Level Of HER2 Amplification Research Articles

Highlights of This Issue

Paclitaxel, a chemotherapeutic agent that induces cell death via microtubule arrest, is the standard of care for metastatic triple-negative breast cancer, however response rates vary, and resistance often develops. Here, Oudin and colleagues report a novel mechanism of resistance to paclitaxel. Expression of pro-metastatic splice variants of the actin regulator Mena allows tumor cells to proliferate, invade and metastasize during Paclitaxel treatment by promoting microtubule dynamics and survival signaling. Paclitaxel treatment also elevates Mena protein levels in vitro and in vivo. These results may lead to improved biomarkers and therapeutic strategies for use of paclitaxel to treat metastatic disease.There is substantial preclinical evidence that drug-to-antibody ratio can have a significant impact on ADC physicochemical and pharmacological properties and has emerged as an important design parameter. Here, Burke and colleagues explored this with PEGylated glucuronide-MMAE linkers conjugated at 8-drugs per antibody, providing homogeneous ADCs that varied in hydrophobic nature. A series of drug-linkers with discrete PEG side chains containing up to 24 ethylene oxide units were evaluated and a threshold length of PEG8 was found to preserve ADC pharmacokinetic properties. Conjugates bearing PEG of sufficient length to preserve ADC exposure provided a wider therapeutic window relative to faster-clearing conjugates bearing smaller PEG side chains.Pretargeted radioimmunotherapy (PRIT) harnesses the tumor-targeting properties of mAbs and the rapid in vivo kinetics of small molecules, offering an attractive approach for moleculary targeted radiotherapy. Houghton and colleagues have adapted a strategy for pretargeted radioimmunotherapy based on the inverse electron demand Diels-Alder cycloaddition between a tetrazine (Tz) bearing radioligand and a trans-cyclooctene (TCO) modified mAb. In this issue, they report the first demonstration of the in vivo efficacy of such a system in a murine model of pancreatic ductal adenocarcinoma. Their PRIT system achieved a dose-dependent reduction in tumor volume and a promising dosimetry profile, suggesting that this approach could be suitable for clinical PRIT applications.The human epidermal growth factor receptor 2 gene (HER2), also known as ERBB2, is amplified and overexpressed in 16% of upper gastro-esophageal (UGI) adenocarcinomas. HER2 amplification levels and HER2 genomic heterogeneity were analyzed in UGI cancers from patients in the LOGiC (TRIO-013) clinical trial of chemotherapy with or without lapatinib, a small molecule inhibitor of EGFR and HER2 to assess a potential role in treatment responsiveness. Press and colleagues found that Asian participants and those less than 60 years of age had significant improvements in progression-free survival, particularly among those whose cancers had 5.01–10.0 and >10.0-fold amplification of HER2.

HER2 amplification level is not a prognostic factor for HER2-positive breast cancer with trastuzumab-based adjuvant treatment: a systematic review and meta-analysis.

BackgroundTrastuzumab-based therapy is a standard, targeted treatment for HER2-positive breast cancer in the adjuvant setting. However, patients do not benefit equally from it and the association between HER2 amplification level and patients' survival remains controversial. A systematic review and meta-analysis was conducted by incorporating all available evidence to evaluate the association between disease free survival (DFS) and HER2 amplification level.ResultsThree cohort studies involving 1360 HER2-positive breast cancer patients stratified by HER2 amplification magnitude were eligible for meta-analysis. The combined HRs for DFS were 1.05 (95% CI: 0.80−1.36, p = 0.74) and 0.97 (95% CI: 0.73−1.29, p = 0.83) for HER2 gene copy number (GCN) and HER2/CEP 17 ratio. No evidence of heterogeneity or public bias was found.MethodsDatabases including PubMed, Embase, Web of Science, and Cochrane Central Register of Controlled Trials (CENTRAL), were searched for eligible literature. HER2 amplification level was evaluated by fluorescence in situ hybridization (FISH) in terms of gene copy number (GCN) and HER2/CEP17 ratio. Hazard ratios (HRs) for DFS with 95% confidence interval (CI) according to the amplification level of HER2 were extracted. The outcomes were synthesized based on a fixed-effects model.ConclusionsHER2 amplification level is not a prognostic factor for HER2-positive breast cancer with trastuzumab-based targeted therapy in the clinical adjuvant setting.

