HER2 Fluorescence In Situ Hybridization Research Articles

ERBB2 amplification status in 67 salivary duct carcinomas assessed by immunohistochemistry, fluorescence in situ hybridization, and targeted exome sequencing

AbstractSalivary duct carcinoma (SDC) is an aggressive salivary gland malignancy with poor survival. Approximately 30% SDC harbor HER2 amplification and response to trastuzumab has been reported. However, a systematic approach for HER2 status assessment in this tumor type has not been established. A total of 67 tumor samples were evaluated for HER2 protein overexpression or ERBB2 gene amplification using at least 2 methods: immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and/or targeted exome next-generation sequencing (NGS). NGS assessed ERBB2 copy number fold change (FC) and total copy number (TCN). HER2 status was first determined by IHC/FISH according to the 2018 ASCO/CAP breast cancer guidelines. FISH results, the “gold standard”, were compared with the NGS results. All (15/15) IHC positive, 35% (6/17) equivocal, and no (0/19) IHC negative SDC were HER2 amplified by FISH. HER2 FISH signal/cell showed a good correlation with FC (Spearman correlation: 0.708, R2: 0.501, p < 0.0001) and TCN (Spearman correlation: 0.763, R2: 0.582, p < 0.0001). Receiver operating characteristics curve estimation showed an area under curve (AUC) of 0.975 for ERBB2 FC. FC cutoff of ≥1.8 corresponded to an accuracy of 95.2% for ERBB2 amplification (Youden's index: 0.84, sensitivity: 89.47%, specificity: 100%). FC < 1.3 could be reliably classified as ERBB2 not amplified and FC ≥ 1.3 and <1.8 as equivocal. TCN estimation showed AUC of 0.981. TCN cutoff of >6.0 corresponded to an accuracy of 92% for HER2 amplification (Youden's index: 0.81, sensitivity: 81.2%, specificity: 100%). TCN < 4 could be reliably classified as ERBB2 not amplified and TCN ≥ 4.0 and ≤6.0 as equivocal. FC and TCN were binarized with respective cutoffs of ≥1.8 and ≥6.0 and the proportion of agreement with FISH were 95% and 92%, respectively. The assessment of ERBB2 copy number by NGS is accurate and reliable with FC or TCN nearly equivalent to FISH in identifying HER2 amplified SDC.

Concordance of breast cancer biomarker testing in core needle biopsy and surgical specimens: A single institution experience.

Accurate diagnostic biomarker testing is crucial to treatment decisions in breast cancer. Biomarker testing is performed on core needle biopsies (CNB) and is often repeated in the surgical specimen (SS) after resection. As differences between CNB and SS testing may alter treatment decisions, we evaluated concordance between CNB and SS as well as associated changes in treatment and clinical outcomes. We performed a retrospective analysis of breast cancer patients at our institution between January 2010 and May 2020. Concordance between CNB and SS was assessed for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Survival in patients, including recurrence, metastatic recurrence, and death, were assessed using chi-squared likelihood ratio. In total, 961 patients met eligibility criteria. Concordance, minor discordance, total concordance (concordance plus minor discordance), and major discordance between CNB and SS were reported for ER (87.7%, 9.2%, 90.8%, and 2.9%), PR (58.1%, 29.1%, 87.2%, and 12.8%), and HER2 IHC (52.5%, 20.9%, 73.4%, 26.6%), respectively. HER2 FISH concordance and major discordance were 58.5% and 1.2%, respectively. Of major discordance, ER (48.2%, p< 0.001) and HER2 FISH (50.0%) led to more management changes than HER2 IHC (2.4%, p= 0.04) and PR (1.6%, p= 0.10). Patients with ER major discordance had increased risk of death (6.7% concordance vs. 22.2% major discordance, p= 0.004). Overall, retesting ER and HER2 was more clinically beneficial than retesting PR. To aid decision-making and minimize healthcare costs, we propose patient-centered guidelines on retesting biomarker profiles.

Characteristics of HER2 Gene Amplification by Fluorescence In Situ Hybridization in Endometrial Serous Carcinoma.

