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Background. 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. Methods. 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. Results. 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 Conclusion. 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.

Background: Amplification of HER2 is seen in 20-30% of breast cancer cases. Measurement of HER2 gene amplification appears to be of vital importance in planning the treatment schedule for patients with breast carcinoma. The aim of our study was to evaluate HER2 amplification status in malignant and benign breast tumors by differential PCR (dPCR). Materials and Methods: The genomic DNA was extracted using the phenol/chloroform extraction procedure from 76 different breast tissues. Differential PCR was performed using the DNA samples isolated from fresh and paraffin- embedded breast cancer tissues. The relative copy number ratio of target gene (HER2) to control gene ( INF-γ ) was measured. dPCR products were then separated by electrophoresis using 2% agarose gel. The intensity of HER2 and INFγ bands were determined for each sample by ImageJ software. Results: According to the ratio between the band intensity of HER2 to INFγ in tumour and also normal samples, 7% and 26% rates of HER2 amplification were observed in benign and malignant samples respectively. The ratio showed a 2-5 fold increase in HER2 gene copy number for tissues with HER2 amplification whereas, a one-fold increase was found in other samples. Conclusion: Differential PCR provides a relatively rapid and inexpensive technique to assess the HER2 gene amplification, especially alongside immunohistochemistry as a routine assessing method .

Determining human epidermal growth factor receptor 2 (HER2) status in breast cancer patients can be attained by fluorescent in situ hybridization (FISH) analysis, which prompts targeted treatment options. This study was performed to compare the staining results of Dako’s manual overnight HER2 FISH pharmDx™ assay with the manual (4 hours) HER2 IQFISH pharmDx™ assay and the Dako Omnis automated (4 hours) HER2 IQFISH assay, for assessment of HER2 amplification.Thirty-four formalin-fixed paraffin-embedded (FFPE) tissue blocks from patients diagnosed with invasive breast cancer were used to compare three FISH assays in three experiments. All assays included Texas Red-labeled HER2 and fluorescein-labeled centromere 17 (CEN-17) probes. First, 20 blocks with known HER2 immunohistochemistry (IHC) and FISH results from a reference laboratory were used in the validation of HER2 FISH pharmDx™ assay. Secondly, 19 of the same blocks were used in a comparison of HER2 FISH pharmDx™ assay and HER2 Instant Quality ...

Aims To determine HER2 amplification status and HER2 overexpression status in neuroendocrine carcinomas (NECs) of the stomach. Methods and results We analysed 51 gastric NECs, including 15 pure NECs and 36 NECs associated with adenocarcinoma and/or dysplasia, for HER2 amplification by dual-colour chromogenic in-situ hybridization, and for HER2 expression by immunohistochemistry. HER2 amplification was observed in three NECs (6%) and in seven (19%) cases of adenocarcinoma/dysplasia associated with NEC. Immunohistochemically, all of the NECs, including those showing HER2 amplification, lacked HER2 expression. On the other hand, positive and equivocal HER2 overexpression was observed in three (8%) and six (17%) cases of adenocarcinoma/dysplasia associated with NEC, respectively. Conclusions HER2 expression is consistently absent in gastric NECs, regardless of HER2 amplification status or association with HER2-positive adenocarcinoma/dysplasia components. Accordingly, HER2 is unlikely to be a valid therapeutic target in gastric NECs. Also, the absence of HER2 expression in NECs could be one of the causes of intratumoral heterogeneity of HER2 expression in gastric cancers.