Abstract 1374: Isolation and analysis of pure intact tumor cell populations from FFPE: Implications for more precise HER2 FISH testing in breast cancer

Abstract Body: Guidelines worldwide focus on the importance of precise, reproducible, and quality assurance of Fluorescent In Situ Hybridization (FISH) methods for testing companion diagnostic markers, including Human Epidermal Growth Factor Receptor 2 (HER2) gene amplification in breast cancer. Despite these guidelines, variations in test results due to pre-analytical sampling and tissue processing are observed. In this study, we demonstrate a unique approach to isolating pure and intact tumor cells from breast cancer Formalin-Fixed, Paraffin-Embedded (FFPE) samples for precise subsequent FISH analysis. Methods: Fifty-micron thick FFPE curls from both HER2 non-amplified breast cancer tumors (n = 4; each with a reported HER2/CEP17 ratio 1.8) and positive control SKBr3 breast cancer cells were tested. Isolation of ∼250 pure and intact cytokeratin-positive/vimentin-negative/DAPI positive tumor cells from each sample was achieved using the DEPArray™ platform, an automated system enabling image-based cell sorting with single-cell resolution for pure cell population isolation and collection. Recovered cells were then cyto-spun onto poly-L coated glass slides prior to standard dual-color HER2/CEP17 FISH (Path Vysion Abbott/Vysis) analysis. Results: Positive HER2 amplification levels for the FFPE derived control SKBr3 cells were observed (HER2/CEP17 ratio >4.4) and consistent with levels reported in the literature. Among the patient samples, ≥75% of the DEPArray™ isolated tumor cells were recovered onto slides prior to FISH. Through routine FISH scoring, an expected non-amplified result was observed for each patient sample, with observed HER2/CEP17 ratios only ranging from 1.1 to 1.4. Conclusion: We demonstrate feasibility in performing FISH for HER2/CEP17 on pure and intact tumor cells isolated from breast cancer derived FFPE using the DEPArray™ platform. Using a 50-micron section permitted recovery of whole, intact, tumor cells based on immunostaining for cytokeratin, vimentin, and DAPI. Efficient recovery of the DEPArray™ sorted cells onto slides further permitted routine FISH analysis of only tumor cells. These preliminary results imply the possibility of more precise FISH analysis when standard FISH results are inconclusive or when insufficient tumor content prohibits downstream analysis. Evaluation of larger numbers of patient samples is underway. Citation Format: Amanda Gerber, Trisky Clarin, Ambica Bhandari, Valeria Sero, Chiara Bolognesi, Gianni Medoro, Nicolò Manaresi, Mathew Moore, Philip D. Cotter, Farideh Bischoff. Isolation and analysis of pure intact tumor cell populations from FFPE: Implications for more precise HER2 FISH testing in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1374.

HER2-amplification level and tumor-infiltrating lymphocytes in breast cancer patients treated with neoadjuvant trastuzumab.

596 Background: Trastuzumab-based neoadjuvant treatment (TNT) has shown clinical benefit in terms of both overall response and pathologic complete response (pCR). In particular, TNT is indicated for women with HER2-positive locally advanced breast cancer and allows for breast-conserving surgery. In addition, some evidences show that TIL may play a role in the response to anti-HER2 therapy. This study aims to investigate the possibility that HER2-amplification level and high level of TIL are predictive factors of response to TNT in locally advanced breast cancer. Methods: 75 HER2-positive breast cancer pts treated from 2009 to 2014 with TNT were included in this retrospective study. Preoperative biopsies or cytological samples were collected and analyzed by dual-color fluorescence in situ hybridization (FISH) to determine HER2-amplification. We used the cut-off recommended by ASCO/CAP 2013. TIL were evaluated in both pre- and post-treatment specimen; TIL score was assessed on hematossilin-eosin slides based on Salgado et al. recommendations. Clinical-pathological characteristics were recorded. Statistical analyses were performed using IBM-SPSS (V21.0). A p-value < 0.05 was considered statistically significant. Results: Among the 75 cases, HER2-amplification level was low (LA) in 14 cases (19%), intermediate (IA) in 17 cases (23%) and high (HA = gene copy number > 10 or clusters of signals) in 44 cases (59%). TNT achieved pCR in 19 of 75 tumors and correlated with HER2-amplification level (36% HA vs 6% IA vs 14% LA, p-value = 0.01). TILs levels were evaluated in 58 biopsies and surgical specimens: only 6 pts were classified as lymphocyte-predominant breast cancer (TILs ≥ 50%). The probability of developing pCR increased with TILs levels, although a statistically significant correlation was not achieved. No correlation was found between TIL and HER2-amplification levels. Age, stage, tumor grade and hormone receptor status were not significantly correlated to pCR. Conclusions: We show that while HER2-amplification level correlates positively with pCR to TNT, high level of TIL is not predictive of better response to trastuzumab in locally advanced HER2-positive disease.

