HER2 Copy Number Research Articles

Abstract PS5-11: Prospective HER-2 testing using simultaneous immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH): Experience of dual testing in a single academic institution

Abstract Background: HER-2 testing informs prognosis and critically guides therapy in breast cancer. Current guidelines for immunohistochemistry (IHC) testing define 3+ as positive, 0 and 1+ as negative, and 2+ as equivocal with additional testing recommended. Guidelines for in situ hybridization (ISH) testing are more complex and generally define a HER-2/CEP17 ratio of ≥2.0 with average HER-2 copy number of ≥4.0 copies/cell as HER-2 positive. Further, HER-2 copy number between 4 and 6 per cell, with a ratio of <2.0, is considered equivocal while >6 per cell is frequently confirmed positive. Limited data suggest that an elevated HER-2 copy number alone has clinical relevance, however most clinical trials addressing HER-2 targeted therapeutics define HER-2 positive based on HER-2/CEP17 ≥2.0. Design: From 2013-2016, all new invasive breast cancer cases diagnosed at the University of Vermont Medical Center were prospectively tested for HER-2 using both IHC and chromogenic in situ hybridization (CISH) simultaneously. All testing was performed on formalin-fixed paraffin-embedded tissue, using the 4B5 Rabbit Monoclonal Antibody (Ventana) for IHC and the INFORM dual ISH DNA probe (Ventana) for CISH. Interpretation was performed using published criteria, and all cases were independently analyzed by two experienced reviewers. Results: 1906 patients underwent HER-2 testing by both IHC and CISH. 1671 (87.7%) patients had a HER-2/CEP17 ratio of <2.0. Of patients considered to be non-amplified by the CISH ratio, 55 (3.2%) had a HER-2 copy number >4. In IHC 0 and 1+ cases with HER-2/CEP17 ratio of <2, 0.4% and 2.3% have equivocal and excess HER-2 copies/cell, respectively, compared to 10.8% for IHC 2+. Additionally, 17 (8.6%) of the IHC 3+ cases had a CISH ratio of <2, of which 41.2% had >4 HER-2 copies/cell (see table). Conclusion: To our knowledge, this is the largest study to date that has examined the concurrent use of both IHC and ISH HER-2 assays prospectively in a real-time clinical setting. In the IHC 2+ cohort, only 12.5% were amplified by ISH testing. Interestingly, of those in the IHC 2+ cohort not amplified by CISH, 10.8% had an equivocal or excess HER-2 copy number. Further, while based on a very small sample size, 41.2% of IHC 3+ patients with a CISH ratio of <2.0 had an equivocal or excess HER-2 average copy number. Finally, data on the clinical outcome and therapeutic benefit of HER-2 targeted therapy in patients with non-amplified ISH testing and equivocal or excess HER-2 copy number remains limited, and warrants further evaluation. Table. Simultaneously tested IHC and CISH data for new invasive breast cancer casesIHCCISH ratio ≥2.0CISH ratio <2.0HER-2 copies/cell (CISH ratio <2.0 cohort)CISH ratio <2.0 with ≥4 HER-2 copies/cellIHCnn (%)n (%)<4 (n)4-6 (n)>6 (n)05010 (0.0)501 (100.0)499200.4%1+94221 (2.2)921 (97.8)8992012.3%2+26533 (12.5)232 (87.5)20625010.8%3+198181 (91.4)17 (8.6)106141.2%Total1906235 (12.3)1671 (87.7)16145323.2% Citation Format: Kara K Landry, Abiy B Ambaye, Donald L Weaver, Peter A Kaufman. Prospective HER-2 testing using simultaneous immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH): Experience of dual testing in a single academic institution [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-11.

Abstract PS6-48: Clinicopathologic features and follow-up outcomes of breast cancers with HER2 FISH group 3 Results: A single institution experience

Abstract INTRODUCTION: There is dearth information on the long-term outcome and role of HER2 targeted therapy in breast cancer patients in the HER2 FISH group 3 category, which is defined as breast cancer with a HER2/CEP17 ratio < 2 and HER2 copy number ≥ 6 signals/cell. In this study, we report the clinicopathologic features and outcomes of breast cancer patients in the HER2 FISH group 3 category at our institution. METHODS: We identified 52/2,874 (1.8%) breast cancer patients with HER2 FISH group 3 results between 1/2007 and 3/2020. 28 of these 52 patients had available detailed clinicopathologic and follow-up data, with an average follow-up of 38.5 months. RESULTS: Most of the cases with group 3 FISH results were high grade ductal carcinomas with positive hormonal receptor expressions and equivocal HER2 expression by immunohistochemistry. Among all the clinicopathologic variables, only tumor size (p=0.048) significantly contributed to the poor clinical outcomes. HER2 copy number failed to show any significant association with histologic grade, tumor size, clinical stage, hormonal receptor status or disease outcomes. There was no statistically significant difference in disease outcome between patients who were treated with HER2 targeted therapy and patients who did not receive HER2 targeted therapy, regardless of the HER2 copy number or clinical stage (Table 1). CONCLUSION: Our preliminary findings suggest that certain patients with HER2 FISH group 3 category breast cancer may not need HER-2 targeted therapy. Larger-scale studies are needed to further evaluate which HER2 FISH group 3 results are more likely to benefit from the HER-2 targeted therapy. Table 1. Response to HER2 targeted therapy in HER2 FISH group 3 breast cancer patients based on HER2 copy numbers and clinical stageHER 2 targeted therapyp-valueGivenNot givenTotal populationGood outcome (n=21)1380.371Bad outcome (n=7)61HER2 copy number < 10Good outcome (n=16)1060.366Bad outcome (n=7)61HER2 copy number ≥ 10Good outcome (n=5)321.000Bad outcome (n=0)00Stage I-IIGood outcome (n=16)970.737Bad outcome (n=3)21Stage III-IVGood outcome (n=5)410.343Bad outcome (n=4)40*Good outcome = No evidence of disease**Bad outcome = Local recurrence, distant metastasis, and/or died of disease Citation Format: Huina Zhang, Rana Ajabnoor, David Hicks, Bradley Turner. Clinicopathologic features and follow-up outcomes of breast cancers with HER2 FISH group 3 Results: A single institution experience [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-48.