Background ZW25, a novel IgG1 bispecific antibody, targets HER2 domains ECD2 and ECD4, resulting in multiple differentiated and unique mechanisms of action, including improved receptor internalization and downregulation relative to trastuzumab. Safety, anti-tumor activity, and biomarker data from an ongoing phase 1 trial of ZW25 monotherapy in solid tumors other than breast cancer, are presented here. Methods Eligible patients with HER2 IHC 3+, IHC 2+/FISH+, or IHC 2+/FISH- tumors confirmed by central review of fresh or archival biopsies, who had progressed on all standard therapies, were enrolled. Assessments included tumor evaluations (RECIST 1.1 Q8W), circulating tumor DNA (ctDNA; pre-dose C1 D1, C2 D15, treatment end), and standard safety evaluations. Results A total of 43 patients, including 17, 6, 10, and 10 with gastroesophageal adenocarcinoma (GEA), biliary tract cancers, colorectal cancer, and other cancers, respectively, received single agent ZW25 at 10 mg/kg QW or 20 mg/kg Q2W. The median number of prior therapies was 3 (range 1-6) for all patients. For GEA and non-GEA patients, 88% and 35% respectively received at least one unique prior HER2 therapy. The most common treatment-related adverse events (all Grade 1 or 2) were diarrhea (49%) and infusion related reaction (34%). The objective response rate (all partial response (PR)) for response evaluable patients was 41% (14/34), stable disease (SD) in 38% (13/34), and progressive disease in 21% (7/34). The majority of patients (74%; 25/34) experienced a decrease in the sum of diameters for their target lesions. Compared to FISH, the positive predictive value of HER2 amplification in pre-dose C1D1 ctDNA was 90% (95% CI 79-96%), negative predictive value 45% (25-67%), and diagnostic accuracy 79% (63-90%). Disease control (PR or SD) > 5 months was associated with lower level of copy number adjusted mutational variant allele frequency in pre-treatment ctDNA (Mann Whitney p = 0.0085). Conclusions ZW25 has been well tolerated with promising single agent activity in heavily pre-treated patients. These data support further clinical development of this bispecific antibody in HER2-expressing solid tumors. Clinical trial identification NCT03929666. Legal entity responsible for the study Zymeworks, Inc. Funding Zymeworks, Inc. Disclosure F. Meric-Bernstam: Research grant / Funding (institution): Novartis; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Taiho; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Calithera; Research grant / Funding (institution): Debio; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Aileron; Research grant / Funding (institution): PUMA; Research grant / Funding (institution): CytomX; Research grant / Funding (institution): Zymeworks; Research grant / Funding (institution): Curis; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): eFFECTOR; Research grant / Funding (institution): Abbvie; Research grant / Funding (institution): Guardant Health; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): GlaxoSmithKline; Honoraria (self): Sumitomo Dainippon Pharma; Dialectica; Advisory / Consultancy: Genentech; Inflection; Pieris; Darwin Health; Samsung Bioepis; Aduro; Spectrum; OrigiMed; Debio; Xencor; Jackson Laboratory; Mersana; Seattle Genetics; Zymeworks; Kolon; Parexel International. M. Beeram: Speaker Bureau / Expert testimony, Speaker’s bureau: Genentech. K. Lee: Honoraria (self): BMS; Honoraria (self): Eli Lilly; Research grant / Funding (institution): ALX Oncology; Research grant / Funding (institution): Array BioPharma; Research grant / Funding (institution): AstraZeneca/Medimmune; Research grant / Funding (institution): Five Prime Therapeutics; Research grant / Funding (institution): Green Cross Corp; Research grant / Funding (institution): LSK BioPharma; Research grant / Funding (institution): Macrogenics; Research grant / Funding (institution): Merck KGaA; Research grant / Funding (institution): MSD; Research grant / Funding (institution): Ono Pharmaceutical; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Pharmacyclics; Travel / Accommodation / Expenses: BMS. Y. Kang: Advisory / Consultancy: Ono; Advisory / Consultancy: BMS; Advisory / Consultancy: Daehwa; Advisory / Consultancy: LSKBiopharma; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Macrogenics; Advisory / Consultancy: Zymeworks; Advisory / Consultancy: Blueprint; Advisory / Consultancy: Merck; Advisory / Consultancy: Serono; Advisory / Consultancy: Novartis; Advisory / Consultancy: Astellas; Research grant / Funding (institution): Roche; Research grant / Funding (institution): Novartis. J. Chaves: Shareholder / Stockholder / Stock options: Northwest Medical Specialties, PLLC. C. Vaklavas: Advisory / Consultancy, no compensaton: Genentech; Advisory / Consultancy: Daiichi-Sankyo; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Roche; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Pharmacyclics; Research grant / Funding (institution): Tracon; Research grant / Funding (institution): Innocrin; Research grant / Funding (institution): Zymeworks; Research grant / Funding (institution): H3 Biomedicine. D. Oh: Advisory / Consultancy: Genentech; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Novartis; Advisory / Consultancy: Merck Serono; Advisory / Consultancy: Bayer; Advisory / Consultancy: Taiho; Advisory / Consultancy: ASLAN; Advisory / Consultancy: Halozyme; Advisory / Consultancy: Zymeworks; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Array; Research grant / Funding (institution): Eli Lilly. E. Elimova: Full / Part-time employment, spouse is employee in vaccine global division: Meric; Advisory / Consultancy: BMS; Honoraria (self): Zymeworks. C. Ferrario: Honoraria (self): Pfizer; Honoraria (self): Bayer; Honoraria (self): Novartis; Honoraria (self): AstraZeneca; Honoraria (self): Merck; Honoraria (self): Astellas Pharma; Advisory / Consultancy: Genomic Health; Advisory / Consultancy: Merck; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Bayer; Speaker Bureau / Expert testimony, Speaker’s bureau: Novartis; Research grant / Funding (institution): Amgen; Research grant / Funding (institution): Astellas Pharma; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Cascadian Therapeutics; Research grant / Funding (institution): Celldex; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): Merck; Novartis; Roche/Genentech; Sanofi; Pfizer; Janssen; Zymeworks; Travel / Accommodation / Expenses: Novartis; Roche. A. Fortenberry: Full / Part-time employment: Zymeworks, Inc.; Shareholder / Stockholder / Stock options: Zymeworks, Inc. G. Rowse: Full / Part-time employment: Zymeworks, Inc.; Shareholder / Stockholder / Stock options: Zymeworks, Inc. T. Gray: Full / Part-time employment: Zymeworks, Inc.; Shareholder / Stockholder / Stock options: Zymeworks, Inc. R. Lai: Full / Part-time employment: Zymeworks, Inc.; Shareholder / Stockholder / Stock options: Zymeworks, Inc. E. Hamilton: Research grant / Funding (institution): Zymeworks; Research grant / Funding (institution): Lilly; Advisory / Consultancy, No personal compensation: Lilly; Research grant / Funding (institution): Pfizer; Advisory / Consultancy, No personal compensation: Pfizer; Research grant / Funding (institution): Genentech/Roche; Speaker Bureau / Expert testimony, No personal compensation: Genentech/Roche; Advisory / Consultancy: Flatiron Health; Research grant / Funding (institution): Cascadian Therapeutics; Research grant / Funding (institution): Hutchinson MediPharma; Research grant / Funding (institution): OncoMed; Research grant / Funding (institution): Medimmune; Research grant / Funding (institution): StemCentrx; Research grant / Funding (institution): Abbvie; Research grant / Funding (institution): Curis; Research grant / Funding (institution): Verastem; Research grant / Funding (institution): Zymeworks; Research grant / Funding (institution): Syndax; Research grant / Funding (institution): Lycera; Research grant / Funding (institution): Rgenix; Novartis; Mersana; TapImmune; BerGenBio; Tesaro; Medivation; Kadmon, Boehringer Ingelheim; Eisai; H3 Biomedicine; Radius Health; Acerta; Takeda; Macrogenics; Immunomedics; FujiFilm; Effector. All other authors have declared no conflicts of interest.