Image-based collection to allow isolation and analysis of pure intact tumor cell populations from FFPE: Implications for more precise HER2 FISH analysis.

e12094 Background: Guidelines worldwide focus on the importance of precise and accurate Fluorescent In Situ Hybridization (FISH) methods for testing companion diagnostic markers, including Human Epidermal Growth Factor Receptor 2 (HER2) gene amplification in breast cancer. Despite these guidelines, variations in test results are observed due to pre-analytical sampling. In this study, we demonstrate a unique approach to isolating pure and intact tumor cells from breast cancer Formalin-Fixed, Paraffin-Embedded (FFPE) samples for precise FISH analysis. Methods: Fifty-micron thick FFPE curls from both HER2 non-amplified breast cancer tumors (n = 4; each with a reported HER2/CEP17 ratio 1.8) and positive control SKBr3 breast cancer cells were tested. Isolation of ∼250 pure and intact cytokeratin-positive/vimentin-negative/DAPI positive cells from each sample were captured using the DEPArray platform, an automated system enabling image-based cell sorting with single-cell resolution for pure cell population isolation and collection. Standard dual-color HER2/CEP17 FISH (Vysis PathVysion) analysis was performed on recovered cells. Results: Positive HER2 amplification levels for the FFPE derived control SKBr3 cells were observed HER2/CEP17 ratio > 4.4. Among the patient samples, ≥ 75% of the DEPArray isolated tumor cells were recovered onto slides. Through routine FISH scoring, an expected non-amplified result was observed for each patient sample, with HER2/CEP17 ratios ranging from 1.1 to 1.4. Conclusions: We demonstrate feasibility in performing FISH on pure and intact tumor cells isolated from breast cancer derived FFPE using the DEPArray platform. Using a 50-micron section permitted recovery of whole, intact, tumor cells based on immunostaining for cytokeratin, vimentin, and DAPI. Efficient recovery of the DEPArray sorted cells onto slides further permitted routine FISH analysis of only tumor cells. These preliminary results imply the possibility of more precise FISH analysis when standard FISH results are inconclusive or when insufficient tumor content prohibits downstream analysis. Evaluation of larger numbers of patient samples is underway

Prognostic role of HER2 amplification based on fluorescence in situ hybridization (FISH) in pancreatic ductal adenocarcinoma (PDAC): a meta-analysis.

BackgroundPrevious studies have shown that human epidermal growth factor receptor 2 (HER2) may play an important role in the invasion and metastasis of pancreatic cancer, but the relationship between HER2 amplification level and prognosis of pancreatic cancer patients is still controversial. Therefore, we performed a meta-analysis to determine the prognostic significance of HER2 amplification based on fluorescence in situ hybridization (FISH) in patients with pancreatic cancer.MethodsPubMed, EMBASE, and Web of Science (Jan 2001 to Jun 2015) were searched. Only articles that detect the HER2 amplification by FISH method were included. RevMan 5.3 and STATA version 12 were used to perform this meta-analysis. Pooled calculations were carried out on hazard ratio (HR) and 95 % confidence interval (CI) to assess the risk of disease.ResultsA total of six eligible studies were enrolled in meta-analysis. The univariate analysis results showed that HER2 amplification was not significantly associated with patients’ overall survival (pooled HR, 1.87, 95 % CI, 0.64–5.46, P = 0.25), which are maintained in one study of multivariate analysis (HR 0.51, 95 % CI, 0.12–2.14, P = 0.358). HER2 amplification also had no correlation with clinicopathological factors such as age, gender, lymph node metastasis, and tumor stage.ConclusionsOur results showed that HER2 amplification based on FISH may not be a good prognostic factor for survival in patients with pancreatic cancer.

Droplet digital polymerase chain reaction detection of HER2 amplification in formalin fixed paraffin embedded breast and gastric carcinoma samples

RationaleHuman epidermal growth factor receptor 2 (HER2) is a key driver of tumorigenesis, and over-expression as a result of HER2 gene amplification has been observed in a number of solid tumors. Recently HER2 has become an important biomarker for the monoclonal antibody treatment of HER2-positive metastatic breast and advanced gastric cancer. The HER2 targeting antibody trastuzumab treatment requires accurate measurement of HER2 levels for proper diagnosis. Droplet digital PCR (ddPCR) with highly direct, precise and absolute nucleic acid quantification could be used to detect HER2 amplification levels. ObjectivesOur objective was to evaluate a robust, accurate and less subjective application of ddPCR for HER2 amplification levels and test the assay performance in clinical formalin-fixed paraffin-embedded (FFPE) breast and gastric carcinoma samples. MethodsGenomic DNA from HER2 amplified cell line SK-BR-3 was used to set up the ddPCR assays. The copy number of HER2 was compared to the chromosome 17 centromere reference gene (CEP17), expressed as HER2:CEP17 ratio. Genomic DNAs of FFPE specimens from 145 Asian patients with breast and gastric carcinomas were assayed using both standard methods, immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), and ddPCR. ResultsBased on 145 clinical breast and gastric carcinoma cases, our study demonstrated a high concordance of ddPCR results to FISH and IHC. In breast cancer specimens, the ddPCR results had high concordance with FISH and IHC defined HER2 status with a sensitivity of 90.9% (30/33) and a specificity of 100% (77/77). In gastric cancer specimens that were concordant in both FISH and IHC, our assay was 95.5% concordant with FISH and IHC (21/22). ConclusionsddPCR has the advantage of automation and also allows levels of HER2 amplification to be easily evaluated in large numbers of samples, and presents a potential option to define HER2 status.

