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Background: There are limited data on the role of multigene tests and their correlation with immunohistochemistry (IHC), especially on core biopsy. MammaTyper is a quantitative conformite Europeeanne (CE) marked, National Institute for Health and Care excellence (NICE) approved, in in vitro diagnostic quantitative real-time polymerase chain reaction (RT-qPCR) test for assessment of mRNA expression of four biomarkers (ESR1, PGR, ERBB2, MKI67)., Methods: We evaluated the concordance of MammaTyper with oestrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 by IHC on 133 core needle biopsies of breast cancer. HER2 was positive if IHC 3+ or 2+ and fluorescence in situ hybridization (FISH)-amplified. Global and hotspot Ki67 expression was analysed using a cutoff of ≥20% assessed manually and by digital image analysis. Agreements were expressed as overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa., Results: RT-qPCR results of ESR1 were highly concordant with IHC with OPA of 94.7% using 1% cutoff and 91.7% when the low ER-positive category was included. The PPA and NPA between RT-qPCR and IHC for PR was 91.5% and 88.0%, respectively, when using the 1% cutoff. For ERBB2/HER2, the OPA was 95% and the PPA was 84.6%. 40 of 72 HER2 IHC score 0 tumours were classified as ERBB2 low. Best concordance between MKI67 by MammaTyper and Ki67 IHC was achieved using hotspot digital image analysis (OPA: 87.2%, PPA: 90.6%, NPA: 80%)., Conclusion: RT-qPCR-based assessment of the mRNA expression of ESR1, PGR, ERBB2, and MKI67 showed high concordance with IHC, suggesting that the MammaTyper test on core needle biopsies represents a reliable, efficient, and reproducible alternative for breast cancer classification and refining HER2 low categorisation., (© 2024 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Human epidermal growth factor receptor 2 ( HER2 ) gene amplification and subsequent protein overexpression is a strong prognostic and predictive biomarker in invasive breast carcinoma (IBC). ASCO/CAP recommended tests for HER2 assessment include immunohistochemistry (IHC) and/or in situ hybridization (ISH). Accurate HER2 IHC scoring (0, 1+, 2+, 3+) is key for appropriate classification and treatment of IBC. HER2-targeted therapies, including anti-HER2 monoclonal antibodies and antibody drug conjugates (ADC), have revolutionized the treatment of HER2-positive IBC. Recently, ADC have also been approved for treatment of HER2-low (IHC 1+, IHC 2+/ISH-) advanced breast carcinoma, making a distinction between IHC 0 and 1+ crucial. In this focused study, 32 IBC with HER2 IHC scores from 0 to 3+ and HER2 FISH results formed a calibration dataset, and 77 IBC with HER2 IHC score 2+ and paired FISH results (27 amplified, 50 non-amplified) formed a validation dataset. H&E and HER2 IHC whole slide images (WSI) were scanned. Regions of interest were manually annotated and IHC scores generated by the software QuantCenter (MembraneQuant application) by 3DHISTECH Ltd. (Budapest, Hungary) and compared to the microscopic IHC score. H-scores [(3×%IHC3+) +(2×%IHC2+) +(1×%IHC1+)] were calculated for semi-automated (MembraneQuant) analysis. Concordance between microscopic IHC scoring and 3DHISTECH MembraneQuant semi-automated scoring in the calibration dataset showed a Kappa value of 0.77 (standard error 0.09). Microscopic IHC and MembraneQuant image analysis for the detection of HER2 amplification yielded a sensitivity of 100% for both and a specificity of 56% and 61%, respectively. In the validation set of IHC 2+ cases, only 13 of 77 cases (17%) had discordant results between microscopic and MembraneQuant images, and various artifacts limiting the interpretation of HER2 IHC, including cytoplasmic/granular staining and crush artifact were noted. Semi-automated analysis using WSI and microscopic evaluation yielded similar HER2 IHC scores, demonstrating the potential utility of this tool for interpretation in clinical practice and subsequent accurate treatment. In this study, it was shown that semi-automatic HER2 IHC interpretation provides an objective approach to a test known to be quite subjective., Competing Interests: FP is a member of the scientific advisory board of Multiplex DX. In addition, FP serves on the diagnostic advisory board and reports receiving consultancy fees from AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liao, Bakoglu, Cesmecioglu, Hanna, Pareja, Wen, D’Alfonso, Brogi, Yagi and Ross.)