Background HER2 (ERBB2) amplification is present in 5-10% of patients (pts) with KRAS and NRAS (RAS) WT mCRC. Tucatinib (Seattle Genetics, Bothell, WA) is a potent, highly selective oral tyrosine kinase inhibitor of the HER2 receptor. In patient-derived xenograft models of HER2+ mCRC, the combination of tucatinib and trastuzumab showed significantly greater antitumor activity compared with tucatinib or trastuzumab alone. Here we present initial results from a trial of tucatinib and trastuzumab in pts with HER2 amplified mCRC. Methods MOUNTAINEER is a multicenter open-label single-arm phase II trial. Pts with RAS WT mCRC had HER2 amplification/overexpression by NGS, FISH, or IHC (3+ or 2+ and amplified by FISH). Prior treatment with 5FU, oxaliplatin, irinotecan, and an anti-VEGF antibody was required. Prior anti-HER2 therapy was excluded. Pts received tucatinib 300mg PO bid and standard doses of trastuzumab (IV, Q3 weeks). The primary endpoint was objective response rate (ORR) per RECIST v1.1. Using a 2-Stage Fleming design, this study compared ORR of 20% (null) vs 40% (alt) in 10 or 25 evaluable pts (1-sided α = 0.11; β=.16). At least 8 responses were needed to meet the primary efficacy endpoint. Results As of 26 April 2019, 26 pts enrolled, and 22 pts completed ≥1 evaluation of response. 16/26 pts (62%) were male. Median age= 52.5 (range 24-70). The primary tumor site of origin included right colon (N = 4), left colon/rectum (N = 17), transverse colon (N = 3), and overlapping (N = 2). Among 22 evaluable pts, ORR= 55% (CR/PR= 12; SD = 5; PD = 5). Clinical benefit rate (CR+PR+SD≥4 months) = 64%. At a median follow-up of 10.6 months, median PFS= 6.2 months (95% CI 3.5-NE). Median OS = 17.3 months (95% CI 12.3-NE). Median duration of response has not been reached. There were 2 (9%) grade 3 treatment-related adverse events (TRAEs) and no grade 4/5 TRAEs. The most common TRAEs were AST elevation (48%; all G1), ALT elevation (30%; all G1), and diarrhea (26%; G1/G2/G3=4%/17%/4%). Conclusions The combination of tucatinib and trastuzumab is well tolerated and has met its primary efficacy endpoint. Based on these results, further expansion of the MOUNTAINEER trial in pts with HER2 amplified mCRC is justified. Updated results will be presented. Clinical trial identification NCT03043313. Legal entity responsible for the study Academic and Community Cancer Research United. Funding Seattle Genetics. Disclosure J.H. Strickler: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Amgen; Honoraria (self), Advisory / Consultancy: Bayer; Honoraria (self): Chugai; Advisory / Consultancy, Research grant / Funding (institution): Genentech/Roche; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Seattle Genetics; Advisory / Consultancy, Research grant / Funding (institution), Shareholder / Stockholder / Stock options, Non-remunerated activity/ies: OncoMed; Advisory / Consultancy: Celgene; Advisory / Consultancy: Proteus Digital Health; Advisory / Consultancy: Chengdu Kanghong Biotechnology Ltd; Research grant / Funding (institution): Exelixis; Research grant / Funding (institution): Sanofi; Research grant / Funding (institution): Nektar; Research grant / Funding (institution): AbbVie; Research grant / Funding (institution): Gilead Sciences; Research grant / Funding (institution): Macrogenics; Research grant / Funding (institution): Leap Therapeutics. A. Cercek: Advisory / Consultancy: Bayer; Advisory / Consultancy: Proteus; Research grant / Funding (institution): Seattle Genetics. C. Wu: Research grant / Funding (institution): Vaccinex; Research grant / Funding (institution): Boston Biomedical; Research grant / Funding (institution): Lycera; Research grant / Funding (institution): Seattle Genetics; Research grant / Funding (institution): Symphogen; Research grant / Funding (institution): FLX Bio. J. Hubbard: Research grant / Funding (institution): Seattle Genetics; Honoraria (institution), Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Merck; Research grant / Funding (institution): Boston Biomedical; Research grant / Funding (institution): Treos Bio; Research grant / Funding (institution): Taiho; Research grant / Funding (institution): Senhwa pharmaceuticals. N. Kemeny: Research grant / Funding (institution): Amgen. P.M. Boland: Research grant / Funding (institution): Merck; Research grant / Funding (institution): Genentech/Roche; Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Hemispherx; Research grant / Funding (institution): Boston Biomedical; Research grant / Funding (institution): Isofol Medical; Research grant / Funding (institution), Travel / Accommodation / Expenses: Ipsen; Research grant / Funding (institution): Athenex; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Clinical Genomics. K. Ng: Advisory / Consultancy, Research grant / Funding (institution): Genentech; Advisory / Consultancy: Eli Lilly; Advisory / Consultancy: Tarrex Biopharma; Advisory / Consultancy: Bayer; Advisory / Consultancy, Research grant / Funding (institution): Seattle Genetics; Research grant / Funding (institution): Gilead Sciences; Research grant / Funding (institution): Trovagene; Research grant / Funding (institution): Celgene; Research grant / Funding (institution): Pharmavite; Research grant / Funding (institution): Consano. T. Bekaii-Saab: Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Seattle Genetics. All other authors have declared no conflicts of interest.

6001 Background: SGCs are rare tumors with no approved therapy for metastatic disease. HER2 amplification occurs in 8% among all SGC histologies, and 25-33% of the aggressive salivary duct carcinoma (SDC) histologic subtype. We hypothesized that ado-trastuzumab emtansine, a HER2 targeted antibody drug conjugate, may be clinically active in these patients. Methods: A cohort of patients with HER2 amplified SGCs were enrolled into a multi-histology basket trial of ado-trastuzumab emtansine, treated at 3.6mg/kg IV every 3 weeks. The primary endpoint was overall response rate (ORR) by RECIST v1.1 or PERCIST. A Simon two-stage optimal design was applied with type I error rate under 2.7%, power of 89%, H0 10%, H1 40%; H0 will be rejected if 6 or more responses are observed in 24 patients. Other endpoints include duration of response (DOR), progression-free survival (PFS), and toxicity. HER2 amplification was identified by next generation sequencing (NGS), and tumors were subsequently tested by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Fluorescence lifetime imaging microscopy - Förster resonance energy transfer (FLIM-FRET) assessed the propensity for HER2-HER3 heterodimerization, which leads to receptor internalization. Results: 10 patients with HER2 amplified SGCs were treated. The median age was 65 (range 36-90 years), 90% were male. The median lines of prior systemic therapy was 2 (range 0-3). ORR was 90% (9/10, 95% CI 56-100%) including 5 complete responses after prior trastuzumab, pertuzumab and anti-androgen therapy. After a median follow up period of 12 months (range 4-20 months), median DOR (range 2-19+) and median PFS (95% CI 4–22+ months) were not reached. Toxicities included grade 1 or 2 infusion reaction, thrombocytopenia and transaminitis; there were no treatment related deaths. HER2 amplification by NGS (fold change 2.8 to 22.8) correlated with HER2/CEP17≥2 by FISH (8/8 tested) or IHC3+ (10/10 tested). FLIM-FRET tested positive in 3/3. Conclusions: Ado-trastuzumab emtansine is highly efficacious in patients with HER2 amplified SGCs as identified by NGS. This study has met its primary endpoint, and cohort expansion is warranted to confirm these results. Clinical trial information: NCT02675829.