A36 - Relationship between levels of HER-2 amplification and pathologic complete response to trastuzumab-based neoadjuvant treatment

A36 - Relationship between levels of HER-2 amplification and pathologic complete response to trastuzumab-based neoadjuvant treatment

Human epidermal growth factor receptor 2 expression in mixed gastric carcinoma.

To investigate human epidermal growth factor receptor 2 (HER2) amplification and protein expression in mixed gastric carcinoma. Fluorescence in situ hybridization and immunohistochemistry were used to detect HER2 amplification and protein expression in 277 cases of mixed gastric carcinoma. Protein staining intensity was rate as 1+, 2+, or 3+. Of the 277 cases, 114 (41.2%) expressed HER2 protein. HER2 3+ staining was observed in 28/277 (10.1%) cases, 2+ in 37/277 (13.4%) cases, and 1+ in 49/277 (17.7%) cases. A HER2 amplification rate of 17% was detected, of which 25/28 (89.3%) were observed in the HER2 3+ staining group, 17/37 (45.9%) in 2+, and 5/49 (10.2%) in 1+. Of the 47 patients with HER2 amplification who received chemotherapy plus trastuzumab, 22 demonstrated median progression-free and overall survivals of 9.1 mo and 16.7 mo, respectively, which were significantly better than those achieved with chemotherapy alone (5.6 mo and 12.1 mo, respectively) in 19 previously treated patients (Ps < 0.05). HER2 detection in mixed gastric carcinoma displays high heterogeneity. Relatively quantitative parameters are needed for assessing the level of HER2 amplification and protein expression.

Quantitative assessment of HER2 amplification in HER2-positive breast cancer: its association with clinical outcomes.

Human epidermal growth factor receptor 2 (HER2) is an effective therapeutic target in breast cancer. However, not all patients benefit from trastuzumab-based therapy. We aimed to investigate whether patients with different levels of HER2 amplification would experience different clinical outcomes with trastuzumab-based chemotherapy. We quantified the HER2 gene copy number (GCN) and HER2/centromere chromosome probe 17 (CEP17) ratio in 291 breast cancer patients with HER2 amplification confirmed by immunohistochemistry and fluorescence in situ hybridization. The optimal cutoff points for HER2 GCN and the HER2/CEP17 ratios for distinguishing positive results were determined by receiver operating characteristic (ROC) curve analyses. ROC analysis identified optimal cutoff points for HER2 GCN and HER2/CEP17 ratios as 11.5 and 6.5 (P = 0.039 and P = 0.012), respectively. The DFS in patients with HER2 GCN <11.5 was significantly longer than in HER2 GCN ≥11.5 patients (P = 0.015) according to Kaplan-Meier survival curves analysis. Similarly, patients with HER2/CEP17 ratios <6.5 had a significantly longer DFS than those with HER2/CEP17 ratios ≥6.5 (P = 0.013). Moreover, patients with HER2 cluster amplification showed a worse survival than those with HER2 non-cluster amplification (P = 0.041). This study demonstrated a significant association between the level of HER2 amplification and survival time in a relatively large cohort of HER2-positive breast cancer patients undergoing trastuzumab-based chemotherapy. Further investigations of more precise quantitative measurements and larger cohorts are required to define this threshold.

Targeting Three Distinct HER2 Domains with a Recombinant Antibody Mixture Overcomes Trastuzumab Resistance.

HER2 plays an important role in the development and maintenance of the malignant phenotype of several human cancers. As such, it is a frequently pursued therapeutic target and two antibodies targeting HER2 have been clinically approved, trastuzumab and pertuzumab. It has been suggested that optimal inhibition of HER2 is achieved when utilizing two or more antibodies targeting nonoverlapping epitopes. Superior clinical activity of the trastuzumab plus pertuzumab combination in metastatic breast cancer supports this hypothesis. Because trastuzumab and pertuzumab were not codeveloped, there may be potential for further optimizing HER2 targeting. The study herein evaluated functional activity of anti-HER2 antibody combinations identifying optimal epitope combinations that provide efficacious HER2 inhibition. High-affinity antibodies to all four extracellular domains on HER2 were identified and tested for ability to inhibit growth of different HER2-dependent tumor cell lines. An antibody mixture targeting three HER2 subdomains proved to be superior to trastuzumab, pertuzumab, or a combination in vitro and to trastuzumab in two in vivo models. Specifically, the tripartite antibody mixture induced efficient HER2 internalization and degradation demonstrating increased sensitivity in cell lines with HER2 amplification and high EGFR levels. When compared with individual and clinically approved mAbs, the synergistic tripartite antibody targeting HER2 subdomains I, II, and IV demonstrates superior anticancer activity.