Human epidermal growth factor receptor 2 (HER2)-low breast cancer has emerged as a subtype of breast cancer, defined by HER2 1+/2+ in immunohistochemistry (IHC) and absence of ERBB2 gene amplification on fluorescence in situ hybridization (FISH). Recent trials showed marked response of HER2-low breast cancer to novel anti-HER2 antibody-drug-conjugates. Data on characteristics of HER2-low breast cancer subtype is limited. Real-world data from the Anatomic Pathology Department of Hotel-Dieu de France, spanning 2017-2023, was retrospectively collected. HER2-positive patients were excluded to compare HER2-low to HER2-zero breast cancer subtypes. Clinicopathological characteristics between the groups were compared using a Chi-Squared test. Out of 1195 patients, we observed 341 (28.5 %) HER2-low breast cancers cases. HER2-positive breast cancer cases (n = 178; 14.9 %) were excluded. There was no significant difference in age and sex between HER2-low and HER2-zero group (p = 0.33 and 0.79, respectively). HER2-low breast cancer was associated with positive estrogen receptor status and positive progesterone receptor status (p < 0.001 and p = 0.01, respectively). Ductal adenocarcinomas were more commonly observed in HER2-low group (p < 0.001). When stratified by hormone (HR) status, 87.4 % of patients had HR-positive status and 12.6 % were HR-negative. Among the HR-negative group, HER2-low tumors tended to show lower proliferation index compared to HER2-zero tumors (25%vs.10 %, p = 0.04). This study showed that HER2-low is distinct from HER2-zero and is common among patients with breast cancer. Clinicopathological features such as histological type differ between HER2-zero and HER2-low breast cancer. Within HR-negative breast cancer, those with low HER2 expression exhibit a less aggressive profile compared to HER2-zero tumors., Competing Interests: Declaration of competing interest No conflict of interest to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Single-molecule fluorescence in situ hybridization (smFISH) represents a promising approach for the quantitative analysis of nucleic acid biomarkers in clinical tissue samples. However, low signal intensity and high background noise are complications that arise from diagnostic pathology when performed with smFISH-based RNA imaging in formalin-fixed paraffin-embedded (FFPE) tissue specimens. Moreover, the associated complex procedures can produce uncertain results and poor image quality. Herein, by combining the high specificity of split DNA probes with the high signal readout of ZnCdSe/ZnS quantum dot (QD) labeling, we introduce QD split-FISH, a high-brightness smFISH technology, to quantify the expression of mRNA in both cell lines and clinical FFPE tissue samples of breast cancer and lung squamous carcinoma. Owing to its high signal-to-noise ratio, QD split-FISH is a fast, inexpensive, and sensitive method for quantifying mRNA expression in FFPE tumor tissues, making it suitable for biomarker imaging and diagnostic pathology.