e12560 Background: Triple negative breast cancer (TNBC) is an aggressive form of breast cancer (BC) which lacks estrogen receptor, progesterone receptor and HER2 amplification. Due to the fact that up to 50% of TNBC express AR and that AR expression has been associated with poor prognosis, targeting AR in TNBC is attracting increasing interest. However, although targeting AR in prostate cancer results in initial response, resistance eventually develops, resulting in treatment failure. We previously identified Serum Response Factor (SRF) as an important transcription factor in a cellular model of castrate-resistant prostate cancer. We showed a relationship between SRF and AR which potentially plays a role in the resistance to AR-targeted therapy. Therefore, we hypothesise that AR resistance mechanisms could be bypassed by targeting SRF, singly or in combination with AR-inhibitors. Methods: In this study three TNBC cell lines with different SRF/ AR levels were selected to investigate the effect of SRF inhibition on cell viability, cell migration and cell invasion. Immunohistochemistry was used to assess SRF expression in 3 BC TMAs: TMA1 (n = 144) and TMA2 (n = 512) with different subtypes of BC and TMA3 with 138 TNBC patients. Results: MTT assays showed response to CCG1423 in the two cell lines positive for SRF (MDA-MB-231 and HS578t) with IC50 values of 20 uM, while MDA-MB-453 (SRF negative) did not respond (IC50 > 80uM). In addition, a synergistic effect on cell viability was demonstrated when CCG1423 was used in combination with MDV3100. Scratch assays to assess cell migration showed a slower gap-closure post- CCG1423 treatment compared to controls. In addition, CCG1423 treatment significantly reduced both cell lines’ ability to invade by approximately 50% at 48 hours post-treatment (p < 0.05). Analysis of SRF expression in clinical samples showed higher SRF protein expression in TNBC vs. non-TNBC patients. In addition, one TMA (n = 144) showed that higher SRF expression was associated with shorter overall survival (HR 1.99 [CI (1.02, 3.87)], P value 0.039) and shorter disease specific survival (HR 2.93 [CI (1.19, 7.21)], P value 0.014). Conclusions: Our data support the rationale for using SRF as an alternative target to AR, alone or in combination with AR-antagonists.

Targeting HER2 for the treatment of HER2-positive breast cancers is now a validated treatment paradigm. However, evidence suggests that this family of receptors may have important roles outside of the realm of HER2 amplification. There is considerable interest in the development of biomarkers to identify such breast cancers. Clin Cancer Res; 21(13); 2886–8. ©2015 AACR. See related article by Leary et al., p. 2932

Introduction The manual fluorescence in situ hybridisation (FISH) Human Epidermal Growth Factor Receptor 2 (HER2)/CEP17 testing method is frequently used, however, it is time consuming and liable to subjectivity. Automation of FISH might increase the throughput and accuracy. Aim To evaluate the agreement between automated and conventional FISH with regard to a reference test silver-enhanced in situ hybridization (SISH) for HER2 amplification, as well as its prognostic significance. Material and methods 154 oesophageal adenocarcinomas were included in a tissue microarray. HER2/CEP17 gene amplification was assessed by automated FISH and was compared with conventional HER2/CEP17 testing methods. Results 46.8% of patients showed HER2 amplified tumours by automated FISH (ratio ≥1.8) compared with 18.1% by conventional FISH. A high automated HER2/CEP17 ratio (≥1.8) was significantly associated with worse survival (HR 1.731; 95% CI 1.075 to 2.786; p=0.024). However, agreement between automated and conventional FISH was only 72.2% and 71.4% between automated FISH and SISH, compared with 94.6% for conventional FISH/SISH. Therefore, thresholds for HER2/CEP17 amplification were sequentially raised from HER2/CEP17 ratio 1.8 till 5.0. A HER2/CEP17 ratio threshold of ≥3.6 had similar prognostic significance as conventional FISH (HR 1.880; 95% CI 1.060 to 3.332; p=0.031 vs HR 1.828; 95% CI 1.102 to 3.033; p=0.020), yielded comparable amplification rates as conventional FISH (14.3% vs 18.1%) and comparable agreement to SISH/Immunohistochemistry (IHC). Conclusions Automation of HER2 FISH analysis in oesophageal cancer has not been performed before. Automated HER2 is feasible, but it seems that the HER2/CEP17 threshold should be adjusted to ≥3.6 to arrive at best comparability with other methods and prognostic value.

Epithelial ovarian cancer is the commonest cause of gynaecological cancer-associated death. The disease typically presents in postmenopausal women, with a few months of abdominal pain and distension. Most women have advanced disease (International Federation of Gynecology and Obstetrics [FIGO] stage III), for which the standard of care remains surgery and platinum-based cytotoxic chemotherapy. Although this treatment can be curative for most patients with early stage disease, most women with advanced disease will develop many episodes of recurrent disease with progressively shorter disease-free intervals. These episodes culminate in chemoresistance and ultimately bowel obstruction, the most frequent cause of death. For women whose disease continues to respond to platinum-based drugs, the disease can often be controlled for 5 years or more. Targeted treatments such as antiangiogenic drugs or poly (ADP-ribose) polymerase inhibitors offer potential for improved survival. The efficacy of screening, designed to detect the disease at an earlier and curable stage remains unproven, with key results expected in 2015.

Aim: We examined an economical method for evaluating the amplification of the human epidermal growth factor receptor 2 (HER2) gene in breast cancer specimens. Materials and Methods: We compared HER2 amplification determined by fluorescence in situ hybridization (FISH) on whole-tissue (WT) blocks used for diagnostic and on tissue microarray (TMA) sections for a cohort of 521 consecutive patients with breast cancer. In a subset of 116 patients, we examined HER2 concordance from the WT section and a TMA section from a randomly chosen additional block (a proxy of the core biopsy). Results: Overall concordance for HER2 amplification between WT and TMA sections was 98.2%, and between sections from WT and from the additional block was 99.0%. Conclusion: The high concordance rates support the use of TMA for the evaluation of HER2 amplification in breast cancer and suggest that FISH can be used to assess HER2 using core biopsies.