HER2 copy number as predictor of disease-free survival in HER2-positive resectable gastric adenocarcinoma

PurposeThe identification of HER2 overexpression in a subset of gastric adenocarcinoma (GA) patients represents a significant step forward in unveiling the molecular complexity of this disease. The predictive and prognostic value of HER2 amplification in advanced HER2 inhibitor-treated GA patients has been investigated. However, its predictive value in resectable patients remains elusive.MethodsWe enrolled 98 treatment-naïve resectable Chinese GA patients with HER2 overexpression assessed using IHC. Capture-based targeted sequencing using a panel consisting of 41 gastrointestinal cancer-related genes was performed on tumor tissues. Furthermore, we also investigated the correlation between HER2 copy number (CN) and survival outcomes.ResultsOf the 98 HER2-overexpressed patients, 90 had HER2 CN amplification assessed using next-generation sequencing, achieving 92% concordance. The most commonly seen concurrent mutations were occurring in TP53, EGFR and PIK3CA. We found HER2 CN as a continuous variable was an independent predictor associated with DFS (p = 0.029). Our study revealed HER2 CN-high patients showed a trend of intestinal-type GA predominant (p = 0.075) and older age (p = 0.07). The median HER2 CN was 15.34, which was used to divide the cohort into CN-high and CN-low groups. Patients with high HER2 CN had a significantly shorter DFS than patients with low HER2 CN (p = 0.002). Furthermore, HER2 CN as a categorical variable was also an independent predictor associated with DFS in patients.ConclusionWe elucidated the mutation spectrum of HER2-positive resectable Chinese GA patients and the association between HER2 CN and DFS. Our work revealed HER2 CN as an independent risk factor predicted unfavorable prognosis in HER2-positive GA patients and allowed us to further stratify HER2-positive resectable GA patients for disease management.

Sensitivity of HER2-amplified colorectal organotypic cancer spheroids at ex vivo resistance to panitumumab and trastuzumab.

68 Background: HER2 amplification is an emerging biomarker in colorectal cancer (CRC) with increased copy number associated with improved clinical outcomes to HER2 targeting. RAS/RAF wildtype CRC also benefit from use of epidermal growth factor receptor inhibition (EGFRi). The sequencing of EGFRi versus HER2 inhibition in low copy number HER2 amplified CRC remains uncertain. Patient-derived cancer organoids (PDCOs) allow an ex vivo method to assess treatment sensitivity. We examined treatment sensitivity of a HER2 amplified PDCO at baseline and following resistance to panitumumab and trastuzumab. Methods: Following IRB-approval, fresh CRC tissue was cultured to maturation. After expansion, subcultures were treated with stepwise (20%) increase to physiologic Cmax of panitumumab (230ug/mL) and trastuzumab (180ug/mL). Threshold for escalation was median relative growth of +20% at 96h. Sensitivity was assessed on primary culture (RC1), panitumumab resistance (RC1-P) and trastuzumab resistance (RC1-T) using 96h of physiologic Cmax panitumumab, trastuzumab, and combination trastuzumab/pertuzumab. Individual sphere response was assessed for change in mean NADH autofluorescence intensity and ratio of NADH/FAD signal. Response was assessed at 96h in comparison to control using effect size of Glass’s Delta (GΔ). Results: Molecular profiling revealed HER2 copy number of 14 with no concurrent alterations in RAS, RAF, or PIK3CA. Time to resistance was similar between panitumumab (55 days) and trastuzumab (51 days). RC1 had baseline growth (+116%) which was reduced with single agent panitumumab (+17%, GΔ=1.40) with intermediate sensitivity to trastuzumab (+48%, GΔ=0.95) and trastuzumab/pertuzumab (46%, GΔ=0.99). Normalized NADH/FAD ratio revealed significant metabolic response to panitumumab (-20%, GΔ=0.66) and trastuzumab/pertuzumab (-35%, GΔ=1.16) with insignificant effect of single agent trastuzumab (-14%, GΔ=0.46). Following resistance to panitumumab, RC1-P had persistent growth with trastuzumab (+68%) which improved in combination trastuzumab/pertuzumab (+34%, GΔ=1.16). Following resistance to trastuzumab, RC1-T was insensitive to EGFRi with panitumumab including persistent growth (+58%, GΔ=0.70) and unchanged metabolism (+2%, GΔ=-0.10). Conclusions: Therapeutic dose escalation in a single PDCO of HER2 amplified CRC suggests improved sensitivity to EGFRi and dual HER2 targeting with trastuzumab/pertuzumab. Resistance to EGFRi resulted in persistent sensitivity to dual HER2 inhibition using trastuzumab/pertuzumab, however resistance to single agent trastuzumab. Resistance to trastuzumab resulted in future insensitivity to EGFRi. Molecular profiling at resistance revealed no pathologic alterations in EGFR or ERBB2 signaling, with ongoing analysis of transcriptional changes by RNAseq.