Targeted anti-human epidermal growth factor receptor 2 (HER2) therapy has recently become the standard for advanced-stage and recurrent HER2-positive endometrial serous carcinoma (ESC) in the United States, and an endometrial carcinoma-specific HER2 testing algorithm has been proposed. However, comprehensive studies on the specific features of HER2 gene amplification in these tumors are lacking. To evaluate the characteristics of HER2 amplification in ESC in the context of breast and gastric HER2 fluorescence in situ hybridization (FISH) guidelines. Ninety-four ESCs with available HER2 immunohistochemistry (IHC) and FISH were included. HER2 IHC was scored according to the proposed endometrial carcinoma-specific algorithm and FISH was evaluated by using the 2018 ESC clinical trial criteria, the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2016 gastric criteria, and ASCO/CAP 2013 and 2018 breast criteria. Most tumors (90.4%; 85 of 94) had a 2+ HER2 IHC score. Polysomy of chromosome 17 was present in 16% (15 of 94) and monosomy 17 was seen in 2% (2 of 94) of tumors. HER2 FISH interpretation per the clinical trial criteria (HER2/CEP17 ratio ≥ 2.0) showed 99% concordance with the current gastric and breast HER2 FISH interpretations. Our results support the clinical trial criteria for HER2 FISH in ESC with a modification to include HER2 IHC 2+ and HER2/CEP17 ratio less than 2.0 and average HER2 copy number of 6.0 or greater in the HER2-positive category. Future prospective clinical investigations are necessary to assess the correlation between specific HER2 FISH result categories and therapeutic response.

Digital imaging correlation of immunohistochemistry and fluorescence in situ hybridization in breast carcinoma cases with HER2 genetic heterogeneity

Genetic heterogeneity (GH) is a rare but important event in the evaluation of HER2 amplification status. We investigated whether HER2 fluorescence in situ hybridization (FISH) GHcorrelated with increased protein expression by immunohistochemistry (IHC) and/or morphologic features using image analyses. Retrospective search of HER2 FISH GH cases 2016-2020 was performed. Cases with both FISH and IHC slides available were considered eligible and were digitally imaged. Additional demographic, histological, and treatment information was compiled from pathology and medical records when available. Overall, 11 of 15cases (73.3%) had HER2 FISH GH that matched to areas of HER2 overexpression or focally different morphology. Nine cases with areas of gene amplification overlapped with <10% of intense circumferential protein expression ("Mini 3+"), and 1 case with focal micropapillary features. Clinical information was available on 6patients (40%), all were alive with no evidence of disease (mean follow-up 30.5 months; range, 12-65 months). One patient with GH and a lymph node metastasis showed nonamplified population in the nodal tumor. GH, when defined as discrete clusters of amplified cells following 2013 ASCO/CAP guidelines, even when less than 10% of the tumor cells, frequently has morphologic correlates such as focal intense protein overexpression or micropapillary morphology. Clinical significance of these focal gene amplification and protein overexpression needs to be further investigated.

RAI1 alternate probe identifies additional gastroesophageal adenocarcinoma cases as amplified following equivocal HER2 fluorescence in situ hybridization testing: experience from a national reference laboratory

The College of American Pathologists/American Society of Clinical Oncology recommends HER2 testing prior to initiation of targeted therapy for patients with advanced Gastroesophageal adenocarcinoma (GEA), using immunohistochemistry (IHC) followed by fluorescence in situ hybridization (FISH) in cases with an equivocal (score 2 + ) result on IHC. The FISH results are considered indeterminate if the HER2/CEP17 ratio is <2.0 with an average CEP17 copy number of ≥3.0 and a HER2 copy number ≥4.0 and ≤6.0 after counting additional tumor cells. Indeterminate results may be resolved by using an alternative chromosome 17 probe such as RAI1. The purpose of this study is to review our experience with RAI1 alternate probe in HER2 FISH testing of GEA in a large reference laboratory setting. Esophageal, gastroesophageal, and gastric adenocarcinomas received for HER2 FISH testing in our lab between 9/2018 and 1/2020 were included. HER2/CEP17 and HER2/ RAI1 ratios, and the average HER2, CEP17, RAI1 signals per cell were recorded. 328 GEA had HER2 testing performed in our lab during the study period. 101 (30.8%) were amplified, 169 (51.5%) were non-amplified and 58 (17.7%) were indeterminate. Following RAI1 testing, 42 (72.4%) of 58 indeterminate cases were reclassified as non-amplified and 16 (27.6%) were reclassified as amplified, increasing the total amplified cases to 117 (35.7%). The correlation between the average CEP17 and RAI1 copy number for all cases was weak (R2 = 0.095). In summary, using the alternate probe RAI1 reclassifies 27.6% of original HER2 FISH indeterminate gastroesophageal carcinomas as amplified, which makes them eligible for targeted therapies.

Abstract P3-09-05: Genomic and clinical characterization of breast tumors with unusual HER2 FISH pattern (ratio &amp;lt; 2, HER2 copy number ≥ 6): Are they mostly HER2 “positive?”