Context.—: Human epidermal growth factor (HER2/neu) gene amplification, a poor prognostic factor in invasive breast cancer, has shown substantial utility as a predictive marker, with significantly improved survival following anti-HER2 therapies like trastuzumab. Dual-color dual in situ hybridization (D-DISH), a recently introduced fully automated assay for HER2/neu evaluation on light microscopy, has several advantages over fluorescence in situ hybridization (FISH)., Objective.—: To standardize and validate the D-DISH assay using FISH as the gold standard and assess interobserver reproducibility in interpreting the D-DISH assay., Design.—: D-DISH was performed using the latest HER2 Dual ISH DNA Probe Cocktail assay (Ventana Medical Systems Inc, Tucson, Arizona) in 148 cases of invasive breast cancer. The same block was used for performing immunohistochemistry by Ventana PATHWAY anti-HER2/neu (4B5) antibody and FISH assay by ZytoLight SPEC ERBB2/CEN17 Dual Color Probe. D-DISH was separately interpreted by 4 pathologists blinded to FISH results., Results.—: Concordance of 98.65% and a Cohen κ value of 0.97 were observed between FISH and D-DISH. Intraclass correlation coefficient (0.93-0.97) and κ values (0.98-1.0) for interobserver reproducibility showed almost perfect agreement by D-DISH. Interobserver reproducibility was also evaluated for genomic heterogeneity, HER2 group categorization, and polysomy (κ values 0.42-0.74, 0.89-0.93, and 0.98-1.0, respectively)., Conclusions.—: We successfully validated the latest version of D-DISH assay as a substitute for FISH in predicting HER2 gene status with significant interobserver reproducibility, concluding that this D-DISH assay may be introduced in routine diagnostic services as a reflex test to ascertain HER2 gene status., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

Context.—: RNA sequencing study has demonstrated that human epidermal growth factor receptor 2 (HER2) RNA levels influence anti-HER2 therapeutic efficacy. However, in situ HER2 RNA expression (isHRE), which evaluates HER2 RNA expression in tissue, has remained unclear in breast cancers (BCs) of various HER2 immunohistochemistry (IHC)/in situ hybridization (ISH) categories., Objective.—: To correlate isHRE with all HER2 IHC/fluorescence ISH (FISH) categories in BC., Design.—: Formalin-fixed, paraffin-embedded tissue sections from 259 BCs, covering all IHC/FISH categories, were analyzed for isHRE by RNAscope., Results.—: We validated HER2 RNAscope scoring as a semiquantitative method to evaluate isHRE and demonstrated significantly higher RNAscope scores in IHC 3+ than in IHC 2+ cases, and in IHC 2+ than in IHC 0/1+ cases. Among the 5 IHC 2+/FISH groups, group 1 (G1) cases had the highest scores. The scores in G3 cases were higher than those in G2, but not significantly different from those in G4 and G5. G4 cases had significantly higher scores than those in G2. Higher HER2 copy numbers and HER2:CEP 17 (centromere 17) copy number ratios were significantly correlated with higher isHRE in G1 cases, but not in G2 to G5 cases. RNAscope scores were significantly lower in HER2-negative (IHC 0) than in HER2-low (IHC 2+/FISH- and IHC 1+) BCs but were not different between IHC 0 and 1+ BCs when analyzed separately., Conclusions.—: We demonstrate the HER2 RNA expression status among BCs of various HER2 IHC/FISH categories in tissue. Such information may be relevant for anti-HER2 treatment decisions considering the role of HER2 RNA expression in predicting anti-HER2 therapeutic efficacy., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

Purpose: Trastuzumab deruxtecan is a new treatment option for patients with advanced human epidermal growth factor receptor 2 (HER2)-low breast cancer (BC). Although HER2-low status has been characterized in early and advanced BC, it has yet to be fully characterized in brain metastases (BrM)., Methods: Patients who underwent surgery for BC BrM at Sunnybrook Health Sciences Centre and for whom HER2 status was available on resected BrM were studied. Estrogen receptor, progesterone receptor, and HER2 status were assessed on the basis of ASCO/College of American Pathologists (CAP) guidelines. HER2-zero was defined as immunohistochemistry (IHC) 0; HER2-low was defined as IHC 1+ or IHC 2+ with fluorescence in situ hybridization (FISH)-negative status. HER2-positive (HER2+) was defined as IHC 3+ or IHC 2+ with positive FISH. Clinicopathologic features were recorded. We also assessed the prognostic association between extent of HER2 expression and (1) brain-specific progression-free survival (bsPFS), as well as (2) overall survival (OS)., Results: In this retrospective cohort of 102 patients with resected BC BrM, 53% (n = 54) were HER2+, 29.4% (n = 30) were HER2-low, and 17.6% (n = 18) had HER2-zero status. Among BrM that were triple-negative on the basis of ASCO/CAP guidelines, 63.6% (n = 14/22) were reclassified as being HER2-low. Sixty percent (n = 15/25) of BrM that were hormone receptor-positive/HER2-negative (HR+/HER2-) were reclassified as being HER2-low. In total, 51 patients had matched primary breast and BrM tissue available; results of HER2 status when categorized as HER2-zero, HER2-low, and HER2+ were concordant in 82.3% (n = 42/51) of cases (Cohen's kappa, 0.58; P = .07). There was no significant association between HER2-zero, HER2-low, and HER2+ status in BrM and either bsPFS or OS., Conclusion: Among patients with surgically resected BrM, a high proportion of those with metastatic triple-negative BC and HR+/HER2- disease have HER2-low BrM with potential to benefit from HER2-targeted therapy.