ER staining levels affect HER2 staining and heterogeneity.

This study explored the relationship of ER expression levels with HER2 staining properties and heterogeneity and discussed the differences in HER2 assessment caused by the 2018 ASCO/CAP guideline updates from that of the 2013 version. HER2-positive breast cancer was divided into three groups of the high hormone receptor expression (LH-high) group, low expression (LH-low) group, or negative (NLH) group to (1) compare differences in the percentage of the HER2 IHC test score of 2 + based on the 2013 ASCO/CAP guideline and in the intratumor heterogeneity of HER2 expression for breast cancer with an IHC score of 3 + among these groups, (2) compare the HER2/CEP17 ratio and the average HER2 copy number, and classified ISH groupings according to the 2018 ASCO/CAP guideline algorithm. (1) Of 244 HER2-positive breast cancers, the cases with a HER2 IHC score of 2 + (n = 54, 22.1%) were significantly more common in the LH-high group (n = 45, P < 0.001). The frequency of heterogeneity was low (n = 25, 10.2%) for the HER2 score of 3 + (n = 190, 77.9%), and significantly higher in the LH-high group (n = 19, 76%, P = 0.002). (2) In a HER2 IHC score of 2 + , Group 2 which is deemed HER2 negative according to the revised 2018 ASCO/CAP guideline was observed in 17 (39.5%) out of 43 cases, of which 16 cases (94.1%) were in the LH-high group. The LH-high group is a heterogeneous group largely consisting of heterogeneous cases with HER2 IHC scores of 2 + or 3 + . NLH, in contrast, is a homogenous group.

Assessment of HER2 Protein Overexpression and Gene Amplification in Renal Collecting Duct Carcinoma: Therapeutic Implication.

Simple SummaryRenal collecting duct carcinoma (CDC) is rare, but very aggressive, variant histology of kidney cancers. Besides surgery, the other therapeutic options, such as pharmacological or radiation therapy, have a poor impact on survival. Therefore, there is an urgent need to identify novel targets that can open up new avenues for alternative treatments. From this perspective, the aim of our study was to assess the HER2 protein expression by immunohistochemistry (IHC) and the gene copy number by fluorescence in-situ hybridization (FISH) in a cohort of 26 CDC. According to the 2018 ASCO/CAP guidelines, 2/26 CDC cases (8%) were HER2-positive. The HER2 protein is a well-established target of anti-HER2 mAbs or kinase inhibitors already used for breast and gastric cancer treatment. Thus, this study provides evidence that supports future biomarker-driven clinical trials that could address the lack of therapy, which is still an unmet clinical need for CDC patients.Collecting duct carcinoma (CDC) is rare and aggressive histology of kidney cancers. Although different therapeutic approaches have been tested, the 2-year survival remains very poor. Since CDC exhibits overlapping features with urothelial carcinoma, the analysis of shared molecular alterations could provide new insights into the understanding of this rare disease and also therapeutic options. We collected 26 CDC cases, and we assessed HER2 protein expression by immunohistochemistry (IHC) and gene amplification by fluorescence in-situ hybridization (FISH) according to 2018 ASCO/CAP HER2-testing recommendations. Six out of twenty-six (23%) tumors showed HER2 positive staining. In particular, 3+ score was present in 2/6 cases (33%), 2+ in 3/6 cases (50%) and 1+ in 1/6 cases (17%). The 6 HER2+ tumors were also analyzed by FISH to assess gene copy number. One out of six CDC with IHC 3+ was also HER2 amplified, showing an average HER2 copy number ≥4.0 (10.85) and a HER2/CEP17 ratio ≥ (5.63), while the 5/6 cases were HER2 negative. Based on the 2018 ASCO/CAP guidelines overall, 2/26 CDC cases (8%) were HER2+. The present study provides evidence for testing, in future studies, HER2 to assess its clinical value as a novel target for the treatment of this highly malignant cancer.

HER2 immunohistochemistry staining positivity is strongly predictive of tumor response to neoadjuvant chemotherapy in HER2 positive breast cancer

BackgroundThe current recommendation is to reflex test HER2 immunohistochemistry (IHC) equivocal breast cancer cases with fluorescence in situ hybridization (FISH) analysis. Either IHC 3+ or FISH positive cancers are considered HER2 positive (HER2+) and treated with HER2 targeted therapy. This study examined the predictive value of HER IHC or FISH positivity in tumor response to HER2 targeted therapy. MethodsBiopsies of 76 HER2+ breast cancer cases were evaluated. All patients were treated with neoadjuvant HER2 targeted therapy and chemotherapy. Tumor response was evaluated on the excisional specimens. Cancers with complete pathologic response (pCR) or MD Anderson residual cancer burden-I (RCB-I) were classified as responders and cancers with RCB-II/III as non-responders. Clinicopathologic parameters were correlated with response. ResultsIn univariate analysis, small tumor size, low nuclear grade, high Ki67, HER2 IHC 3+, homogenous strong HER2 IHC staining, high HER2/CEP17 ratio, and high HER2 copy number were significantly associated with pCR/RCB-I. In multivariate analysis, homogenous strong HER2 IHC staining pattern was significantly associated with pCR/RCB-I. The receiver operating characteristics (ROC) model showed either high HER2/CEP17 ratio or HER2 copy number individually was predictive of tumor response. ConclusionHER2 IHC staining pattern is significantly associated with tumor response to neoadjuvant chemotherapy, reiterating the importance of HER2 IHC evaluation. The ROC model shows either high HER2/CEP17 ratio or high HER2 copy number individually is predictive of tumor response to neoadjuvant HER2 targeted therapy in HER2+ breast cancer.