Abstract Background: Breast tumors with HER2/CEP17 fluorescent in situ hybridization (FISH) ratio &amp;lt; 2 and HER2 copy number ≥ 6, defined as Group 3 FISH pattern by the 2018 ASCO/CAP HER2 testing guidelines, are clinically rare. Their biologic and molecular characteristics are under-characterized. They require only a concomitant HER2 immunohistochemistry score of at least 2+ to merit HER2 “positive” status by the ASCO/CAP guidelines. We seek to characterize the genomic and tumor microenvironment landscape of breast tumors with this unique HER2 FISH pattern. Our second aim is to assess the clinicopathologic features with emphasis on HER2-targeted therapy response.Method: Breast cancers with Group 3 FISH pattern were evaluated by the following methods: 1) High-resolution genome-wide copy number alterations by molecular inversion probe (MIP) array; 2) molecular profiling of tumor immune microenvironment, tumor signaling pathways, and PAM50-based intrinsic subtypes by Nanostring nCounter Breast Cancer 360 Panel; 3) tumor infiltrating lymphocytes (TIL) histologic quantitation, and 4) clinical chart review. Classically amplified HER2 breast tumors (Group 1 FISH pattern; ratio ≥ 2 and HER2 copy number ≥ 2) were used as comparison. Results: Thirty-five (1.3%) cases were identified from 2731 consecutive clinical cases that underwent HER2 FISH testing from 2014 to 2019. Of those, thirteen consecutive cases (spanning 2014 - 2017) with sufficient genomic material were analyzed using MIP array. Group 3 tumors had a more complex karyotype and greater chromosomal instability, compared to classically amplified HER2 breast tumors. None of the Group 3 tumors showed HER2 locus amplification at 17q12. Instead, most showed gain of the 17q arm. Six of the Group 3 tumors were profiled by Nanostring nCounter. Compared to HER2 classically amplified tumors, Group 3 tumors were more immune cold, enriched in ER signaling and TGF-beta signaling pathways. In contrast, HER2 classically amplified tumors were enriched in immune infiltration, cytokine and chemokine signaling, PI3K and MAPK signaling, epithelial-mesenchymal transition signaling, and proliferation (P &amp;lt; 0.5 for all). PAM50 analysis showed that classically amplified tumors were more enriched for HER2-subtype (2/4; 50%), while the majority of the Group 3 tumors were enriched for Luminal B-subtype (5/6; 83%). TIL percentage was statistically higher in HER2 classically amplified tumors compare to Group 3 tumors (avg 53% vs 3%; P = 0.02). Clinicopathologic correlation revealed a high rate of ER positivity and high tumor grade in Group 3 tumors. Group 3 FISH pattern can occur as de novo or in the context of FISH status change following therapy. In the 17 evaluable patients for HER2-targeted treatment efficacy, none of the eight patients who received HER2-targeted neoadjuvant therapy achieved complete pathologic response. Nine of ten patients who received TDM-1 in the metastatic setting progressed with minimal treatment response. Significantly, most of these patients (16/17; 94%) were considered overall HER2 positive by the latest ASCO/CAP guideline. Conclusion: Breast tumors with Group 3 HER2 FISH pattern are molecularly and clinically dissimilar from classically amplified HER2 positive breast tumors. HER2-targeted therapy did not appear efficacious in either the neoadjuvant or metastatic/recurrent settings. The lack of apparent efficacy of HER2-targeted therapy, in the context of their HER2 positive status by the current HER2 guideline assessment, warrants further investigation of this HER2 FISH subtype. Citation Format: Christina H Wei, Lixin Yang, Daphne Stewart, Victoria Bedell, Daniel Schmolze, Sophia Apple, Joyce L. Murata-Collins, Raju Pillai, Joanne E. Mortimer. Genomic and clinical characterization of breast tumors with unusual HER2 FISH pattern (ratio &amp;lt; 2, HER2 copy number ≥ 6): Are they mostly HER2 “positive?” [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-05.

Clinical and Pathologic Features Associated With Invasive Breast Carcinoma With 2018 American Society of Clinical Oncology/College of American Pathologists In Situ Hybridization Group 2 Results (Human Epidermal Growth Factor Receptor 2 [HER2]/Chromosome 17 Centromere [CEP17] Ratio ≥2.0 and Average HER2 Copy Number