Background: Accurate quantification of human epidermal growth factor receptor 2 (HER2) gene amplification is important for predicting treatment response and prognosis in patients with breast cancer. Fluorescence in situ hybridization (FISH) is the gold standard for the diagnosis of HER2 status, particularly in cases with equivocal status on immunohistochemistry (IHC) staining, but has some limitations of non-classical amplifications and such cases are diagnosed basing on additional IHC and FISH. This study investigated the clinical utility of a novel super-resolution fluorescence microscopy technique for the better FISH signal visualization and HER2 FISH classification., Methods: Fourteen breast cancer tissue samples were retrospectively collected between September 2018 and February 2022, and FISH HER2 signal quantification was evaluated by determining the HER2/chromosome 17 centromere (CEP17) ratio and the number of HER2 signals per nucleus in super- versus conventional-resolution images., Results: Super-resolution images maintained the same overall HER2 diagnosis from routine, but HER2 FISH amplification changed negative to monosomy in two cases. Two Letrozole non-response relapses coincided to monosomy samples. The median number of HER2 signals per nucleus was 7.5 in super-resolution images and 4.0 in conventional-resolution images in HER2-positive samples and 2.8 and 2.1 signals per nucleus, respectively, in HER2-negative samples., Conclusions: Super-resolution images improved signal visualization, including a significant difference in the number of countable HER2 and CEP17 signals in a single nucleus compared with conventional-resolution images. Increased accuracy of signal quantification by super-resolution microscopy may provide clinicians with more detailed information regarding HER2 FISH status that allows to better FISH classification such as HER2-low samples., (© 2024. The Author(s).)

Since the release of the DESTINY-Breast04 (DB-04) trial findings in June 2022, the field of pathology has seen a renaissance of HER2 as a predictive biomarker in breast cancer. The trial focused on patients with metastatic breast cancer who were classified as "HER2-low," i.e., those with immunohistochemistry (IHC) HER2 1 + or 2 + and negative in situ hybridization (ISH) results. The study revealed that treating these patients with trastuzumab deruxtecan (T-DXd) instead of the oncologist's chosen chemotherapy led to outstanding improvements in survival. This has challenged the existing binary HER2 pathological classification system, which categorized tumors as either positive (overexpression/amplification) or negative, as per the ASCO/CAP 2018 guideline reaffirmed by ASCO/CAP 2023 guideline update. Given that DB-04 excluded patients with HER2 IHC score 0 status, the results of the ongoing DB-06 trial may shed further light on the potential benefits of T-DXd therapy for these patients. Roughly half of all breast cancers are estimated to belong to the HER2-low category, which does not represent a distinct or specific subtype of cancer. Instead, it encompasses a diverse group of tumors that exhibit clinical, morphological, immunohistochemical, and molecular variations. However, HER2-low offers a distinctive biomarker status that identifies a specific therapeutic regimen (i.e., T-DXd) linked to a favorable prognosis in breast cancer. This unique association emphasizes the importance of accurately identifying these tumors. Differentiating between a HER2 IHC score 0 and score 1 + has not been clinically significant until now. To ensure accurate classification and avoid misdiagnosis, it is necessary to adopt standardized procedures, guidelines, and specialized training for pathologists in interpreting HER2 expression in the lower spectrum. Additionally, the utilization of artificial intelligence holds promise in supporting this endeavor. Here, we address the current state of the art and unresolved issues in assessing HER2-low status, with a particular emphasis on the score 0. We explore the dilemma surrounding the exclusion of HER2-zero patients from potentially beneficial therapy based on traditional HER2 testing. Additionally, we examine the clinical context, considering that DB-04 primarily involved heavily pretreated late-stage metastatic breast cancers. We also delve into emerging evidence suggesting that extrapolating HER2-low status from the original diagnosis may lead to misleading results. Finally, we provide recommendations for conducting high-quality testing and propose a standardized pathology report in compliance with 2023 ASCO/CAP updates and 2023 ESMO consensus statements on HER2-low breast cancer., (© 2023. The Author(s).)

