽

Vol 56 (2) ◽

pp. 230-238 ◽

Cited By ~ 2

Author(s):

Luisa Vera Muscatello ◽

Enrico Di Oto ◽

Giuseppe Sarli ◽

Valentina Monti ◽

Maria Pia Foschini ◽

...

Keyword(s):

Protein Expression ◽

Gene Amplification ◽

Her2 Status ◽

Tyrosine Kinase Receptor ◽

Her2 Amplification ◽

Her2 Gene ◽

Her2 Protein ◽

Mammary Carcinomas ◽

Her2 Gene Amplification

Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor overexpressed in a subset of breast cancer due to HER2 gene amplification. HER2 protein is expressed in feline mammary carcinomas, but little is known about its cytogenetic alterations. The aim of this study was to evaluate HER2 gene amplification status and its correlation with HER2 protein expression in feline mammary carcinomas. Feline mammary carcinomas were retrospectively selected and immunohistochemically (IHC) evaluated for HER2 protein expression. All the HER2 IHC-positive (3+) and equivocal (2+) cases and a subset of negative cases (0/1+) were selected for fluorescence in situ hybridization (FISH). Dual-core tissue microarrays were prepared for FISH. IHC and FISH were evaluated according to the 2013 American Society of Clinical Oncology/College of American Pathologists guidelines. The study included 107 feline mammary carcinomas from 88 queens. HER2 protein expression was positive (3+) in 7 cases (6.5%), equivocal (2+) in 48 cases (45%), and negative (0/1+) in 52 cases (48.5%). HER2 status was indeterminate in 8 feline mammary carcinomas (12%), amplified in 3 (4%), equivocal in 4 (6%), and nonamplified in 53 (78%). HER2 gene amplification and protein expression were significantly positively correlated ( R = 0.283; P < .0001). HER2 gene is amplified in a subset of feline mammary carcinomas despite the HER2 positive or equivocal protein expression, but it remains to be determined if the HER2 amplification is a gene alteration that drives mammary tumor carcinogenesis or only a bystander passenger mutation.

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