Smoking is considered an important risk factor for bladder cancer (BC), yet molecular characterization of BC in nonsmokers has not been extensively studied. Here, we compare molecular differences between smokers and nonsmokers with BC. BC specimens (676) profiled at a Clinical Laboratory Improvement Amendments-certified laboratory from 2006 to 2014 were retrospectively evaluated for molecular differences between smokers and nonsmokers. Protein expression was determined with immunohistochemistry. In situ hybridization was used for ERBB2 ( HER2/neu) and EGFR evaluation. Genes were evaluated using Sanger or next-generation sequencing. Thirty patients were confirmed lifetime nonsmokers (NS) and 39 were reformed or current smokers (RCS). There was a trend for increased PIK3CA mutations in NS versus RCS (43% vs 11%, p =0.1760), whereas TP53 alterations were higher in RCS versus NS (63% vs 53%, p =0.6699). EGFR amplification was observed more in NS versus RCS (22% vs 11%, p =0.5815), while HER2 was amplified only in RCS (23% vs 0%, p =0.05). The molecular differences between RCS and NS with BC suggest a different oncogenesis with potentially different treatment options. Patient summary Bladder cancer patients with no history of smoking have different molecular characteristics than those with smoking history. We found that smokers tend to have higher incidence of HER2 amplification, whereas nonsmokers seemed to have higher PIK3CA mutation. This knowledge provides essential information, which can bear relevance to treatment options.

Objectives: The purpose of this study was to determine the optimum number of cells that should be counted when scoring human epidermal growth factor receptor 2 (HER2) brightfield dual-color in situ hybridization (BDISH), including cases with HER2 /chromosome 17 (Chr17) ratios in the 1.80 to 2.20 range. Methods: In total, 131 cases of breast carcinoma with HER2 immunohistochemistry and BDISH were included. For cases with a HER2 /Chr17 ratio of less than 1.80 or more than 2.20 (n = 115), BDISH scoring was performed for 60 cells using three tumor fields, and for cases with a HER2 /Chr17 ratio of 1.80 to 2.20 (n = 16), scoring was performed for 120 cells using six tumor fields. Mean HER2 /Chr17 ratio and HER2 copy number were calculated for cumulative cell counts. Results: The HER2 status as determined by the HER2 /Chr17 ratio or HER2 copy number was unchanged following counting of additional cells in 100% of cases with ratio of less than 1.80 or more than 2.20. The HER2 status of two cases with ratios of 1.80 to 2.20 changed from positive to negative following counting of 120 cells. Conclusions: Our findings support recommendations to score 20 nuclei in conjunction with careful assessment of immunohistochemistry and scan of the BDISH slide to identify areas of heterogeneity. Scoring of additional cells/fields is likely not of benefit and might be a disadvantage since the scorer moves out of the area of strongest signal.

Aims GATA3 has been reported as a specific urothelial marker among organs in the pelvic region, and has been classified as highly sensitive and specific for urothelial and breast carcinomas. Our aim was to verify GATA3 expression in extramammary Paget disease, and to determine whether it can be use to differentiate primary vulvar Paget disease from pagetoid urothelial intraepithelial neoplasia (PUIN). We also analysed HER2 protein expression and HER2 gene amplification and their roles as prognostic factors in extramammary Paget disease. Methods and results We analysed GATA3 and HER2 expression in 11 primary vulvar Paget disease cases and two PUIN cases. All cases showed nuclear expression of GATA3. Of 13 cases, five were equivocal for HER2 expression (score 2+) and one was positive (3+). Fluorescence in-situ hybridization results showed amplification in two of these six cases. Both HER2-amplified cases were invasive. Conclusion GATA3 was positive in all extramammary Paget disease cases tested (13 cases), and it has no value for differentiating between primary and secondary vulvar Paget disease from the urological tract. HER2 amplification might confer an aggressive and invasive pattern in primary vulvar Paget disease, as both amplified cases showed an invasive pattern.

Aim: The primary aim of the study was to assess the association between risk of recurrence and HER2 equivocal gene status through immunohistochemistry in patients with early breast cancer. Patients and Methods: We retrospectively analyzed clinical and pathological data of 455 consecutive patients with early breast cancer (BC) who were HER2+ and had a HER2/CEP17 ratio

Background: HER2 in situ hybridization (ISH) has become a common test in breast cancer. Abbott FISH was used in most clinical studies showing the efficacy of anti-HER2 treatment in HER2 positive carcinomas. Only reports comparing one or two of newly developed ISH assays with Abbott FISH have been published previously. We conducted a comprehensive concordance study of 5 ISH methods with Abbott FISH in a large series of breast carcinomas. Methods: Tissue Micro Arrays (TMA) were constructed by taking three 0.6 mm tissue cores from formalin-fixed/paraffin-embedded tissue-blocks from 402 primary breast carcinomas diagnosed in 2007 (supported by the Dutch Pathological Society). Up to 384 cases were analyzable in the TMA. ISH was performed after ample experience with 6 ISH assays. Scoring was performed by two independent observers without knowledge of the other ISH data according the ASCO-guidelines for HER2−testing. HER2 and chromosome 17 (Chr17) signals were counted separately, the HER2:Chr17 ratio was calculated and considered positive when the ratio was ≥ 2.0 In cases with a ratio was between 1.8 and 2.2, additional enumeration was performed. The discordant cases were reviewed and scores were reassigned on consensus of opinion. Concordance and Cohen's kappa score were calculated. Conclusion: Concordance of 5 HER2 ISH assays with Abbott FISH were shown to be 98% or higher. Each of these tests might be used for a reliable assessment of HER2 amplification. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-11-08.

ER81 is a transcription factor that may contribute to breast cancer; however, little known about the role of ER81 in breast carcinogenesis. To investigate the role of ER81 in breast carcinogenesis, we examined ER81 expression in IDC, DCIS, ADH, HUT, and normal breast tissues by immunohistochemical staining. We found that ER81 overexpression was detected in 25.7% (9/35) of HUT, 41.2% (7/17) of ADH, 54.5% (12/22) of DCIS, and 63.0% (51/81) of IDC. In 20 of breast cancer tissues combined with DCIS, ADH, and HUT, ER81 expression was found in 14/20 (70%) IDC. In these 14 cases all cases were ER81 positive expression in DCIS, 13 of 14 cases were positively expressed of ER81 in ADH and 8 of 14 were positive for ER81 in HUT components. A statistical significance was found between NBT and HUT ( 𝑃 < . 0 5 ) and HUT and ADH ( 𝑃 < . 0 5 ). Clinical-pathological features analysis of breast cancer revealed that ER81 expression was significantly associated with Her2 amplification and was negatively associated with ER and PR expression. Our results demonstrated that ER81 overexpression was present in the early stage of breast development that suggested that ER81 overexpression may play an important role in breast carcinogenesis.