Evaluation of PD-L1, tumor-infiltrating lymphocytes, and CD8+ and FOXP3+ immune cells in HER2-positive breast cancer treated with neoadjuvant therapies.

The tumor immune microenvironment plays a critical role in the prognosis and outcome of breast cancers. This study examined the role of tumor-infiltrating lymphocytes (TILs), CD8+, FOXP3+ lymphocytes, PD-L1 expression, and other clinicopathological parameters in HER2+ breast cancer and correlate with tumor response to neoadjuvant therapy. We included 173 HER2+ patients treated with neoadjuvant HER2-targeted chemotherapy regimens from 2010 to 2016. 67 cases had biopsy blocks to evaluate TIL, CD8, FOXP3, and PD-L1 immunohistochemistry staining. Tumors were classified as pCR vs non-pCR group. Clinicopathological parameters, TIL, CD8+ and FOXP3+ cell count, and PD-L1 expression were correlated with pCR rate. Univariate analyses showed that pCR rate was significantly correlated with low PR, low ER, high Ki-67, high FOXP3, HER2 IHC3+ , high HER2 ratio and copy number. By multivariate analysis, Ki-67 was the only variable significantly correlated with pCR. PD-L1 expression was detected in 9.2% cases. TIL hotspot has a non-significant correlation with pCR rate (p = 0.096). High Ki-67 is a strong predictor for pCR in HER2+ breast cancer. TIL and FOXP3 T cells may play a role in tumor response in HER2+ cancer. PD-L1 is expressed in a subset of HER2+ breast cancer, supporting a role of immunotherapy in treating a subset of HER2+ breast cancers. The role of PD-L1, TIL, and other markers of immunogenicity as predictors of response to neoadjuvant chemotherapy in HER2+ breast cancer should be further evaluated.

Evaluation of Next Generation Sequencing for Detecting HER2 Copy Number in Breast and Gastric Cancers

Amplicon-based next generation sequencing (NGS) approaches have been preferentially adopted by the clinical laboratories on the basis of a short turnaround time (TAT) and small DNA input needs. However, little work has been done to assess the amplicon-based NGS methods for copy number variation (CNV) detection in comparison with current standard methods like immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The correlation between NGS based CNV detection and the later standard methods has remained unexplored. We developed an amplicon-based panel to detect human epidermal receptor growth factor (HER2) amplification in formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 280 breast cancer and 50 gastric cancer patients. Assessment by IHC and FISH was conducted in parallel, and descriptive statistics were used to assess the concordance. The copy number detected by NGS was correlated with either the average HER2 copy number (signals/cell) (r = 0.844; p < 0.001) or the HER2/CEP17 ratio (r = 0.815; p < 0.001). We determined a cut-off value for NGS to categorize HER2 amplification status by using 151 HER2 non-amplified FFPE samples. In breast cancer patients, the cut-off value was 2.910, with 95.35%, 98.67% and 97.29% sensitivity, specificity and concordance, respectively. However, this cut-off value displayed low sensitivity in gastric cancer patients (64.71%), and the following macrodissection procedure was not effective for increasing sensitivity (57.14%). Evaluation of HER2 copy number with NGS in our study was comparable with IHC and FISH in breast cancer patients, but concordance in gastric cancer was only moderate. The greater discordance in gastric cancer may reflect the underlying biological mechanisms, and further study is warranted. NGS-based HER2 assessment may decrease the equivocal HER2 determinations in breast cancer patients assessed by FISH/IHC.

Breast Cancer With a HER2 IHC2+ and FISH HER2/CEP17 Ratio ≥2.0 and an Average HER2 Gene Copy Number

Purpose: For breast cancer, accurately illustrating HER2 characteristics is a critical precondition for evaluating the prognosis and predicting the efficacy of anti-HER2 therapy. Our purpose was to expose HER2 mRNA expression through an in situ hybridization assay (RNAscope), to aid the identification of HER2 status in breast cancers with a previously controversial classification for patients suffering from a HER2 IHC2+ and HER2/CEP17 ≥2.0 and a <4.0 mean HER2 gene copy number/cell (entitled FISH group 2 by update 2018 HER2 testing guideline).Methods: A total of 8,983 cases of breast cancer with a known HER2 status detected by initial IHC, and a necessary reflex FISH assay for those with IHC2+, were retrospectively analyzed. Then, 41 cases of HER2 IHC2+ in the FISH group 2 were collected and a RNAscope was performed.Results: The incidence of breast cancers with IHC2+ and in the FISH group 2 was 0.46% (41/8,983) in our single-institutional study cohort. In most of the cases (27/41, 65.9%), low levels of HER2 mRNA expression (score 1 and 2 by RNAscope) were demonstrated. Only one case (1/41, 2.4%) of high-level HER2 mRNA expression (score 4 by RNAscope), harboring a FISH HER2/CEP17 ratio of 2.06 and an average HER2 copy number of 3.70, was revealed. One case with the highest FISH HER2/CEP17 ratio of 3.90, showed the lowest level of HER2 mRNA expression (score 1 by RNAscope). Two cases with the same highest average HER2 signals/cell (3.95) by FISH possessed score 3 and score 2 with RNAscope, respectively. No cases with a score of 0 by RNAscope occurred in our sample. In the majority of cases (35/41, 85.4%), hypodisomy of chromosome 17 (average CEP17 signals/cell ≤1.75) was observed. There was no significant relationship between the mRNA expression and FISH results (average HER2 signals/cell, average CEP17 copy number, or HER2/CEP17 ratio) and clinicopathological features (ER and PR statuses, Ki 67 index, tumor size, and lymph node metastasis) in our population.Conclusion: HER2 mRNA overexpression was not a feature in our group of patients. Based on our data, breast cancers with HER2 IHC2+ and in FISH group 2 support a categorization of HER2 negative.