HER2 heterogeneity affects trastuzumab responses and survival in patients with HER2-positive metastatic breast cancer.

Heterogeneity of HER2 gene amplification is found in a subset of breast cancers. We investigated the impact of HER2 heterogeneity on trastuzumab responses and clinical outcomes in 112 patients with HER2-positive metastatic breast cancer. Regional and genetic heterogeneity of HER2 gene amplification was determined in three different areas of each tumor by immunohistochemistry and silver in situ hybridization. We also assessed the overall levels of HER2 amplification and the proportion of tumor cells with a HER2/CEP17 ratio of more than 2.2 or strong and complete membranous (3+) expression of HER2 protein. HER2 regional and genetic heterogeneity based on the HER2/CEP17 ratio was confirmed in 8.7% and 2.7% of cases, respectively. Poor response to trastuzumab was associated with overall low-level or equivocal amplification, HER2 regional heterogeneity by the HER2/CEP17 ratio, the HER2/CEP17 ratio of more than 2.2 in less than 80% of tumor cells, and HER2 immunohistochemical expression of 3+ in less than 75% of tumor cells. In survival analyses, low-level or equivocal HER2 amplification, HER2 regional heterogeneity based on the HER2/CEP17 ratio, and the HER2/CEP17 ratio of more than 2.2 in less than 80% of tumor cells were associated with shorter time to progression and lower overall survival in univariate and multivariate analyses. These results suggest that accurate assessment of HER2 status, including HER2 heterogeneity, is important in predicting trastuzumab responses and outcomes in patients with HER2-positive metastatic breast cancer.

Abstract CT228: A phase II study of afatinib (A) in patients (pts) with metastatic human epidermal growth factor receptor (HER2)-positive trastuzumab (T) refractory esophagogastric (EG) cancer

Abstract Background: Trastuzumab (T) combined with chemotherapy has been the standard of care for pts with HER2+ EG cancer. Resistance to T is now emerging in this population. Afatinib (A), a potent ErbB Family Blocker, induced nearly complete tumor regression in MSKCC HER2+ patient derived xenografts (PDX). We report the initial results of a phase II study of afatinib in patients with T refractory EG cancer. Methods: Pts with HER2+ (IHC 3+ or FISH&amp;gt;2.0) EG cancer -progressive on trastuzumab -received A 40 mg. Archival pre-T tissue, tumor biopsy after progression on T and after 1 week on A mandated on protocol. The primary endpoint-overall disease control: stable disease (SD) or partial response (PR). Results: 14 pts treated with A; median duration 5.1 mos (1.7 to 12.1 mos), median 2 (1 to 4) prior T regimens, 64% of tumors IHC3+; 36% IHC2+/FISH&amp;gt;2.2. Adverse events included: diarrhea (Grade 1/2:69%), fatigue (Grade1/2: 54%), rash (Grade 1/2:54%), mucositis (Grade1: 23%), paronychia (Grade 1/2:15%). 4 of 14 pts (28%) with disease control (PR or SD); including 1 pt with confirmed PR - a durable 75% regression of biopsy proven metastases. Median OS 6.6 mos (1.9 to NR). PDXs established from biopsies of T refractory tumors of 5 pts. EGFR amplification was detected in the tumor of the patient with PR on afatinib, suggesting EGFR amplification as a predictor of response to afatinib. High level of HER2 amplification was retained in post-trastuzumab resistance sample suggesting ongoing dependence on HER2. PIK3CA mutations were observed in tumors of 33% (2 of 6) patients, ERBB3 mutations in 33% cases (2 of 6), and MTOR or FBXW7 mutations in 50% (3 of 6) patients. Conclusions: Afatinib shows clinical efficacy in patients with T refractory EG cancer. The study met the criteria for expansion of the accrual based on original Simon's minimax two-stage design. Efforts to elucidate the mechanisms of T resistance including validation of potential drivers of T resistance using HER2+ PDXs are ongoing. Updated molecular and clinical data will be presented. Citation Format: Yelena Y. Janjigian, Tooba Imtiaz, David H. Ilson, David B, Solit, Michael F. Berger, Efsevia Vakiani. A phase II study of afatinib (A) in patients (pts) with metastatic human epidermal growth factor receptor (HER2)-positive trastuzumab (T) refractory esophagogastric (EG) cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT228. doi:10.1158/1538-7445.AM2014-CT228