Y-box binding protein-1 (YB-1) expression in the mammary gland promotes breast carcinoma that demonstrates a high degree of genomic instability. In the present study, we developed a model of pre-malignancy to characterize the role of this gene during breast cancer initiation and early progression. Antibody microarray technology was used to ascertain global changes in signal transduction following the conditional expression of YB-1 in human mammary epithelial cells (HMEC). Cell cycle-associated proteins were frequently altered with the most dramatic being LIM kinase 1/2 (LIMK1/2). Consequently, the misexpression of LIMK1/2 was associated with cytokinesis failure that acted as a precursor to centrosome amplification. Detailed investigation revealed that YB-1 localized to the centrosome in a phosphorylation-dependent manner, where it complexed with pericentrin and γ-tubulin. This was found to be essential in maintaining the structural integrity and microtubule nucleation capacity of the organelle. Prolonged exposure to YB-1 led to rampant acceleration toward tumorigenesis, with the majority of cells acquiring numerical and structural chromosomal abnormalities. Slippage through the G(1)/S checkpoint due to overexpression of cyclin E promoted continued proliferation of these genomically compromised cells. As malignancy further progressed, we identified a subset of cells harboring HER2 amplification. Our results recognize YB-1 as a cancer susceptibility gene, with the capacity to prime cells for tumorigenesis.

We performed a multicenter assessment of a new HER2 dual-color chromogenic in situ hybridization (CISH) test and herein report on concordance of CISH data with fluorescence in situ hybridization (FISH) data and intraobserver and interlaboratory scoring consistency. HER2 results were evaluated using duplicate cores from 30 breast cancers in 5 laboratories using the Ventana HER2 dual-color ISH assay (Ventana Medical Systems, Cambridgeshire, England) and in 1 central laboratory using a standard FISH assay. Overall 93.3% of cases were successfully analyzed by CISH across the 5 participating laboratories. There was excellent concordance (98.0% overall) for diagnosis of HER2 amplification by CISH compared with FISH. Intraobserver variability (7.7%) and intersite variability (9.1%) of absolute HER2/chromosome enumeration probe 17 ratios were tightly controlled across all participating laboratories. The Ventana HER2 dual-color ISH assay is robust and reproducible, shows good concordance with a standard FISH assay, and complies with requirements in national and international guidelines for performance of ISH-based diagnostic tests.

In August 2006, the Australian government announced a decision to subsidize trastuzumab therapy for early breast cancer, to commence 6 weeks later. It was mandated that HER2 gene amplification, determined by in situ hybridization (ISH), be shown, and that the sponsor company, Roche Products Pty Ltd, should fund this testing. This announcement potentially required provision of ISH testing for HER2 for every newly diagnosed breast cancer, where previously HER2 testing had been performed by immunohistochemistry with support from a single fluorescence ISH (FISH) reference laboratory for indeterminate cases. The Australian HER2 Testing Advisory Board, an independent expert group, responded to the challenge of rapidly providing accurate nationwide ISH testing. Bright-field ISH was selected as the testing platform and a decentralized testing model, with support from a central FISH laboratory, was adopted. An implementation plan was developed addressing standards for training, accreditation, and quality assurance. Within 6 weeks, 8 pathology laboratories were accredited for ISH testing and by September 2008, 2 years after the announcement, 22 ISH testing laboratories were taking part in the national program and almost 20,000 ISH tests had been performed. This article describes the design and rapid implementation of a nationwide program of bright-field ISH as the first-line testing platform for HER2 status in early breast cancer. We believe that this model for the coordinated and large-scale implementation of a new biomarker test has wide application, given that accurate assessment of a range of novel biomarkers is being used increasingly to determine eligibility for new targeted treatment modalities.

PURPOSE While HER2 testing is well established in directing appropriate treatment for breast cancer, a small percentage of cases show equivocal results by IHC and FISH. Alternative probes may be used in equivocal cases. We present a single community-based institution's experience in further evaluating these cases. PATIENTS AND METHODS Between 2014-2016, 4255 samples were submitted for HER2 amplification testing by alternative probes, TP53, RAI1, and RARA. Of patients tested by FISH, 505/3908 (12.9%) also had IHC data. RESULTS Most (73.9%) FISH equivocal cases remained equivocal after IHC testing. Duplicate testing by IHC using a different paraffin block resulted in poor reproducibility as 44% of IHC negative became equivocal and 40% of IHC equivocal became negative. Of the equivocal cases by IHC, 52.3% became positive by alternative FISH. However, 50.5% of cases equivocal by conventional FISH were classified as HER2 amplified by alternative probes. Most cases were positive by more than one probe; 78% of positive cases were positive by RAI1 and 73.9% by TP53. There was a significant difference between IHC and FISH alternative testing (P CONCLUSION The prevalence of double HER2 equivocal cases and the discrepancy between IHC and alternative FISH testing suggest that FISH alternative testing using both RAI1 and TP53 probes is necessary for conclusive classification. Due to the significant difference between IHC repeat testing, reflexing on IHC alone in this group of cases may not be reproducible. Because almost half of FISH equivocal cases converted to HER2 amplified upon alternative testing, clinical studies to determine benefit of anti-HER2 therapy in these patients is urgently needed. Alternative FISHIHC Score 0 NegativeIHC Score 1 NegativeIHC Score 2 EquivocalIHC Score 3 PositiveNo%No%No%No%Negative1463.65454.517847.7436.4Positive836.44545.519552.3763.6Total22100.099100.0373100.011100.0 Citation Format: Sally Agersborg, Christopher Mixon, Thanh Nguyen, Sramila Aithal, Forrest Blocker, Lawrence Weiss, Robert Gasparini, Shiping Jiang, Wayne Chen, Gregory Hess, Maher Albitar. Immunohistochemistry and alternative FISH testing in breast cancer with HER2 equivocal amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4524.

e13025Background: HER2 amplification/overexpression is a prognostic and predictive factor for the development of breast cancer (BC) metastases to central nervous system. Anti-HER therapy improve ov...

3594Background: HER2 amplification (HER2+) in metastatic colorectal cancer (mCRC) is associated with resistance to anti-EGFR antibodies and response to HER2 targeted therapies. This study assessed ...