The American Society of Clinical Oncology/College of American Pathologists updated the human epidermal growth factor receptor 2 (HER2) breast carcinoma testing guideline in 2018 to address issues from uncommon HER2 fluorescence in situ hybridization (FISH) results. Based on the 2013 American Society of Clinical Oncology/College of American Pathologists guideline, cases wherein the HER2/chromosome 17 centromere (CEP17) ratio of 2.0 or more with an average HER2 copy number of less than 4.0 were considered in situ hybridization (ISH) positive. Under the 2018 guideline, such cases are classified as ISH Group 2 and are no longer considered eligible for anti-HER2 therapy when the corresponding HER2 immunohistochemistry result is 0, 1+, or 2+. To assess the clinical, pathologic, and treatment aspects of patients with ISH Group 2 results. We retrospectively reviewed HER2 FISH results at our center between January 2012 and December 2014 and identified and characterized cases with ISH Group 2 results. Thirty-nine cases with ISH Group 2 results from 39 patients were reviewed. Twenty of 39 (51%) patients received anti-HER2 therapy. Patients treated with HER2-targeted therapy were less likely to have hormone receptor-positive tumors, compared with patients without anti-HER2 treatment, though not significantly (P = .30). The only significant difference between the 2 patient groups was receipt of cytotoxic chemotherapy treatment (P < .001). Overall, clinical outcome was similar between the 2 groups (P > .99). This retrospective study with median follow-up of at least 6 years shows patients with ISH Group 2 tumors had similar clinical outcomes, irrespective of HER2-targeted therapy. Further analysis in the prospective setting would provide valuable data that would potentially inform clinical decision making.

HER2 Testing: Insights From Pathologists' Perspective on Technically Challenging HER2 FISH Cases.

College of American Pathologists and the American Society of Clinical Oncology guidelines provide straightforward criteria for HER2 interpretation in breast carcinomas; however, a subset of cases present unusual diagnostic dilemmas. Ten challenging HER2 fluorescence in situ hybridization (FISH) cases were selected for analysis. The study included a variety of problematic cases such as those with discordant immunohistochemistry (IHC) and FISH results, cases with high intratumoral variability in HER2 copy number, a case with a highly amplified clone in 5% to 10% of the tumor sample, and a case with tumor cells containing tightly clumped HER2 signals. Six high volume HER2 FISH laboratories performed and interpreted HER2 FISH (adding HER2 IHC if necessary). Interpretation strategies were discussed. There was 100% concordance between laboratories in 4/10 cases. Tumors with increased intratumoral variability (tumors with high variability in HER2 copy number per cell but which otherwise do not fulfill College of American Pathologists and the American Society of Clinical Oncology criteria for heterogeneity) exhibited 100% concordance in 3/4 cases, but 1 case had only 50% agreement. Low positive HER2 cases (group 1 cases with <6 average HER2 copies/cell) had 1 laboratory disagreeing with the majority in 4/4 cases, and this was the only category with discordance between IHC and FISH methodologies. All laboratories identified the case with heterogeneity and interpreted it as positive. Five of the 6 laboratories interpreted the case with tightly clustered HER2 signals as positive. This study offers specific observations and interpretation strategies that laboratories can use when confronted with difficult HER 2 cases. It then highlights communication strategies a laboratory may use to discuss these unusual HER2 results with the clinical team.

Reappraisal of HER2 Amplification in High-Grade Urothelial Carcinoma Based on 2018 ASCO/CAP Clinical Practice Guidelines.

To examine and compare human epidermal growth factor receptor 2 (HER2) amplification status in high-grade urothelial carcinoma (HGUCa), using both 2013 and 2018 HER2 reporting guidelines for breast carcinoma from the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP). HER2 status by fluorescence in situ hybridization (FISH) assay in 78 cases of HGUCa was compared using 2013 and 2018 HER2 reporting guidelines. HER2 amplification was observed in 22 (28.2%) of 78 tumors, of which 17 were in group 1, 1 in group 2, and 2 each in groups 3 and 4 (FISH assay, 2018). The remaining 14 HER2-amplified tumors (FISH assay, 2013) became negative, falling into group 2 (FISH assay, 2018) and were either negative or equivocal on immunohistochemistry (IHC, 2018). All FISH-negative tumors (n = 37) using 2013 criteria remained negative (group 5, 2018). FISH-equivocal tumors (2013) were further categorized into HER2 amplified (n = 1) and HER2 negative (n = 4) (2018). Overall, 20 (25.6%) tumors had discordant HER2 FISH results (2018 vs 2013). Implementing 2018 guidelines, HER2 amplification decreased from 36 to 22 cases. The group with a HER2/CEP17 ratio of 2 or more and average HER2 copy number less than 4 (group 2) were predominantly negative by IHC, suggesting a biologically distinct group of HGUCa that is different from HER2-amplified tumors, which may not respond to HER2-targeted therapy.

HER2 Testing Characteristics Can Predict Residual Cancer Burden following Neoadjuvant Chemotherapy in HER2-Positive Breast Cancer.

