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

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

Journal of Clinical Oncology ◽

10.1200/jco.20.02761 ◽

2021 ◽

pp. JCO.20.02761

Author(s):

Ivonne Haffner ◽

Katrin Schierle ◽

Elba Raimúndez ◽

Birgitta Geier ◽

Dieter Maier ◽

...

Keyword(s):

Gastric Cancer ◽

Tumor Cells ◽

Growth Factor Receptor ◽

Metastatic Gastric Cancer ◽

Her2 Status ◽

Her2 Expression ◽

Her2 Amplification ◽

Her2 Gene ◽

Amplification Ratio ◽

Impact On Survival

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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Metastatic Breast Cancer Survival according to HER2 and Topo2a Gene Status

Disease Markers ◽

10.1155/2009/808947 ◽

2009 ◽

Vol 26 (4) ◽

pp. 171-180 ◽

Cited By ~ 6

Author(s):

N. Todorović-Raković ◽

Z. Nešković-Konstantinović ◽

D. Nikolić-Vukosavljević

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Cancer Survival ◽

Growth Factor Receptor ◽

Metastatic Breast ◽

Her2 Amplification ◽

Potential Marker ◽

Starting Point ◽

Significant Difference

The aim of this study was to determine the relationship between amplification of HER2 (Human epidermal growth factor receptor 2) and Topo2a (topoisomerase 2a) and their influence on prognosis in metastatic breast cancer (MBC) patients. Amplification of both HER2 and Topo2a genes was determined by chromogenic in situ hybridization (CISH) in primary tumor tissue of 71 MBC patients. Starting point for follow-up was the time of diagnosis of metastatic disease. Although there was significant correlation between HER2 amplification and Topo2a alterations, Topo2a amplification was not strictly related to HER2 amplification. Follow-up of patients showed that there was no difference in MBC survival between HER2-nonamplified and HER2-amplified patients for subgroup as whole, but there was significant difference in MBC survival between patients with and without Topo2a amplification. HER2 amplification showed prognostic value in subgroups of patients, as well as Topo2a. Combination of these two genes with different status (nonamplified, amplified, coamplified) indicated that they might have additive effect. Also, it has been shown that Topo2a-amplified cases have poorer survival than Topo2a-nonamplified, when treated with CMF therapy.Topo2a amplification seems to be more promising biomarker of MBC survival, than HER2, and potential marker of resistance to CMF therapy.

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Adverse prognostic impact of heterogeneous HER2 gene amplification in patients with esophageal adenocarcinoma (EAC).

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.4009 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 4009-4009

Author(s):

Harry H. Yoon ◽

Qian Shi ◽

William R. Sukov ◽

Christopher A. Sattler ◽

Anne E. Wiktor ◽

...

Keyword(s):

Genetic Heterogeneity ◽

Multivariable Analysis ◽

Chromosome 17 ◽

Prognostic Impact ◽

Her2 Amplification ◽

Her2 Gene ◽

Digestive Cancer ◽

Cox Models ◽

Her2 Heterogeneity ◽

Risk Of Cancer

4009 Background: HER2 expression in upper digestive cancer is reported to be heterogeneous, which substantially affects interpretation of HER2 positivity in the clinic. Yet the frequency and prognostic impact of HER2 genetic heterogeneity and polysomy 17 (poly17) are unknown in EAC. Methods: HER2 amplification (fluorescence in situ hybridization) and protein expression were examined in untreated surgical EAC specimens (N = 661) at Mayo Clinic. HER2 genetic heterogeneity was defined per ASCO/CAP as amplification (HER2/CEP17 ratio ≥ 2) in 5-50% of cancer cells; poly17 refers to ≥ 3 copies of chromosome 17. Most tumors were T3-4 (68%) or lymph node (LN)-positive (73%). Cox models were used to assess disease-specific (DSS) and overall survival (OS). Results: HER2 amplification was detected in 117 of 661 EACs (18%), of which 20 (17%) showed HER2 heterogeneity. HER2 heterogeneous tumors had a significantly higher frequency of poly17 and high tumor grade. HER2 heterogeneity by amplification vs expression were correlated. Since heterogeneity was limited to HER2-amplified tumors, survival analysis was stratified by amplification status. In multivariable analysis, only HER2 heterogeneity and metastatic LN number were prognostic (Table). Conclusions: Among HER2 amplified EACs, 17% show HER2 heterogeneity, which is associated with increased poly17 and independently predicts 2-fold higher risk of cancer-specific death. Among HER2-nonamplified cases, poly17 is independently associated with worse survival. These novel findings demonstrate aggressive subgroups in HER2-amplified and -nonamplified EACs that have important implications for HER2 analysis and evaluation of benefit from HER2 targeted therapy. [Table: see text]