TPS3620Background: Despite advances in systemic therapy, few patients are cured, creating a large unmet need for strategies targeting novel signaling and resistance pathways. The HER2 pathway, whic...

e24264Background: ERBB2 (HER2) is a member of the human epidermal growth factor receptor family. HER2 amplification or over-expression has been shown to play an important role in the development an...

García-Caballero T, Grabau D, Green A R, Gregory J, Schad A, Kohlwes E, Ellis I O, Watts S & Mollerup J (2010) Histopathology56, 472–480 Determination of HER2 amplification in primary breast cancer using dual-colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens Aims: Fluorescence in situ hybridization (FISH) can be used to reveal several genomic imbalances relevant to proper cancer diagnosis and to the correct treatment regime. However, FISH requires expensive and advanced fluorescence microscopes in addition to expertise in fluorescence microscopy. To determine whether a newly developed dual-colour chromogenic in situ hybridization (CISH) method is a suitable alternative to FISH, we analysed the human epidermal growth factor receptor 2 gene (HER2) amplification level of 168 breast cancer specimens using dual-colour CISH and FISH and compared the results. Methods and results: We found 100% agreement between HER2 status determined by FISH and dual-colour CISH. Furthermore, we observed that the time used to score slides was significantly reduced by 28% in dual-colour CISH compared with the FISH protocol. Concordance between HER2 protein status and dual-colour CISH or FISH was equally good with an overall agreement of 96.8%. Correlation between the HER2/centromere 17 gene ratios obtained with dual-colour CISH and FISH was highly significant with an overall correlation coefficient (ρ) of 0.96. Conclusions: We conclude that dual-colour CISH and bright field microscopy are excellent alternatives to FISH when analysing the HER2 status of primary breast cancer.

714 Background: Addition of cetuximab has shown clinical benefit in RAS and BRAF wild-type (wt) patients with metastatic colorectal cancer (mCRC). Human epidermal growth factor receptor 2 ( HER2) amplification was reported to be one of the resistance mechanisms to cetuximab. We investigated whether monitoring of HER2 amplification in circulating DNA allows early detection of progression and informs about resistance to cetuximab. Methods: We analyzed HER2 amplification in circulating DNA by 8 week-interval using droplet digital polymerase chain reaction (ddPCR) from 30 RAS and BRAF wt patients, who progressed after failure of cetuximab-containing regimens between Jul 2015 and Jun 2017. Results: 16.7% (5/30) of patients exhibited dynamic fluctuations of HER2 amplification in plasma, one of whom was found to be positive for HER2 amplification in matched tumor tissue sample using FISH. Two patients had HER2 and c-MET co-amplification. All 5 primary sites were left side, 4 rectums and 1 descending colon. 3 patients received cetuximab as first line therapy, whereas 2 patients in the 2nd line setting. Among these 5 patients, changes in circulating DNA levels showed good agreement with changes in tumor volume. Furthermore, quantifications of HER2 amplification showed obvious increase with an average lead time of 2 months compared with CT documented progress. But interestingly, there was no difference in progression-free survival (PFS) between these 5 patients and the others without HER2 amplification (p > 0.05). Conclusions: Plasma HER2 amplification detected by ddPCR was showed dynamic changing over time and would predict resistance to cetuximab-containing treatment. Average 2 months lead time was observed and need to validate in the further study.

61 Background: HER2 amplification in gastrointestinal (GI) cancers, beyond gastric cancer, is uncommon and as a result infrequently tested. In breast cancer, HER2 amplification has been associated with brain metastases (BM) with associated prognostic value and therapeutic opportunity. The purpose of this project was to determine if HER2 amplification is more frequent in GI cancer patients with brain metastases. Methods: Retrospective review of our institution’s medical record system identified all patients with GI primary malignancies who had resection of BM between 1999-2015 with tissue available. Fluorescence in-situ hybridization for HER2 amplification and immunohistochemistry for HER2 expression was performed on each sample and quantified by a board-certified pathologist. Results: Twenty-five GI cancer patients with BM were identified: 40% esophageal adenocarcinoma (AC), 12% esophageal squamous cell carcinoma (SCC), 44% colorectal AC and 4% pancreatic AC. At diagnosis 36% had metastatic disease. A BM was the isolated first site of metastasis in: 11/13 esophageal cancers, 1/11 colorectal cancers and 1/1 pancreatic cancer. Two esophageal cancer patients and one colon cancer patient had BM at diagnosis but otherwise median time to BM was 30 months (range 4-127 months). There was a median of 1 BM with a maximum of 4 lesions per patient. The most common intracranial site was the cerebellum (40%) with a median metastasis size of 27 mm (range 5-55 mm). Overall, HER2 was amplified in 24% (n=6) with 4 of these patients having paired primary tumor tissue available. None of the primary tumor tissue was HER2 amplified. No colorectal cancer patients had HER2 amplification but 67% of esophageal SCC, 30% of esophageal AC and the only pancreatic AC patient had Her2 amplified and overexpressed brain metastases. Conclusions: HER2 amplification was enriched in esophageal SCC and AC patients who developed resectable BM despite none of these patients having HER2 amplification in their primary tissue. HER2 amplification testing in esophageal cancer patients with BM (regardless of histology) may lead to additional therapeutic options, even if primary tissue was negative for HER2 amplification.

This study evaluated efficacy of single-agent trastuzumab against advanced or recurrent HER2-positive endometrial carcinoma (EC), and explored predictors for HER2 amplification.Eligible patients had measurable stage III, IV, or recurrent EC. There was no limit on prior therapy although total prior doxorubicin dose was limited to 320 mg/m(2). Tumors were required to have HER2 overexpression (2+ or 3+ immunohistochemical staining) or HER2 amplification (FISH HER2/CEP 17 ratio2.0). Trastuzumab was administered intravenously at a dose of 4 mg/kg in week 1, then 2 mg/kg weekly until disease progression. The primary endpoint was tumor response.Of the 286 tumors centrally screened by LabCorp, 33 (11.5%) were HER2-amplified. Three of 8 clear (38%) cell carcinomas and 7 of 25 serous carcinomas (28%) screened exhibited HER2 amplification compared with 7% (2/29) of endometrioid adenocarcinomas. HER2 overexpression was correlated with HER2 amplification (r=0.459; p0.0001). Thirty-four women were enrolled; 1 was excluded (refused treatment); and 18 had tumors with known HER2 amplification. No major tumor responses were observed. Twelve women experienced stable disease, 18 had increasing disease, and 3 were indeterminate for tumor response. Neither HER2 overexpression nor HER2 amplification appeared to be associated with progression-free survival or overall survival.Trastuzumab as a single agent did not demonstrate activity against endometrial carcinomas with HER2 overexpression or HER2 amplification, although full planned accrual of women with HER2 amplified tumors was not achieved due to slow recruitment. Serous and clear cell endometrial carcinomas appear to be more likely to demonstrate HER2 amplification.