Objectives The response to HER2-targeted neoadjuvant chemotherapy (NAC) in HER2-positive (+) breast cancer can be quantified using residual cancer burden (RCB) pathologic evaluation to predict relapse free/overall survival. However, more information is needed to characterize the relationship between patterns of HER2 testing results and response to NAC. We evaluated clinicopathologic characteristics associated with RCB categories in HER2+ patients who underwent HER2-directed NAC. Methods A retrospective chart review was conducted with Stage I–III HER2+ breast cancer cases following NAC and surgical resection. HER2 immunohistochemistry (IHC) staining and fluorescence in situ hybridization (FISH), histologic/clinical characteristics, hormone receptor status, and RCB scores (RCB-0, RCB-I, RCB-II, and RCB-III) were evaluated. Results 64/151 (42.4%) patients with HER2+ disease had pathologic complete response (pCR). Tumors with suboptimal response (RCB-II and RCB-III) were more likely to demonstrate less than 100% HER2 IHC 3+ staining (p < 0.0001), lower HER2 FISH copies (p < 0.0001), and lower HER2/CEP17 ratios (p = 0.0015) compared to RCB-I and RCB-II responses. Estrogen receptor classification using ≥10% versus ≥1% staining showed greater association with higher RCB categories. Conclusions HER2+ characteristics show differing response to therapy despite all being categorized as positive; tumors with less than 100% IHC 3+ staining, lower HER2 FISH copies, and lower HER2/CEP17 ratios resulted in higher RCB scores.

Clinicopathological Features of Breast Cancer with Polysomy 17 and Its Response to Neoadjuvant Chemotherapy.

The interpretation of human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) results may be challenging in tumors with polysomy 17, which is defined as increased signals of chromosome enumeration probe 17 (CEP17). The effect of polysomy 17 on HER2 protein expression and tumor treatment response has not been established. In this retrospective study, we investigated the clinicopathological features of breast cancer with polysomy 17 and determined the tumors' response to neoadjuvant chemotherapy (NACT). The study included 366 patients with primary breast cancer whose tumors had a CEP17 count of ≥ three/nucleus based on HER2 FISH studies. These cases were categorized according to HER2/CEP17 ratio and HER2 signals/nucleus using the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. We compared the clinicopathological characteristics and tumor response to NACT among different groups. There was a statistically significant difference in patients' age at diagnosis, tumor pathological grade, estrogen and progesterone receptor status, and NACT response among different HER2 FISH groups. Polysomy 17 tumors in group 1 had a higher rate of response (pathological complete response and residual cancer burden class I) to NACT containing anti-HER2 reagent than did those in other groups (p = 0.004), whereas polysomy 17 tumors in group 3 did not show a significant response to anti-HER2 treatment. Polysomy 17 tumors in different HER2 FISH groups have different pathological features and respond to NACT differently. These results may help us identify patients who will benefit from anti-HER2 therapy.

Abstract PS18-02: Highly multiplexed tissue-based cyclic immunofluorescence (t-CyCIF) for precision oncology identifies novel patterns of HER2 heterogeneity in breast cancer