Next-generation sequencing (NGS) identifies a new breast cancer subtype with HER2 low-amplification status as a candidate for targeted therapy.

553 Background: HER2 expression or amplification qualify patients to receive targeted therapeutics against HER2; however, traditional methods of quantifying HER2 amplification using fluorescence in situ hybridization (FISH) do not include a reliable definition for low level amplification. With the promising response rate of patients with low HER2 amplified-metastatic breast cancer to subsequent-line trastuzumab deruxtecan (DS-8201a) therapy, there is a need to improve the existing criteria to accurately identify patients with low HER2. In our study, we investigate whether HER2 amplification quantified by NGS could provide a method to stratify patients into subgroups. Methods: A total of 774 patients diagnosed with breast cancer from Guangdong Provincial People's Hospital (GDPH) who underwent targeted NGS using 520 or 33 cancer-related genes and had their HER2 status evaluated with either FISH or IHC were included in this study. HER2 status were defined as per 2018 ASCO/ACP guidelines. Results: Our results demonstrate that NGS could quantify HER2 amplification with high sensitivity and specificity, with area under the curve of 0.990 [95%CI: 0.982-0.999]. The receiver operating curve indicated an optimal cut-off of 2.62 copy number (CN) for identifying IHC/FISH HER2-negative status with 97.8% specificity. Meanwhile, the cut-off of ≥ 3.62 CN identified patients with IHC/FISH HER2-positive status with 99.8% specificity. Among the 774 patients, 65.8% (n = 509) had HER2 CN of ≤ 2.62 and were classified as HER2 non-amplified, while 25.8% (n = 199) had HER2 CN of ≥ 3.62, classified as HER2-amplified. The remaining 66 patients (8.5%) had HER2 CN between 2.62 and 3.62, and were the patients with heterogeneous IHC/FISH results, classified using NGS as HER2 low-amplified. Patients with low-amplified (49.0% vs. 38.8%, P &lt; 0.001) and amplified (50.3% vs. 38.8%, P &lt; 0.001) HER2 had significantly more number of copy number amplifications in other gene, including CDK12, RARA, and SPOP (P &lt; 0.001, P &lt; 0.001) than patients with HER2 non-amplified, indicating distinct mutation profile. Conclusions: Our results demonstrate that NGS could provide a more accurate stratification of patients based on their HER2 amplification levels. Patients with low levels of HER2 amplification has a distinct mutation profile, suggesting that NGS could serve as a robust tool to identify patients with HER2 amplification, whether high or low, who could benefit treatment with targeted agents designed against heterogeneous HER2 expression.

A multiparameter classifier to predict response to lapatinib plus trastuzumab (LT) without chemotherapy in HER2+ breast cancer (BC).

1011 Background: Several trials have shown 25-30% pathologic complete response (pCR) rates in patients with HER2+ BC treated with LT therapy (+ endocrine therapy if ER+), but no chemotherapy (CTX). We hypothesize that a multiparameter classifier, comprised of HER2 gene and protein levels, intratumor heterogeneity (ITH), HER2-enriched (E) subtype, and PIK3CA mutation status can identify patients whose tumors are “addicted” to HER2 signaling and are likely to achieve pCR from a CTX-sparing de-escalation strategy. Methods: Baseline specimens from 2 trials (TBCRC023 [NCT00999804] and PAMELA [NCT01973660]) of neoadjuvant CTX-sparing LT (+ endocrine therapy if ER+) in HER2+ BC were used. HER2 protein and ITH (scored for % 3+ by IHC), and gene amplification (HER2:CEP17 ratio and copy number (CN) by CISH) were measured on the same slide by the dual gene protein assay (GPA). HER2-E and PIK3CA mutation status were assessed by research-based PAM50 and MSK-IMPACT platforms, respectively. A decision tree algorithm was used to determine the GPA cutoffs and to construct the classifier of response (by pCR) in TBCRC023, which was then validated in PAMELA. Results: Of the evaluable patients from TBCRC023 (N = 130) and PAMELA (N = 151), GPA data were available for 121 and 94 cases, respectively. Both cohorts exhibited similar distributions for HER2 ratio, CN, and % 3+, and a strong correlation between HER2 ratio and CN (R &gt; 0.92). In TBCRC023, 73 cases had data from GPA, PAM50, and IMPACT, of which 15 had pCR. Recursive partitioning identified cutoffs of HER2 ratio &gt; 4.6 and % 3+ &gt; 97.5% in both the GPA data cohort (N = 121) and complete data cohort (N = 73). With PAM50 and IMPACT data, the model added HER2-E and PIK3CA wild-type (wt). For practical reasons, the classifier was locked as HER2 ratio ≥ 4.5 AND % 3+ ≥ 90% AND PIK3CA-wt AND HER2-E, which yielded a PPV of 55% and NPV of 94%. Validation in PAMELA using 45 cases with data for all 3 assays yielded PPV of 44% and NPV of 82%. 29 TBCRC023 cases without IMPACT data could be predicted to be non-pCR, of which 26 were correct (NPV = 89%). In PAMELA, 66 additional cases could be predicted to be non-pCR, of which 54 were correct (NPV = 81%). Conclusions: We have constructed a multiparameter classifier that can predict pCR with targeted therapy alone that compare to pCR rates of CTX + dual anti-HER2 in unselected patients. Prospective validation in a clinical trial is warranted. [Table: see text]