Our purposes were to perform a robust assessment of a new HER2 chromogenic in situ hybridization test and report on concordance of silver in situ hybridization (SISH) data with fluorescence in situ hybridization (FISH) data and on intraobserver and interlaboratory scoring consistency. HER2 results were scored from 45 breast cancers in 7 laboratories using the Ventana (Tucson, AZ) INFORM HER-2 SISH assay and in 1 central laboratory using a standard FISH assay. Overall, 94.8% of cases were successfully analyzed by SISH across the 6 participating laboratories that reported data. Concordance for diagnosis of HER2 amplification by SISH compared with FISH was high (96.0% overall). Intraobserver variability (8.0%) and intersite variability (12.66%) of absolute HER2/chromosome 17 ratios appear to be tightly controlled across all 6 participating laboratories. The Ventana INFORM HER-2 SISH assay is robust and reproducible, shows good concordance with a standard FISH assay, and complies with requirements in national guidelines for performance of diagnostic tests.

Background and aims: HER2 status should be precisely evaluated in breast cancer to ensure appropriate patient management. Method: We evaluated HER2 status in 755 breast cancers including 92 consecutively resected cases by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Results: For the 92 consecutive cases, the positive rate of HER2 IHC (2+ and 3+) was 25.0%, and HER2 amplification (HER2/CEP17 ratio ≥ 2.0) was observed in 21.7%. Of the 755 cases, 88% of IHC 3+ cases; 57% of IHC 2+ cases; 15% of IHC 1+ cases; and 1% of IHC 0 cases showed HER2 amplification. Notably, 20 (2.6%) of the 755 cases exhibited a HER2/CEP17 ratio between 1.8 and 2.2, which was recently classified equivocal by the ASCO/CAP. Most of them revealed HER2 high polysomy with an average copy number per cell between 3.29 and 7.24. Immunohistochemically, 4 of the 20 cases were luminal A, 12 were luminal B, and 3 were HER2 subtype. Conclusions: The relatively high rate of HER2 amplification among IHC 1+ or 2+ cases at our institute emphasizes the importance of validating HER2 testing in different laboratories and the need for complementary HER2 FISH in IHC 1+ patients. Most patients equivocal for HER2 amplification had HER2 high polysomy and expressed hormone receptor.

The aim of this study is to delineate more clearly the prevalence of HER2+ breast cancer in women with germline BRCA1/2 mutations. For this purpose, we analysed primary mutation screens on women with breast cancer with unequivocal HER2 amplification and assessed the proportion of BRCA1 and BRCA2 breast cancers that were HER2+ comparing this with the existing literature. The results are that 1063 primary BRCA screens had confirmed tumour HER2 status. If HER2+ only 2.5 % (4/156) and 3.2 % (5/156) of women had a BRCA1 or BRCA2 mutation identified respectively; compared to 27.7 % (115/415) and 8.2 % (34/415) with triple negative tumours. Only 2.1 % (4/195) women with BRCA1-related breast cancer had HER2 amplified breast cancers rising to 6.8 % (n = 12, p = 0.04) in BRCA2. These rates are in keeping with most of the existing literature except a recent large multicenter report which documented higher rates but with no control group. The study concluded that true HER2-amplified breast cancers are rare amongst BRCA1 mutation carriers and are less common in BRCA2 than background rates.

The aim of this study is to assess the impact of changes of the 2013 ASCO/CAP guideline on the results of HER2 testing in breast cancer. A series of 916 primary invasive breast cancer cases, assessed as HER2 2+ by IHC in part using the 2007 and in part the 2013 ASCO/CAP criteria, was evaluated for HER2 amplification status by SISH and classified according to both 2007 and 2013 ASCO/CAP ISH guideline criteria. We observed a significant increase of HER2-positive cases (12.4 to 16.8 %) and a decrease of HER2-equivocal cases (3.6 to 0.7 %). Of the cases studied, 52.1 % fulfilled both criteria of HER2/CEP17 ratio and average HER2 copy number per cell to be classified as HER2-positive. Reclassification of the cases from before the introduction of the new ASCO/CAP guideline with the 2013 ISH criteria resulted in an increase of cases with a HER2-positive status (12.4 to 14.2 %) and in a decrease of HER2-equivocal cases (3.6 to 1.6 %). The 2013 ASCO/CAP guideline selects more patients for anti-HER2 targeted therapy, mostly based on the modifications of criteria to evaluate ISH-HER2.

Background Human epidermal growth factor receptor-2 (HER2) overexpression and gene amplification are currently established by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. This study investigates whether high-density single nucleotide polymorphism (SNP) arrays can provide additional diagnostic power to assess HER2 gene status. Methods DNA from 65 breast tumor samples previously diagnosed by HER2 IHC and FISH analysis were blinded and examined for HER2 copy number variation employing SNP array analysis. Results SNP array analysis identified 24 (37%) samples with selective amplification or imbalance of the HER2 region in the q-arm of chromosome 17. In contrast, only 15 (23%) tumors were found to have HER2 amplification by IHC and FISH analysis. In total, there was a discrepancy in 19 (29%) samples between SNP array and IHC/FISH analysis. In 12 of these cases, the discrepancy towards FISH could be attributed to concomitant amplification or deletion of the centromeric region, which harbors the FISH reference probe sequence. In 3 tumors, repeated IHC/FISH analysis revealed that the original IHC/FISH analysis had failed to indicate the correct HER2 expression level. Finally, the SNP array analysis revealed that more than two thirds of the samples exhibited polyploidy that was unrecognized by conventional FISH. Conclusions Collectively, the data show that determination of HER2 copy number variations by SNP array-based genomic segmentation analysis is an effective supplement to IHC/FISH HER2 analysis that, by providing additional diagnostic sensitivity and accuracy, may elect more women for targeted treatment with HER2 inhibitors. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1035-1) contains supplementary material, which is available to authorized users.