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Quantitative Analysis of HER2 Amplification by Droplet Digital PCR in the Follow-Up of Gastric Cancer Patients Being Treated with Trastuzumab after Surgery

Gastroenterology Research and Practice ◽

10.1155/2019/1750329 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Yanzhuo Liu ◽

Maozhu Yang ◽

Tao Jiang ◽

Chunbin Lan ◽

Hao Yuan ◽

...

Keyword(s):

Gastric Cancer ◽

Growth Factor Receptor ◽

Elongation Factor ◽

Progression Free Survival ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Great Promise ◽

Therapeutic Effects ◽

Her2 Amplification

Background. Circulating tumor DNA (ctDNA) derived from tumors is a promising biomarker for monitoring tumor status and evaluating therapeutic effects and prognosis. We studied the plasma human epidermal growth factor receptor 2 (HER2) amplification in gastric cancer (GC) patients by droplet digital PCR (ddPCR) during therapy with trastuzumab. Methods. A total of 12 patients were recruited after surgery. All patients received FOLFOX chemotherapy combined with trastuzumab as a treatment regimen. During the 12 months of the follow-up period, using elongation factor Tu GTP binding domain containing 2 (EFTUD2) as a reference gene, plasma HER2 to EFTUD2 ratios (the HER2 ratio) were determined for each patient every 2 months by ddPCR. Results. The concordance rate of HER2 amplification examined in plasma and formalin-fixed paraffin-embedded (FFPE) samples with ddPCR was 81.4%, with a sensitivity of 76.5% and a specificity of 83.8%. Plasma HER2 ratios were correlated with the primary tumor size (p<0.01). A significant decrease in the plasma HER2 ratio was found after two months of treatment (p<0.0001). Nine patients experienced partial response, and three patients had stable disease. Seven patients had progressive disease (PD) during follow-up, and four of them had died. The median progression-free survival (PFS) was 9.8 months. For each patient who developed PD, the plasma HER2 ratio was approximately 2.3-4.1 times higher than the cut-off value at the time of PD, which was the highest during the whole follow-up period. Conclusion. Longitudinal monitoring for the plasma HER2 ratio by ddPCR in the clinical courses of GC patients holds great promise for use as an indicator of tumor progression and treatment efficacy.

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A Reliable and Standardizable Differential PCR and qPCR Methodology Assesses HER2 Gene Amplification in Gastric Cancer

Biology ◽

10.3390/biology10060516 ◽

2021 ◽

Vol 10 (6) ◽

pp. 516

Author(s):

Ignacio Juarez ◽

Juan Francisco Toro-Fernandez ◽

Christian Vaquero-Yuste ◽

Marta Molina-Alejandre ◽

Inmaculada Lasa ◽

...

Keyword(s):

Gastric Cancer ◽

Genomic Dna ◽

Housekeeping Gene ◽

Her2 Overexpression ◽

Her2 Amplification ◽

Conventional Pcr ◽

Her2 Gene ◽

The Mean ◽

Pcr Techniques ◽

Reliable Methods

We have applied two PCR techniques, differential PCR (diffPCR) and qPCR for the identification of HER2 gene amplifications in genomic DNA of tumor and distal gastric samples from patients with gastric cancer. The diffPCR technique consists of the simultaneous amplification of the HER2 gene and a housekeeping gene by conventional PCR and the densitometric analysis of the bands obtained. We established a cut-off point based on the mean and standard deviation analyzing the DNA of 30 gastric tissues from patients undergoing non-cancer gastrectomy. diffPCR and qPCR yielded consistent results. HER2-overexpression was detected in 25% of patients and was further confirmed by immunohistochemistry and immunofluorescence. The approaches herein described may serve as complementary and reliable methods to assess HER2 amplification.