Abstract P1-08-38: HER2 heterogeneity affects trastuzumab responses and survival in patients with HER2-postive metastatic breast cancer

Abstract Purpose: Heterogeneity of HER2 gene amplification is found in a subset of breast cancers. However, it is not known whether breast cancers with heterogeneous HER2 amplification respond differently to HER2-targeted therapy than those with homogeneous amplification. In this study, we investigated the relationship between HER2 heterogeneity and trastuzumab resistance and clinical outcomes in patients with HER2-positive metastatic breast cancer. Patients and methods: We studied tumor tissues from 127 patients with HER2-positive metastatic breast cancers who had received trastuzumab-based chemotherapy. Regional and genetic heterogeneity of HER2 amplification was determined in three different areas of each tumor by immunohistochemistry (IHC) and silver in situ hybridization. We also assessed the overall levels of HER2 amplification, and the proportion of tumor cells with a HER2/CEP17 ratio &amp;gt;2.2 or HER2 overexpression (IHC score of 3+). HER2status including HER2 heterogeneity was correlated with trastuzumab responses and survival of the patients. Results: HER2regional and genetic heterogeneity was confirmed in 7.8% and 3.6% of cases, respectively. Poor response to trastuzumab was associated with overall low-level amplification, HER2regional heterogeneity, HER2/CEP17 ratio &amp;gt;2.2 in &amp;lt;80% of tumor cells, and HER2 IHC score of 3+ in &amp;lt;75% of tumor cells. In survival analyses, low-level HER2 amplification, HER2regional heterogeneity, and HER2/CEP17 ratio &amp;gt;2.2 in &amp;lt;80% of tumor cells were associated with shorter time to progression and lower overall survival in univariate and multivariate analyses. Conclusion: Accurate assessment of HER2 status including HER2 heterogeneity is important in predicting trastuzumab responses and outcomes in patients with HER2-positive metastatic breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-38.

Abstract P4-12-02: High HER2 gene amplification and clinical outcomes in localized HER2-positive breast cancer

Abstract BACKGROUND Anti-HER2 therapy with trastuzumab is associated with a significant improvement in disease-free survival as compared to chemotherapy alone, and is considered the standard of care for localized HER2 positive breast cancer. However, a subset of HER2 positive breast cancers do not respond to trastuzumab. While various mechanisms have been proposed for trastuzumab resistance, one potential contributor could be very high level of HER2 gene amplification. Since trastuzumab is a HER2 receptor antagonist, it is possible that single agent trastuzumab might be unable to block HER2 downstream signaling thresholds efficiently in the presence of very high HER2. The clinical outcomes for tumors with high HER2 gene amplification treated with trastuzumab have not been well studied. METHODS With IRB approval, we reviewed the clinical records of all Stage I-III breast cancer patients with HER2+ breast cancer at our institution from 2008-2012. HER-2 to Chromosome 17 FISH ratio was determined by two pathologists with high inter-person reliability using the PathVysion dual color probe (Abbott Laboratories). We abstracted data on demographics, tumor characteristics including tumor size (T), lymph node involvement, grade, DCIS, HER2 amplification levels, and clinical outcomes from the clinical charts. We defined high HER2 amplification as FISH ratio &amp;gt; 8.0, as used in the HERA trial. Categorical data are summarized by frequency and percentage and comparisons between groups are performed by chi-square tests. In addition, we conducted a meta-analyses and systematic review to evaluate the association between high HER2 gene amplification and clinical outcome with/without trastuzumab in the large adjuvant HER2 clinical trials. RESULTS A total of 503 patients with HER2+ breast cancer were seen between the years of 2008-2012, and 16% (N = 82) had tumors with high HER2 levels. The median age was 50.5 years (range 29-89). The majority of tumors were T1 (56.79%) or T2 (34.57%), and had HER2 IHC staining of 3+ (94.37%). Tumors with high HER2 levels were more likely to be ER-/PR- (48.4%) than ER+/PR+ (32.8%) or ER+/PR- (18.8%), and likely to have concomitant DCIS (82.5%) and high grade (grade 3 = 74%). Women (n = 16) with high HER2+ breast cancer treated with standard neoadjuvant therapy with single agent trastuzumab (AC-TH or TCH) had a low pathological complete response (pCR) rate of 7.14%. In addition, this group had a high recurrence risk of 42.9%. Two patients with recurrence had mutation profiling by multiplexed genotyping platform (SNaPshot) and mutations in PIK3CA and TP53 oncogene were identified.One patient with grade 3, high HER2+ (FISH 8.2) microinvasive DCIS, treated with mastectomy, developed pulmonary metastases 3 years after original diagnosis. The meta-analysis revealed adjuvant trastuzumab with chemotherapy did not result in improved disease free survival as compared to chemotherapy alone among tumors with high FISH ratio (Hazard Ratio: 0.89, 95% CI: 0.57, 1.38; p = 0.60). CONCLUSIONS: Our results suggest that tumors with high HER2 amplification, including small tumors, have an aggressive biology, are less likely to respond to standard trastuzumab based therapy, and more likely to have a recurrence, compared with historical HER2 controls. High FISH may predict a clone of cells that have resistance to single agent trastuzumab warranting more aggressive HER2 directed therapy such as dual HER2 or combined HER2 and PI3K/Akt/mTOR blockade. These findings need confirmation in additional studies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-02.