Abstract Background Immunohistochemical (IHC) evaluation has shown that human epidermal growth factor receptor 2 (HER2) may not be expressed homogeneously among all cancer cells within a given tumor. The clinical significance of intratumoral HER2 heterogeneity is unclear. Exploration of tumor heterogeneity is facilitated by tissue imaging technologies such as t-CyCIF, a highly multiplexed immunofluorescence microscopy technique that permits visualization of up to 60 antigens and analysis on a single cell level from formalin-fixed, paraffin-embedded tissue. To utilize t-CyCIF for the evaluation of breast tumors, this study was undertaken to: 1) validate antibodies to be used against the clinically relevant markers HER2, estrogen receptor (ER) and progesterone receptor (PR), and 2) use these antibodies along with other validated antibodies to define the tumor microenvironment (TME) to interrogate breast tumors at a single cell level. Methods T-CyCIF is an iterative whole-slide imaging process, in which successive four-channel images, each involving different antibodies, are collected from the same sample and then merged to generate a high-dimensional representation used for visualization and analysis. In phase one of this study, 948 tissue cores (representing 295 patients in triplicate) were used to validate HER2, ER, and PR antibodies against a single antibody commonly used in clinical practice as a reference. Analyses were performed at the level of tissue cores, cells and pixels. Inter-assay analyses were performed comparing: t-CyCIF vs. IHC, the latter assessed by digital pathology and two pathologists; and also, t-CyCIF vs. fluorescence in situ hybridization (FISH) for HER2. In the second phase, following selection of validated HER2, ER and PR antibodies, expression of CD45, CD68, PD-L1, p53, Ki67, pRB and the androgen receptor (AR) were evaluated at a single cell level in 312 HER2+ invasive breast cancer samples, representing 104 patients, to better understand the TME, cancer cell heterogeneity and the cell identities/states present in breast carcinomas. Results In the first phase of the study, 13 different ER, PR or HER2 antibodies were analyzed. The pixel-to-pixel evaluation, which evaluates concordance in staining, resulted in r scores of 0.86 (ER; Pearson correlation), 0.93 (PR) and 0.94 (HER2) and correlation scores in single-cell comparisons ranged from 0.76 to 0.81. Correlation scores on the tissue core level were high in the inter-assay analyses, i.e. t-CyCIF vs. IHC (e.g. r scores up to 0.87 and 0.91 for ER and HER2, respectively, on t-CyCIF vs. Aperio; and 0.85 to 0.94 by pathology review) and t-CyCIF vs. HER2 FISH (r scores up to 0.71). This resulted in validated fluorophore-conjugated antibody panels for use in t-CyCIF that correspond well to established standards. In the second phase, single cell analysis of HER2+ breast cancer was performed. Cancer cells were defined as keratin positive and using t-Distributed Stochastic Neighbor Embedding (t-SNE) seven cancer cell clusters were identified including two HER2hi clusters differing in ER, p53, AR and PD-L1 expression, two HER2lo clusters differing in PR, Ki67, pRB, p53 and AR and three HER2neg clusters differing in PR, Ki67, ER, PD-L1 and AR. Heterogeneity scores were calculated based on diversity among clusters. Conclusion This study is the first to evaluate the performance of breast cancer-specific antibodies in a highly multiplexed imaging platform such as t-CyCIF. Using the validated antibody panel, we uncovered patterns of expression of markers relevant to breast cancer biology that correlate with HER2 high, low and negative states. Ongoing studies are looking at correlations between HER2 heterogeneity, responses to therapy and clinical outcomes. Citation Format: Jennifer L Guerriero, Jia-Ren Lin, Ricardo G Pastorello, Ziming Du, Shaolin Mei, Krishan Taneja, Stuart J Schnitt, Deborah A Dillon, Peter K Sorger, Sandro Santagata, Elizabeth A Mittendorf. Highly multiplexed tissue-based cyclic immunofluorescence (t-CyCIF) for precision oncology identifies novel patterns of HER2 heterogeneity in breast cancer [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 PS18-02.

HER2 fluorescent in situ hybridization signal degradation: a 10-year retrospective study.

Fluorescence in situ hybridization (FISH) analysis is recommended for invasive breast carcinomas with equivocal (2+) immunohistochemical expression of human epidermal growth factor receptor 2 (HER2). However, existing guidelines for the retention and storage requirements for HER2 FISH slides vary widely among countries and laboratories. To determine the degradation rateof HER2 FISH signals, and the optimal retention time and storage conditions for HER2 FISH slides. Dual-probe HER2 FISH slides from March 2009 to June 2019 were retrieved from the archive to assess the presence, intensity and quantity of the green chromosome enumeration probe 17 (CEP 17) and orange HER2 signals. Per the institutional policy, FISH slides are placed in slide boxes and stored in -80°C freezers for up to 4years, whereas older slides are stored at room temperature. After excluding HER2 FISH slides that were deemed uninterpretable due to technical issues, a total of 6255 slides were assessed. Slides from 2009 to 2014 were stored at room temperature, while slides from 2015 to 2019 were stored in -80°C freezers. Slides stored in freezers showed retention of both the green and the orange signals. Slide stored at room temperature demonstrated significant decrease in the signal retention rate and the loss of signal did not progress in a linear fashion. The CEP17 signal was quenched much faster than the HER2 signal. Our study is the first to demonstrate HER2 FISH signal degradation with time and slide storage conditions. Storing HER2 FISH slides in a -80°C freezer allows for retention of both HER2 and CEP17 signals. At room temperature, the signals start to degrade with CEP17 signals lost at a faster rate. The results of the study may be used in official guidelines for storage conditions and retention time for HER2 FISH slides.

HER2 Testing for Breast Cancer in the Genomics Laboratory: A Sea Change for Fluorescence In Situ Hybridization.