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Clinical difference between fibroblast growth factor receptor 2 subclass, type IIIb and type IIIc, in gastric cancer

Scientific Reports ◽

10.1038/s41598-021-84107-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Masakazu Yashiro ◽

Kenji Kuroda ◽

Go Masuda ◽

Tomohisa Okuno ◽

Yuichiro Miki ◽

...

Keyword(s):

Gastric Cancer ◽

Overall Survival ◽

Growth Factor ◽

Fibroblast Growth Factor ◽

Fibroblast Growth Factor Receptor ◽

Clinical Stage ◽

Growth Factor Receptor ◽

Fish Analysis ◽

Fibroblast Growth ◽

Clinical Difference

AbstractFibroblast growth factor receptor 2 (FGFR2) has two isoforms: IIIb type and IIIc type. Clinicopathologic significance of these two FGFR2 subtypes in gastric cancer remains to be known. This study aimed to clarify the clinicopathologic difference of FGFR2IIIb and/or FGFR2IIIc overexpression. A total of 562 patients who underwent gastrectomy was enrolled. The expressions of FGFR2IIIb and FGFR2IIIc were retrospectively examined by immunohistochemistry or fluorescence in situ hybridization (FISH) using the 562 gastric tumors. We evaluated the correlation between clinicopathologic features and FGFR2IIIb overexpression and/or FGFR2IIIc overexpression in gastric cancer. FGFR2IIIb overexpression was observed in 28 cases (4.9%), and FGFR2IIIc overexpression was observed in four cases (0.7%). All four FGFR2IIIc cases were also positive for FGFR2IIIb, but not in the same cancer cells. FGFR2IIIb and/or FGFR2IIIc overexpression was significantly correlated with lymph node metastasis and clinical stage. Both FGFR2IIIb and FGFR2IIIc were significantly associated with poor overall survival. A multivariate analysis showed that FGFR2IIIc expression was significantly correlated with overall survival. FISH analysis indicated that FGFR2 amplification was correlated with FGFR2IIIb and/or FGFR2IIIc overexpression. These findings suggested that gastric tumor overexpressed FGFR2IIIc and/or FGFR2IIIb at the frequency of 4.9%. FGFR2IIIc overexpression might be independent prognostic factor for patients with gastric cancer.

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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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Genomic somatic alterations of human epidermal growth factor-2 (HER2) gene: A pan-cancer analysis.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.3112 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 3112-3112

Author(s):

Xinhua Zhu ◽

Yong Zhou ◽

Yuzi Zhang ◽

Shangli Cai

Keyword(s):