HER‐2/neu gene amplification in relation to expression of HER2 and HER3 proteins in patients with esophageal adenocarcinoma

Human epidermal growth factor receptor 2 (HER2) is a therapeutic target in patients with esophageal adenocarcinoma (EAC), with gene amplification used as a selection criterion for treatment, although to the authors' knowledge the concordance between amplification and HER2 protein expression remains undefined in EAC. Furthermore, the association between HER2 and its interacting partner, human epidermal growth factor receptor 3 (HER3), is unknown yet appears to be of potential therapeutic relevance. Patients with untreated EACs (N = 673) were analyzed for HER2 amplification and polysomy 17 by fluorescence in situ hybridization in parallel with immunohistochemistry (IHC) (IHC scores of 0-1+, 2+, and 3+). Amplification was defined as HER2/CEP17 ≥ 2. HER3 expression by IHC was analyzed in randomly selected cases (n = 224). IHC and fluorescence in situ hybridization results were compared using least squares linear regression. Overall, 17% of the EACs (116 of 673 EACs) were HER2-amplified with an amplification frequency that was highest among IHC3+ cases (89%) and declined among IHC2+ cases (13%) and IHC0 to IHC1+ cases (4%). Among HER2-amplified cases, the level of amplification increased linearly with HER2 membranous expression (HER2/CEP17 ratio: 7.9 in IHC3+ and 5.5 in IHC2+ vs 2.8 in IHC0 to IHC1+ [P < .0001]), with 14% of amplified tumors demonstrating absent/faint expression (IHC0 to IHC1+). Polysomy 17 was not found to be associated with HER2 expression. Cytoplasmic HER3 expression was detected in 87% of tumors (195 of 224 tumors) and was found to be significantly associated with better differentiation (P < .0001). Stepwise increases in HER3 expression were associated with higher HER2 expression levels (P = .0019). Levels of HER2 protein expression and amplification were found to be linearly associated and highly concordant. Among amplified tumors with absent/faint expression, the level of amplification was low. Frequent expression of HER3 suggests its relevance as a therapeutic target, and its significant association with HER2 supports ongoing efforts to inhibit HER2/HER3 in patients with EAC.

Level of HER2 Gene Amplification Predicts Response and Overall Survival in HER2-Positive Advanced Gastric Cancer Treated With Trastuzumab

Previous studies have highlighted the importance of an appropriate human epidermal growth factor receptor 2 (HER2) evaluation for the proper identification of patients eligible for treatment with anti-HER2 targeted therapies. Today, the relationship remains unclear between the level of HER2 amplification and the outcome of HER2-positive gastric cancer treated with first-line chemotherapy with trastuzumab. The aim of this study was to determine whether the level of HER2 gene amplification determined by the HER2/CEP17 ratio and HER2 gene copy number could significantly predict some benefit in overall survival and response to therapy in advanced gastric cancer treated with trastuzumab-based chemotherapy. Ninety patients with metastatic gastric cancer treated with first-line trastuzumab-based chemotherapy were studied. The optimal cutoff values for HER2/CEP17 ratio and HER2 gene copy number (GCN) for discriminating positive results in terms of response and prolonged survival were determined using receiver operating characteristic curves analyses. In this study, a median HER2/CEP17 ratio of 6.11 (95% CI, 2.27 to 21.90) and a median HER2 gene copy number of 11.90 (95% CI, 3.30 to 43.80) were found. A mean HER2/CEP17 ratio of 4.7 was identified as the optimal cutoff value discriminating sensitive and refractory patients (P = .005). Similarly, the optimal cutoff for predicting survival longer than 12 months was 4.45 (P = .005), and for survival longer than 16 months was 5.15 (P = .004). For HER2 GCN, the optimal cutoff values were 9.4, 10.0, and 9.5, respectively (P = .02). The level of HER2 gene amplification significantly predicts sensitivity to therapy and overall survival in advanced gastric cancer treated with trastuzumab-based chemotherapy.