A clinicopathological study of 30 breast cancer cases with a HER2/CEP17 ratio of ≥2.0 but an average HER2 copy number of

The American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) have recently issued updated guidelines on human epidermal growth factor receptor 2 (HER2) testing by fluorescence in situ hybridization (FISH) in invasive breast cancers. Cases with a HER2/chromosome enumeration probe 17 (CEP17) ratio of ≥2.0 but an average HER2 copy number of <4.0 signals per cell (ISH group 2) are no longer automatically classified as ISH positive. Herein, 30 cases in ISH group 2 were collected. Another 100 patients with a HER2/CEP17 ratio <2.0 and <4.0 HER2 signals per cell (ISH group 5) and 100 patients with a HER2/CEP17 ratio of ≥2.0 and an average HER2 copy number of ≥4.0 signals per cell (ISH group 1) were also recruited for comparison. According to the 2018 ASCO/CAP guidelines, all the cases in ISH group 2 were categorized as HER2 negative. The clinicopathological characteristics of the patients in ISH group 2 were intermediate between ISH group 1 and group 5. Survival analyses revealed that there was no significant disease-free survival (DFS) and overall survival (OS) difference between patients with or without targeted therapy in ISH group 2, as well as between patients with targeted therapy in ISH group 1 and patients in ISH group 2. Patients without targeted therapy in ISH group 2 had a significantly worse OS than patients with targeted therapy in ISH group 1 and patients in ISH group 5. In conclusion, patients in ISH group 2 represent a biologically heterogeneous subset, which are different from those in ISH group 1 and 5. A larger cohort of patients in ISH group 2 should be included for future researches to define the efficacy of HER2-targeted therapy.

Integrated genomic characterization of ERBB2/HER2 alterations in invasive breast carcinoma: a focus on unusual FISH groups.

In patients with invasive breast cancer, fluorescence in situ hybridization (FISH) testing for HER2 typically demonstrates the clear presence or lack of ERBB2 (HER2) amplification (i.e., groups 1 or 5). However, a small subset of patients can present with unusual HER2 FISH patterns (groups 2-4), resulting in diagnostic confusion. To provide clarity, the 2018 CAP/ASCO HER2 testing guideline recommends additional testing using HER2 immunohistochemistry (IHC) for determining the final HER2 status. Despite this effort, the genomic correlates of unusual HER2 FISH groups remain poorly understood. Here, we used droplet digital PCR (ddPCR) and targeted next-generation sequencing (NGS) to characterize the genomic features of both usual and unusual HER2 FISH groups. In this study, 51 clinical samples were selected to represent FISH groups 1-5. Furthermore, group 1 was subdivided into two groups, with groups 1A and 1B corresponding to cases with HER2 signals/cell ≥6.0 and 4-6, respectively. Overall, our findings revealed a wide range of copy number alterations in HER2 across the different FISH groups. As expected, groups 1A and 5 showed the clear presence and lack of HER2 copy number gain, respectively, as measured by ddPCR and NGS. In contrast, group 1B and other uncommon FISH groups (groups 2-4) were characterized by a broader range of HER2 copy levels with only a few select cases showing high-level gain. Notably, these cases with increased HER2 copy levels also showed HER2 overexpression by IHC, thus highlighting the correlation between HER2 copy number and HER2 protein expression. Given the concordance between the genomic and protein results, our findings suggest that HER2 IHC may inform HER2 copy number status in patients with unusual FISH patterns. Hence, our results support the current recommendation for using IHC to resolve HER2 status in FISH groups 2-4.

Impact of the Updated 2018 American Society of Clinical Oncology/College of American Pathologists Guideline for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer.

The updated American Society of Clinical Oncology/College of American Pathologists guideline for human epidermal growth factor receptor 2 (HER2) testing in breast cancer requires pathologists to re-evaluate HER2 status. To define HER2 status of breast cancer using immunohistochemistry and fluorescence in situ hybridization. Diagnostic reports of invasive breast cancers made between 2014 and 2018 with HER2 immunohistochemistry and fluorescence in situ hybridization results were retrieved. HER2 status was re-defined using the updated recommendations. Of 2514 tumors, 89.7% (2254 of 2514) suggested for fluorescence in situ hybridization assay were HER2 immunohistochemistry 2+. Approximately 8.9% (225 of 2514) and 1.4% (35 of 2514) of tumors were of immunohistochemistry 0/1+ and 3+, respectively. Based on the average HER2 copy number and HER2:CEP17 ratio, tumors were assigned into 5 groups, including 13.1% (330 of 2514) group 1 tumors, 2.1% (52 of 2514) group 2 tumors, 1.1% (27 of 2514) group 3 tumors, 7.0% (175 of 2514) group 4 tumors, and 76.8% (1930 of 2514) group 5 tumors. In combination with immunohistochemistry, all tumors in group 2 and group 4 changed HER2 status, from positive and equivocal into negative, respectively, while group 3 tumors remained positive. Comparative analyses of clinicopathologic features of tumors in different groups revealed that group 2 and 4 tumors displayed worse clinicopathologic features than those of group 5, while group 3 tumors shared similar clinicopathologic features to those of group 1. Following the updated guideline, HER2 status is clearly designated. Significant differences regarding clinical features were observed between tumors in different groups but they share the same HER2 status, suggesting further validation of the accuracy of this diagnostic approach is warranted.

