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Context.— The American Society of Clinical Oncology/College of American Pathologists HER2 testing guideline in breast cancer was updated in 2018 to address issues on interpretation of uncommon results using dual-probe in situ hybridization according to the 2013 guideline. Objective.— To assess impact of the 2018 guideline on breast cancer with equivocal HER2 immunohistochemistry results. Design.— We retrospectively reviewed HER2 fluorescence in situ hybridization (FISH) data (HER2/CEP17 ratio and average HER2 copy number per cell) of HER2 immunohistochemistry–equivocal (2+ or 1+ to 2+) breast cancers at our center between January 2014 and May 2018 and compared HER2 FISH results according to 2013 and 2018 guidelines. Results.— A total of 1666 HER2 FISH results from 1421 patients with equivocal HER2 immunohistochemistry were reviewed. Based on the 2013 guideline, HER2 FISH results were amplified in 346 cases (20.8%), equivocal in 242 (14.5%), and nonamplified in 1078 (64.7%). Using the 2018 guideline, 258 cases (16%) were reclassified, including 242 previously equivocal test results (15%) and 16 previously positive results (1%) reclassified as negative. The subset of 2013 HER2-equivocal and 2018 HER2-nonamplified cases with HER2/CEP17 ratio lower than 2.0 and average HER2 copy number 4.0 or higher and lower than 6.0 showed higher incidence of micropapillary morphology compared with HER2-amplified cases. Despite most patients in this group not receiving HER2-targeted treatment, 96% had no evidence of disease at follow-up. Conclusions.— The 2018 guideline eliminated HER2 FISH–equivocal cases by reclassifying HER2-equivocal cases and cases with nonclassical amplification without HER2 overexpression as HER2 negative. As a consequence, we observed a considerable increase in HER2 FISH–negative cases and a slight decrease in HER2 FISH–positive cases.

Background: The current ASCO/CAP guidelines for the determination of HER2 status indicate that dual-probe in-situ hybridization (ISH) samples demonstrating a HER2/CEP17 ratio ≥ 2.0 and average HER2 copy number Methods: All invasive mammary carcinomas demonstrating a HER2/CEP17 ratio ≥2.0 and average HER2 copy number Results: A total of 96 (1.4%) of 6,976 consecutive FISH cases met criteria for inclusion into the study. The majority of cases tested negative (67%) or equivocal (29%) by IHC. HER2 protein overexpression was observed in 4% of cases. The results of HER2 IHC testing are summarized below. HER2 IHC results for HER2 ISH positive tumors showing ratio≥2.0 and average HER2 copy number Conclusions: HER2 ISH positive breast cancers demonstrating a HER2/CEP17 ratio ≥2.0 and average HER2 copy number Citation Format: Livasy C, Johnson N, Domfeh A. HER2 in-Situ hybridization positive breast cancers with HER2/CEP17 ratio ≥2.0 and average HER2 copy number

Purpose: To evaluate whether pathologic complete response (pCR) to neoadjuvant trastuzumab is dependent on the level of HER2 amplification. Experimental Design: 114 HER2-overexpressing early breast cancer patients who had received neoadjuvant trastuzumab were included in this study. Absolute HER2 and chromosome 17 centromere (CEP17) were measured by in situ hybridization analysis, and associations were examined between HER2/CEP17 ratio and tumor pCR status (commonly defined by ypT0 ypN0, ypT0/is ypN0, and ypT0/is). Results: In trastuzumab-treated patients, ypT0 ypN0 was achieved in 69.0% of patients with high-level amplification (HER2/CEP17 ratio > 6), but only in 30.4% of tumors with low-level amplification (ratio ≤ 6; P = 0.001). When pCR was defined by ypT0/is ypN0 or ypTis, 75.9% and 82.8% of tumors with high-level amplification had a complete response, whereas only 39.1%, and 38.3% with low-level amplification achieved pCR (P = 0.002 and P < 0.001, respectively). Logistic regression revealed that tumors with high-level amplification had a significantly higher probability achieving ypT0 ypN0 (OR, 5.08; 95% confidence interval, 1.86–13.90; P = 0.002) than tumors with low-level amplification, whereas no other clinicopathologic parameters were predictive of pCR. The association between high-level HER2 amplification and pCR was almost exclusively confined to hormone receptor (HR)–positive tumors (ypT0 ypN0: 62.5% vs. 24.0%, P = 0.014; ypT0/is ypN0: 75.0% vs. 28.0%, P = 0.005; and ypT0/is: 87.5% vs. 28.0%, P < 0.001), and was largely absent in HR-negative tumors. Conclusions: An HER2/CEP17 ratio of >6 in the pretherapeutic tumor biopsy is associated with a significantly higher pCR rate, particularly in HER2/HR copositive tumors, and can be used as a biomarker to predict response before neoadjuvant trastuzumab is initiated. Clin Cancer Res; 23(14); 3676–83. ©2017 AACR.