Guidelines for HER2 testing in breast cancer have changed over time, from the US Food and Drug Administration guideline to the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines published in 2007, 2013, and 2018. To investigate the change in assignment of HER2 status in breast cancers with equivocal (2+) immunohistochemistry (IHC) results by fluorescence in situ hybridization (FISH) following implementation of the ASCO/CAP 2018 guideline. The study included 3556 invasive breast cancers that were HER2 equivocal (2+) by IHC and were submitted to our FISH laboratory after July 2018. Reflex testing (with repeat IHC staining) was performed on certain categories of FISH results known as groups 2, 3, and 4. Concomitant review of IHC and FISH was performed on these reflex cases per 2018 guideline recommendations. The FISH data were analyzed to compare US Food and Drug Administration and ASCO/CAP 2007, 2013, and 2018 interpretations. Of 3548 invasive breast cancers with complete data available, the percentage agreement for FISH according to different guidelines was highest for ASCO/CAP 2018 versus US Food and Drug Administration (96.5%), followed by ASCO/CAP 2018 versus 2007 (93.8%), and lowest with ASCO/CAP 2018 versus 2013 (83.7%). Per the 2018 guideline, reflex IHC testing was performed on 633 breast cancers (17.8%); the majority of reflex testing results were negative (541 of 633; 85.5%). The overall distribution of HER2 FISH results (per the 2018 guideline) was 88.5% negative and 11.5% positive. By eliminating the equivocal FISH category, the 2018 ASCO/CAP guideline significantly reduced the HER2 FISH-positive rate in tumors with equivocal (2+) IHC results.

Next-generation assessment of human epidermal growth factor receptor 2 gene (ERBB2) amplification status in invasive breast carcinoma: a focus on Group 4 by use of the 2018 American Society of Clinical Oncology/College of American Pathologists HER2 testing guideline.

The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) updated the testing guideline in 2018 to address issues arising from uncommon human epidermal growth factor receptor 2 (HER2) fluorescence in-situ hybridisation (FISH) results according to the 2013 guideline. Next-generation sequencing (NGS) may be used to better classify patients. The aim of this study was to assess the ERBB2 amplification status of invasive breast carcinoma with equivocal HER2 immunohistochemistry (IHC) results by using NGS, focusing on Group 4 (HER2/CEP17 ratio of <2.0; average HER2 signals/cell of ≥4.0 and <6.0). We retrospectively reviewed HER2 FISH and NGS data of HER2 IHC-equivocal breast carcinomas at our centre between January 2009 and September 2019, wherein all three assays were performed on the same tissue block, and compared HER2 FISH results, according to the 2018 ASCO/CAP guideline, and the ERBB2 amplification status determined with NGS. A total of 52 HER2 FISH and NGS results from 51 patients with HER2 IHC-equivocal breast carcinomas were reviewed. The cohort included eight cases classified as 2018 ASCO/CAP in-situ hybridisation Group 1, three classified as Group 2, three classified as Group 3, 14 classified as Group 4, and 24 classified as Group 5. Thirteen of 14 (92.9%) Group 4 (HER2-negative) cases were classified as ERBB2-non-amplified by the use of NGS; the discordant case was later classified as Group 1 with alternative sample FISH testing. NGS revealed no significant difference in somatic mutations or copy number alterations between Groups 4 and 5. Our NGS findings support the reclassification of HER2 FISH-equivocal cases as HER2-negative under the 2018 ASCO/CAP guideline.

373 Poster - Evaluation of FISH of ER-positive breast cancer with low-HER2 expression could improve their survival

Background: Several prognostic and predictive biomarkers for decision-making regarding neo-adjuvant (NAC) and adjuvant treatment in estrogen receptor (ER)-positive breast cancer patients have been developed. However, their ability to predict patient prognosis and treatment outcomes is poor. Hence, validated markers for predicting prognosis and treatment outcomes need to be developed. Expression of human epidermal growth factor receptor 2 (HER2) is evaluated by immunohistochemistry (IHC), and its gene amplification by Fluorescence in situ Hybridization (FISH) is not always examined. In our hospital, HER2 FISH has been assessed in almost all patients including those with HER2 status 0 and 1 (i.e., low-HER2 expression). Moreover, anti HER2 chemotherapy is administered if the patient’s status is low-HER2 FISH-positive. In this report, we retrospectively investigated the ratio and characteristics of low-HER2 FISH-positive patients and compared their recurrence-free survival (RFS) with that of luminal A FISH-negative patients to examine whether evaluation of HER2 FISH in luminal A breast cancer could improve survival.