Comparative analysis of HER2 copy number between plasma and tissue samples in gastric cancer using droplet digital PCR

In this study, we measured the human epidermal growth factor receptor 2 (HER2) copy number in both tissue and plasma samples of gastric cancer patients by using droplet digital polymerase chain reaction (ddPCR) method. Eighty gastric cancer patients were enrolled and both formalin-fixed and paraffin-embedded tissue and preoperative plasma samples were collected. HER2 status was determined by HER2 immunohistochemistry (IHC)/silver in situ hybridization (SISH) in tissue samples and ddPCR of the target gene HER2 and the reference gene eukaryotic translation initiation factor 2C, 1 in both tissue and plasma. The concordance rate of tissue HER2 status determined by IHC/SISH and HER2 ddPCR was 90.0% (72/80), and the sensitivity and specificity of tissue ddPCR were 85.0% and 95.0%, respectively. The concordance rate of plasma ddPCR and IHC/SISH was 63.8% (51/80). The sensitivity, specificity, positive predictive value, and negative predictive value of plasma HER2 ddPCR were 37.5%, 90.0%, 79.0%, and 59.0%, respectively. As HER2 measurement by tissue ddPCR showed a high concordance rate with HER2 status by IHC/SISH, it could replace tissue IHC/SISH testing in gastric cancer. These findings may contribute to the development of tissue and plasma HER2 testing that would be useful in daily practice.

Abstract P3-08-63: Predictors of pathologic complete response in HER2 positive breast cancer patients treated with neoadjuvant targeted therapy

Abstract Background: Anti-HER2 therapy is a standard treatment for HER2-positive tumors, however, the response to targeted therapy varies with a pathologic complete response rate of approximately 40%. Additionally, the relationship between the level of HER2 gene amplification and the clinical outcome is unclear. Purpose: The aim of this study was to determine clinico-pathologic predictors of pathologic complete response after neoadjuvant targeted therapy in HER2 positive breast cancers. Materials and Methods: We performed a retrospective review of HER2-positive breast cancer patients treated with Neoadjuvant Systemic Therapy (NST). PCR was defined as no residual invasive carcinoma in the breast and axillary lymph nodes (ypT0/is, pN0). We divided our cohort into two groups: patient with pathologic complete response (pCR) and patients without complete responses (non-pCR). We compared the HER2 copy number, HER2/CEP-17 ratio, and clinical and pathologic features using univariate and multivariate analyses. Results: A total of 163 patients with HER2 gene amplification (copy number ≥4) or HER2/CEP17 ratio ≥ 2.0 who received neoadjuvant chemotherapy along with Trastuzumab or Trastuzumab-Pertuzumab therapy were included in the study. Only 33.3% of patients achieved pCR. Patients who achieved pCR had tumors with higher HER2 copy numbers (median: 19.1, range: 4.5 - 45.6), compared to those who did not achieve pCR (median:15.22, range: 4.12 - 35.0, p=0.003). The HER2/CEP17 ratio was higher in the pCR group (median 7.43, range 2.1 - 17.94) compared with non-pCR group (median 6.1, range 1.72 - 12.6, p= 0.32). A receiver operating characteristic curve (ROC) to identify the optimal cutoff point to predict pCR revealed the area under the curve for HER2 copy number and HER2/CEP17 ratio to be 0.639 and 0.600, respectively. Univariate analysis showed significant correlation of pCR with HER2 copy number of ≥14 (p=0.003) and an HER2/CEP17 ratio of 4 or greater (p=0.044), and Ki-67 ≥ 40 % (p=0.002). Consistent with previous studies, tumors with negative hormone receptor (HR) statuses had higher pCR rates (p&amp;lt;0.005). In contrast, variables including age (&amp;lt;50 years), and tumor size (&amp;gt;5cm), were not associated with pCR. In our study, all patients with who negative lymph nodes, achieved pCR. In multivariate regression analysis, only HR negativity (OR=2.60, p=0.008), Ki-67 ≥ 40 % (p=0.015 OR=2.625) and HER 2 copy number ≥14 (p=0.009 OR=2.689) remained as a significant factors associated with pCR. Conclusion: Patients with PCR tend to have higher HER/CEP-17 ratios, smaller tumors and negative nodal statuses. Based on our result, HER2 copy number, negative HR status and high KiI-67 are one of the strongest predictors of pCR in HER2 positive tumors. Citation Format: Mariela Huerta Rosario, Sunati Sahoo. Predictors of pathologic complete response in HER2 positive breast cancer patients treated with neoadjuvant targeted therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-63.

Abstract P4-10-21: Utility of HER2 IHC intensity in selecting areas for FISH testing in equivocal (2+) cases