Quantification of EGFR autoantibodies in the amplification phenomenon of HER2 in breast cancer

Gene amplification or overexpression of human epidermal growth factor receptor HER2/ErB2 is seen in 25-30% of patients with breast cancer and is related to an aggressive disease. The mechanism behind the HER2 gene amplification is unknown, but it may be caused by continuous stimulation and activation. We hypothesised that autoantibodies against EGFR might have a stimulatory effect. To investigate this we developed a quantitative method to measure autoantibodies against EGFR in serum (S-EGFRAb). Serum samples from primary breast cancer patients were selected based on the degree of HER2 protein and gene amplification in the cancer tissue. Fifty patients had low levels of HER2 (≤ 16 ng/mg total protein) and no HER2 gene amplification; 43 patients had high levels of HER2 (≥ 200 ng/mg total protein) and HER2 gene amplification. Serum was also collected from controls consisting of 50 healthy age-matched women. An ELISA was developed to measure S-EGFRAb quantitatively. No significant differences in S-EGFRAb concentrations were seen between patients with high and low levels of HER2 or between the patients and the controls. Furthermore, no significant correlations were observed between S-EGFRAb and stage, differentiation state, age or prognosis. A negative correlation (p=0.0022) was found between S-EGFRAb and disease free survival in the group of patients with relapse or death. S-EGFRAb can be measured accurately using the ELISA we developed. We conclude that autoantibodies against EGFR do not seem to be associated with the HER2 gene amplification phenomenon.

Droplet Digital PCR Measurement of HER2 Copy Number Alteration in Formalin-Fixed Paraffin-Embedded Breast Carcinoma Tissue

Human epidermal growth factor receptor 2 (HER2) testing is routinely performed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) analyses for all new cases of invasive breast carcinoma. IHC is easier to perform, but analysis can be subjective and variable. FISH offers better diagnostic accuracy and added confidence, particularly when it is used to supplement weak IHC signals, but it is more labor intensive and costly than IHC. We examined the performance of droplet digital PCR (ddPCR) as a more precise and less subjective alternative for quantifying HER2 DNA amplification. Thirty-nine cases of invasive breast carcinoma containing ≥30% tumor were classified as positive or negative for HER2 by IHC, FISH, or both. DNA templates for these cases were prepared from formalin-fixed paraffin-embedded (FFPE) tissues to determine the HER2 copy number by ddPCR. ddPCR involved emulsifying hydrolysis probe-based PCR reaction mixtures containing the ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); also known as HER2] gene and chromosome 17 centromere assays into nanoliter-sized droplets for thermal cycling and analysis. ddPCR distinguished, through differences in the level of HER2 amplification, the 10 HER2-positive samples from the 29 HER2-negative samples with 100% concordance to HER2 status obtained by FISH and IHC analysis. ddPCR results agreed with the FISH results for the 6 cases that were equivocal by IHC analyses, confirming 2 of these samples as positive for HER2 and the other 4 as negative. ddPCR can be used as a molecular-analysis tool to precisely measure copy number alterations in FFPE samples of heterogeneous breast tumor tissue.

Efficacy of Primed In Situ Labelling in Determination of HER-2 Gene Amplification and CEN-17 Status in Breast Cancer Tissue

Considerable attention has been given to the accuracy of HER-2 testing and the correlation between the results of different testing methods. This interest reflects the growing importance of HER-2 status in the management of patients with breast cancer. In this study the detection of HER-2 gene and centromere 17 status was evaluated using dual-colour primed in situ labelling (PRINS) in comparison with fluorescence in situ hybridization (FISH). These two methods were evaluated on a series of 27 formalin fixed paraffin embedded breast carcinoma tumours, previously tested for protein overexpression by HercepTest (grouped into Hercep 1+/0, 2+ and 3+). HER-2 gene amplification (ratio ≥ 2.2) by PRINS was found in 3:3, 6:21 and 0:3 in IHC 3+, 2+ and 1+/0 cases, respectively. Comparing FISH and IHC (immunohistochemistry), showed the same results as for PRINS and IHC. Chromosome 17 aneusomy was found in 10 of 21 IHC 2+ cases (47.6%), of which 1 (10%) showed hypodisomy (chromosome 17 copy number per cell ≤ 1.75), 7 (70%) showed low polysomy (chromosome 17 copy number per cell=2.26 - 3.75) and 2 (20%) showed high polysomy (chromosome 17 copy number per cell ≥ 3.76). The overall concordance of detection of HER-2 gene amplification by FISH and PRINS was 100% (27:27). Furthermore, both the level of HER-2 amplification and copy number of CEN17 analysis results correlated well between the two methods. In conclusion, PRINS is a reliable, reproducible technique and in our opinion can be used as an additional test to determine HER-2 status in breast tumours.