Breast Cancer ◽

Gastric Cancer ◽

Biliary Tract ◽

Biliary Tract Cancer ◽

Circulating Tumor Dna ◽

Her2 Amplification ◽

Her2 Gene ◽

Tract Cancer ◽

Epidermal Growth ◽

Tumor Types

3112 Background: Human epidermal growth receptor 2 (HER2) is a well-known oncogenic drive gene with multiple targeted therapeutic options. In this study, we aim to assess the landscape of HER2 alterations in solid tumors and evaluate the feasibility of circulating tumor DNA (ctDNA) tested by next-generation sequence (NGS) as a tool to detect HER2 alterations. Methods: Alterations of HER2 by NGS (Illumina NextSeq 500) were queried in 3D Medicines database. The mean depth of tissue and circulating tumor DNA (ctDNA) test was 500X and 5000X, respectively. 11,013 patients tested using tumor tissue and 6,970 patients tested using ctDNA were included in this analysis. Results: Of 11,013 patients tested using tumor tissue, any HER2 and known or likely deleterious HER2 mutations were identified in 739 (6.7%) and 531 (4.8%) patients, respectively. Of 531 patients who carried known or likely deleterious HER2 mutations, 263 (49.5%) had HER2 amplification and 259 (48.8%) had single nucleotide variations (SNVs). Across all tumor types, breast cancer was found to have the highest frequency of HER2 amplification (14.9%, 48/323), followed by gastric cancer (6.6%, 31/470) and biliary tract cancer (5.8%, 33/571). Moreover, 11% (8/73) of duodenal cancer, 4.5% (7/154) of urothelial cancer, 3.8% (18/470) of gastric cancer, 3.1% (142/4555) of lung cancer, 2.9% (17/571) of biliary tract cancer, 2.8% (44/1562) of colorectal cancer and 2.7% (9/323) of breast cancer carried known or likely deleterious HER2 SNVs. Of 6970 patients tested using ctDNA, any HER2 and known or likely deleterious HER2 mutations were identified in 592 (8.5%) and 277 (4.0%) patients, respectively. In the ctDNA cohort, 15.7% (36/230) of breast cancer and 3.1% (5/161) of biliary tract cancer carried HER2 amplification. However, 11.6% (20/173) of gastric cancer had HER2 amplification tested by ctDNA which was higher than that tested using tissue. Furthermore, 5.6% (13/230) of breast cancer, 4.5% (2/44) of urothelial cancer, 3.4% (6/173), 2.5% of biliary tract cancer and 2.0% (94/4586) lung cancer harbored known or likely deleterious HER2 SNVs in ctDNA cohort. Conclusions: HER2 alterations existed across tumor types and the landscape of genomic alterations in HER2 gene varied according to different type of tumor. In addition, ctDNA can be used as a potential tool to detect HER2 alterations.

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Adverse Prognostic Impact of Intratumor HeterogeneousHER2Gene Amplification in Patients With Esophageal Adenocarcinoma

Journal of Clinical Oncology ◽

10.1200/jco.2012.43.1890 ◽

2012 ◽

Vol 30 (32) ◽

pp. 3932-3938 ◽

Cited By ~ 61

Author(s):

Harry H. Yoon ◽

Qian Shi ◽

William R. Sukov ◽

Mark A. Lewis ◽

Christopher A. Sattler ◽

...

Keyword(s):

Targeted Therapy ◽

Esophageal Adenocarcinoma ◽

Gene Amplification ◽

Prognostic Impact ◽

Her2 Amplification ◽

Her2 Gene ◽

Cox Models ◽

Node Number ◽

Polysomy 17 ◽

Her2 Heterogeneity

PurposeThere is increasing recognition of the existence of intratumoral heterogeneity of the human epidermal growth factor receptor (HER2), which affects interpretation of HER2 positivity in clinical practice and may have implications for patient prognosis and treatment. We determined the frequency and prognostic impact of heterogeneous HER2 gene amplification and polysomy 17 in patients with esophageal adenocarcinoma (EAC).Patients and MethodsHER2 amplification (by fluorescence in situ hybridization) was examined in surgical EAC specimens (n = 675). HER2 heterogeneity was defined according to consensus guidelines as gene amplification (HER2/CEP17 ratio ≥ 2.0) in more than 5% but less than 50% of cancer cells. No patient received neoadjuvant or HER2-targeted therapy. Cox models were used to assess disease-specific survival (DSS) and overall survival (OS).ResultsOverall, 117 EACs (17%) demonstrated HER2 amplification, of which 20 (17%) showed HER2 heterogeneity. All HER2-heterogeneous tumors were amplified. Among HER2-amplified tumors, heterogeneous tumors had significantly higher frequency of poor histologic grade and polysomy 17. In multivariable models that included number of metastatic lymph nodes, grade, tumor stage, and polysomy 17, only HER2 heterogeneity and node number were prognostic among HER2-amplified tumors, with heterogeneity showing worse DSS (hazard ratio, 2.04; 95% CI, 1.09 to 3.79; P = .025) and OS (P = .026). Among HER2-nonamplified EACs, polysomy 17 was independently associated with worse DSS (P = .012) and OS (P = .023).ConclusionAmong HER2-amplified EACs, 17% show HER2 heterogeneity, which independently predicts for worse cancer-specific death. Among HER2-nonamplified EACs, polysomy 17 is independently associated with worse survival. These novel findings demonstrate aggressive subgroups in HER2-amplified and -nonamplified EACs that have important implications for HER2 analysis and determination of benefit from HER2-targeted therapy.

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