Abstract Intro: The 2018 ASCO/CAP HER2 guidelines eliminated the equivocal category for in situ hybridization (ISH). Additional workup is recommended for definitive classification of tumors that are sorted into ISH groups 2, 3 or 4. Evaluation of HER2 IHC using sections from the same tissue sample used for ISH is recommended to guide the selection of areas to score by ISH. Here we seek to evaluate the utility of selecting the area for ISH testing in IHC equivocal (2+) cases based on the staining intensity of the IHC staining. Methods: A total of 66 cases with equivocal HER2 IHC (2+) and reflexed to FISH were prospectively collected from 8/22/18 to 6/26/2019. The areas of most and least intense HER2 IHC staining were separately identified and scored by FISH. The mean HER2:CEP17 ratio and mean HER2 signals/tumor cell were calculated for both populations and statistical significance was evaluated using the Wilcoxon Ranked Sum test. Results: The distribution of the high intensity area was 58 ISH group 5, 4 ISH group 4, and 8 ISH group 1. The distribution of the low intensity area was 51 ISH group 5, 9 ISH group 4, 2 ISH group 2, and 5 ISH group 5. In the high and low intensity areas, 59 of 66 (95.5%) cases sorted into the same ISH group and 7 (10.6%) sorted into different ISH groups. Of these 7, 3 (4.5%) had different final FISH categorizations. All 3 cases were ISH Group 1 (amplified) in the high intensity area and ISH group 2, 4 or 5 (not amplified in the absence of 3+ IHC) in the low intensity area. The other 4 cases with different ISH groups did not have different final HER2 categorizations. The mean HER2/CEP17 ratios for the high and low intensity areas were 1.50 and 1.39 (p = 0.075). There was no statistically significant difference in mean HER2 copy number for the high and low intensity areas (p = 0.92). Discussion: When areas with high and low HER2 IHC intensity were compared, there was a small difference in the mean HER2:CEP17 ratio with a trend toward statistical significance. However, the absolute difference in the means was small (0.11) and only a minority of cases (4.5%) showed discordant final HER2 categorization between the two groups. All of the cases with discordant HER2 categorization were amplified (ISH Group 1) in the IHC high intensity area and not amplified (ISH Groups 2, 4 and 5) in the low intensity area. The data suggests that using HER2 IHC to guide selection of areas for ISH testing may identify amplified tumors (or clones) in a minority of cases (4.5%). Additional analysis of a larger number of cases is required to validate these findings. Citation Format: Johann D Hertel, Kathleen A Kaiser-Rogers, Siobhan O'Connor, Benjamin Calhoun. Utility of HER2 IHC intensity in selecting areas for FISH testing in equivocal (2+) cases [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-21.

The updated 2018 American Society of Clinical Oncology/College of American Pathologists guideline on human epidermal growth factor receptor 2 interpretation in breast cancer: comparison with previous guidelines and clinical significance of the proposed in situ hybridization groups

The aim of the study was to evaluate the impact of the updated 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline on human epidermal growth factor receptor 2 (HER2) interpretation in breast cancer compared with that of the previous guidelines and also the significance of in situ hybridization (ISH) groups proposed by the updated guideline. HER2 ISH reports and immunohistochemistry (IHC) data from 1,348 invasive breast cancers diagnosed at a single institution were included in this study. HER2 IHC was reassessed using the 2018 guideline, and HER2 ISH status was determined by the 2007, 2013, and 2018 guidelines. When applying the updated guideline, most of the HER2 ISH-equivocal cases as per the previous guidelines were reclassified as ISH negative, and 0.8% of HER2 ISH-positive tumors as per the 2007 guideline and 2.5% of those as per the 2013 guideline were changed to ISH negative. Accordingly, the negative HER2 ISH results significantly increased in the 2018 guideline compared with the 2013 guideline. HER2 ISH-positive tumors in ISH group 3 (HER2/chromosome enumeration probe 17 [CEP17] ratio <2.0 and average HER2 copy number ≥6.0 per cell) were characterized by equivocal HER2 protein expression, CEP17 copy number gain, and low HER2 copy numbers compared with classic HER2 ISH-positive tumors in ISH group 1 (HER2/CEP17 ratio ≥2.0 and average HER2 copy number ≥4.0 per cell). HER2 ISH-negative tumors in ISH group 4 (HER2/CEP17 ratio <2.0 with average HER2 copy number ≥4.0 and<6.0 per cell) revealed more aggressive clinicopathologic features and poorer clinical outcomes than those in ISH group 5 (HER2/CEP17 ratio <2.0 and average HER2 copy number <4.0 per cell), especially in the hormone receptor-positive subgroup. In conclusion, implementation of the updated 2018 ASCO/CAP guideline leads to a significant increase in HER2 ISH-negative results compared with the 2013 guideline, mainly via reclassification of the ISH-equivocal cases to ISH-negative ones. ISH groups proposed by the updated guideline provide additional information on the clinicopathologic characteristics of the tumors.

Quantitative digital imaging analysis of HER2 immunohistochemistry predicts the response to anti-HER2 neoadjuvant chemotherapy in HER2-positive breast carcinoma.

Patients with HER2-positive breast cancer commonly receive anti-HER2 neoadjuvant chemotherapy and pathologic complete response (pCR) can be achieved in up to half of the patients. HER2 protein expression detected by immunohistochemistry (IHC) can be quantified using digital imaging analysis (DIA) as a value of membranous connectivity. We aimed to investigate the association HER2 IHC DIA quantitative results with response to anti-HER2 neoadjuvant chemotherapy. Digitized HER2 IHC whole slide images were analyzed using Visiopharm HER2-CONNECT to obtain quantitative HER2 membranous connectivity from a cohort of 153 HER2+ invasive breast carcinoma cases treated with anti-HER2 neoadjuvant chemotherapy (NAC). HER2 connectivity and other factors including age, histologic grade, ER, PR, and HER2 fluorescence in situ hybridization (FISH) were analyzed for association with the response to anti-HER2 NAC. Eighty-three cases (54.2%) had pCR, while 70 (45.8%) showed residual tumor. Younger age, negative ER/PR, higher HER2 DIA connectivity, higher HER2 FISH ratio and copy number were significantly associated with pCR in univariate analysis. Multivariate analysis demonstrated only age, HER2 DIA connectivity, PR negativity, and HER2 copy number was significantly associated with pCR, whereas HER2 DIA connectivity had the strongest association. HER2 IHC DIA connectivity is the most important factor predicting pCR to anti-HER2 neoadjuvant chemotherapy in patients with HER2-positive breast cancer.