Purpose To evaluate whether pathological complete response to neoadjuvant trastuzumab is dependent on the level of HER2 amplification. Patients and Methods 114 women with HER2-overexpressing early breast cancer who had received neo-adjuvant trastuzumab in the prospective ABCSG-24 and ABCSG-32 trials, and for whom the HER2/CEP17 ratio was available, were included in this analysis. The ratio was correlated with tumor response as measured by the three most commonly used definitions of pathological complete response: ypT0 ypN0, ypT0/is ypN0, and ypT0/is. Results In trastuzumab-treated patients, ypT0 pN0 was achieved in 69.0% of patients with a HER2/CEP17 ratio of >6, but only in 30.4% of tumors with a ratio of ≤6 (p=0.001, Chi Square test). When pCR was defined by ypT0/is pN0 or by ypTis, 75.9% and 82.8% of tumors with a high ratio had a complete remission, while only 39.1%, and 38.3% with a low ratio achieved a pCR (p=0.002 and p Conclusion A HER2/CEP17 ratio of >6 in the pre-therapeutic tumor biopsy is associated with a significantly higher pCR rate particularly in HER2 / HR co-positive tumors, and can be used to predict outcome before neoadjuvant trastuzumab is initiated. Citation Format: Singer CF, Tan YY, Fitzal F, Steger GG, Egle D, Reiner A, Rudas M, Gruber C, Bartsch R, Fridrik M, Seifert M, Exner R, Balic M, Bago-Horvath Z, Filipits M, Gnant M, For the Austrian Breast and Colorectal Cancer Study Group. Pathological complete response to neoadjuvant trastuzumab is dependent on HER2/CEP17 ratio in HER2-amplified early breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-10.

HER2-positive breast cancer is an aggressive subtype of breast cancer that overexpresses human epidermal growth factor receptor 2 promoting cancer cell growth. Monoclonal antibodies targeting the HER2 receptor have improved survival for this patient population. Achieving pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) has correlated with disease-free survival in multiple trials, but we do not know why some HER2-positive tumors respond better to these therapies. We evaluated the correlation between HER2/CEP17 ratio and partial versus complete response following NAC. We evaluated whether patients with higher HER2/CEP17 ratios would have higher rates of pCR after NAC. Using the National Cancer Database (NCDB), we performed a retrospective review comparing pCR rates after NAC based on HER2 ratio between 2005 and 2014. Patients were excluded if they were HER2 negative, did not undergo NAC, or if the HER2 ratio was not recorded. Trends in percentage of pCR versus partial response were analyzed using SPSS. The NCDB included 237,118 patients with HER2 equivocal or HER2-positive breast tumors. 29,291 of these patients underwent NAC, and HER2/CEP17 ratios were recorded in 14,597 of the NAC cases. A pCR was noted in 9752 patients and 11,402 patients had a partial response. The ratios were significantly different between complete vs. partial response groups (include ratios), P

College of American Pathologists published guidelines for the assessment of HER2/neu genetic heterogeneity. When 20 cells are counted for evaluation of HER2/neu amplification, a single 3:1 HER2/CEP17 ratio cell characterizes the sample as heterogneous. Heterogeneity for HER2/neu amplification may indicate biologically important characteristics including likelihood of amplification in metastases. We performed fluorescence in situ hybridization on 1546 cases. For each case, 20 cells of invasive carcinoma were analyzed for HER2/CEP17 ratio. Cases were assessed as nonamplified (ratio1.8), borderline amplified (1.8 ≤ ratio ≤ 2.2), or amplified (ratio2.20). Heterogeneity was present when the percentage of cell with ratios2.20 was ≥5% but50%. Individual cells were typed by probe ratios and distribution of cell types determined. The distribution of HER2/CEP17 ratio was determined with the number of 3:1 HER2/CEP17 cells plotted against the number of amplified cells. 3:1 HER2/CEP17 ratio cells occur with low frequency (2.2%) but are the determining factor for heterogeneity in 46% of heterogenous cases. Thirty-five percent of heterogenous cases were due to a single 3:1 cell. Single 3:1 cells are a poor predictor for additional amplified cells. Inclusion of cells with a 3:1 HER2/CEP17 ratio in the definition of heterogeneity may be too broad as these cells are the determining factor in approximately one third of diagnoses of heterogeneity but are not strongly associated with other measures of amplification. Moreover, 3:1 HER2/CEP17 ratio cells are a poor predictor for the presence of additional amplified cells in a sample.

e12632Background: Human epidermal growth factor receptor gene 2 (HER2) amplification status in breast cancers provides prognostic information and predicts response to HER2 targeted therapies. Breas...

The aim of this study was to compare HER2 amplification, as determined by the HER2 copy number (CN) and the HER2/CEP17 ratio, with protein expression in gastric and gastro-oesophageal junction (G/GOJ) adenocarcinoma.HER2 immunohistochemistry (IHC) and silver in situ hybridisation (SISH) were performed in 185 cases. Modified gastric criteria were used for IHC scoring. HER2 and CEP17 CNs were counted in at least 20 cancer cells and the ratio calculated as per previously defined protocols. These two SISH methods were statistically compared against the different IHC scores.Thirty-four cases showed amplification, by both methods in 29, and either method in five. IHC score was 3+ in 29 cases; 26 showed amplification by both methods, one by ratio only and two were not amplified. IHC score was 2+ in 24 cases; three showed amplification by both methods and two by either. One each of IHC 1+ and 0 showed an increased ratio but not CN. The HER2 CN and ratio for IHC score 3+ compared to scores 2+, 1+ and 0 were significantly different (all p