HER2 FISH for Breast Cancer: Advances in Quantitative Image Analysis and Automation

Quantitative image analysis of the status of human epidermal growth factor receptor 2 (HER2) by both immunohistochemistry staining and fluorescent in situ hybridization (FISH) is important for the treatment of breast cancer. Guidelines of the American Society for Clinical Oncology and College of American Pathologists, for HER2 FISH, have evolved over time to improve test accuracy, and efforts have been made to better address the problems with the interpretation that are encountered with borderline-positive cases. Standardization and automation of HER2 sample preparation, processing, and digital quantitation are being considered. We compared the manual quantitation of HER2 FISH with automated scoring and reviewed the history and current status of automated scoring of HER2 FISH. We explored areas for the possible automation of the process of HER2 FISH and discussed the latest improvements in quantitative image analysis. We conclude that an integrated review of hematoxylin and eosin staining, immunohistochemistry, and FISH by digital image analysis technology would help pathologists to readily identify tumor areas, differentiate invasive from in situ carcinoma, and to recognize HER2 signal patterns (even in clustered heterogeneity). An integrated system would also allow automatic alerts for discrepancies in results for FISH versus immunohistochemistry, and for tumor histology and grade.

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.

Abstract P5-02-03: Highly multiplexed tissue-based cyclic immunofluorescence in breast cancer for precision oncology

Abstract Background: Tissue-based cyclic immunofluorescence (t-CyCIF) is a recently described technique for highly multiplexed immunofluorescence microscopy of formalin-fixed, paraffin-embedded (FFPE) specimens. Via an iterative process, successive four-channel images are collected from the same sample and then registered to each other to generate a high-dimensional representation that is used for visualization and analysis. This technique can be used to capture up to 60 different antigens on a single FFPE tumor section and permits quantification of cell lineage and state markers, intracellular signaling proteins, drug targets and immune cell antigens, thereby promoting biomarker discovery efforts that are fundamental to precision oncology. As with most technologies that utilize immunostaining, proper antibody validation is key to reliable performance. In this study we used t-CyCIF to evaluate multiple antibodies directed against proteins commonly used to characterize breast carcinomas and their associated microenvironment. Our goal was to validate these antibodies on the t-CyCIF platform prior to the more widespread use of this technology to propel novel discoveries on breast cancer initiation, progression and treatment. Methods: To choose the optimal antibody candidate for each biomarker in t-CyCIF, we compared multiple fluorophore-conjugated antibodies for each of the following three proteins routinely evaluated in breast carcinomas: estrogen receptor (ER), progesterone receptor (PR) and HER2. For each of these, a single antibody commonly used in clinical practice was used as a reference. Analyses were performed at the level of pixels, cells and tissue cores. In addition, inter-assay analyses were performed comparing: (1) t-CyCIF vs. immunohistochemistry (IHC), the latter assessed both by digital pathology and by two independent pathologists; and (2) t-CyCIF vs. fluorescence in situ hybridization (FISH) for HER2. Following validation of these antibodies, we evaluated the expression of CD45, CD68, PD-L1, p53, Ki67 and androgen receptor along with ER, PR and HER2, to better understand both the tumor microenvironment and the cell identities/states in breast carcinomas. Results: A total of 948 tissue cores were included in the study. In the first phase, 13 different antibodies were analyzed: three raised against ER and five each against PR and HER2. The pixel-to-pixel evaluation resulted in r scores using Pearson correlation equal to 0.86 for both ER markers tested; ranging from 0.88 to 0.93 for PR; and from 0.56 to 0.94 for HER2. The correlation scores in single-cell comparisons ranged from 0.76 to 0.88 for ER, 0.54 to 0.81 for PR, and from 0.56 to 0.76 for HER2. Comparisons were then performed at the tissue core level. In light of the data generated through these multiple levels of analyses, we identified fluorophore-conjugated candidates for use in t-CyCIF. Correlation scores on the tissue core level were high in the inter-assay analyses, i.e. t-CyCIF vs. IHC (e.g. r scores up to 0.91 for HER2 on t-CyCIF vs. IHC) and t-CyCIF vs. HER2 FISH (r scores up to 0.71). In the second phase, we characterized the tumor microenvironment and cell identities present in 260 breast carcinomas. With a qualified panel of antibodies, we performed single-cell analyses of 589,343 cells and identified unexpected patterns of PD-L1 expression in distinct populations of tumor cells. Conclusion: This study is the first to evaluate the performance of breast cancer-specific antibodies in a highly multiplexed imaging platform such as t-CyCIF. This work demonstrates a step-by-step approach for qualifying reagents to be used in a multiplexed, spatially resolved tissue imaging modality. This validation study will facilitate the use of t-CyCIF for additional studies in breast cancer to evaluate both tumor elements and components of the microenvironment. Citation Format: Ricardo G Pastorello, Jia-Ren Lin, Ziming Du, Shaolin Mei, Krishan Taneja, Deborah A Dillon, Stuart J Schnitt, Peter K Sorger, Elizabeth A Mittendorf, Sandro Santagata, Jennifer L Guerriero. Highly multiplexed tissue-based cyclic immunofluorescence in breast cancer for precision oncology [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 P5-02-03.

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.