HER2 status is now routinely assessed in endometrial serous carcinoma (ESC) due to the reported predictive value of HER2 protein overexpression and/or gene amplification. Herein the authors compare 2 proposed testing and interpretation guidelines for HER2 in ESC. Forty-three consecutive cases of ESC that had been dually tested by both HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were interpreted using 2 sets of guidelines. Guideline set 1 (GS1) is the 2018 American Society of Clinical Oncology/College of American Pathologists guidelines for breast cancer. Guideline set 2 (GS2) is a recent proposal that is a slight modification of the enrollment criteria for the clinical trial (NCT01367002) that demonstrated a survival benefit for anti-HER2 therapy in ESC. By IHC, GS1 and GS2, respectively classified 39.5% (17/43) and 28% (12/43) of ESC as HER2-negative, 37.2% (16/43) and 53.4% (23/43) as HER2 equivocal, and 23.2% (10/43) and 18.6% (8/43) as HER2-positive ( P > 0.05 for all). IHC and FISH were highly concordant at the extremes using either set of guidelines, as no cases were found to be IHC3+/FISH-negative or IHC 0-1+/FISH-positive. GS1 and GS2 were comparable regarding the proportion of IHC equivocal cases that were HER2 amplified by FISH (19% vs 23% respectively; [ P = 0.71]). GS1 and GS2 displayed 98% (42/43) concordance regarding the final (IHC and/or FISH-based) classification of tumors as being HER2-positive or negative, and the same 13 cases were ultimately classified as HER2 amplified using either GS1 or GS2. One "discordant" case was classified as HER2-positive using GS2 but HER2-negative using GS1 (HER2 IHC score 2+ using both guidelines, HER2:CEP17 signal ratio of 3, HER2 signal number of 3.4). Six (14%) of the 43 cases (FISH Groups: 2, 3, and 4) would require IHC results to interpret the FISH findings using GS1. Because GS1 requires that the HER2 IHC staining be observed within a homogeneous and contiguous invasive cell population, and this is not a requirement in GS2, GS2 may be better suited for ESC given its frequently heterogeneous staining pattern. Additional studies may be required on the optimal interpretation of problematic dual-probe FISH scenarios in GS2 and the necessity for IHC correlation in such scenarios. Using either set of guidelines, our findings support a reflex testing strategy of restricting FISH testing to cases that are IHC equivocal., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 by the International Society of Gynecological Pathologists.)

Background: Approximately 20% of breast cancers express HER2-positive receptors in the USA. HER2 receptor immunohistochemistry (IHC) staining with equivocal (2+) results commonly undergoes fluorescence in-situ hybridization (FISH) for further classification. Current guidelines do not recommend routine FISH testing in IHC-negative (0 or 1+) cases. This study investigates an institution that performs both IHC and FISH testing on all cases to identify the true HER2-positive rate., Patients and Methods: A retrospective chart review from 2015 to 2021 was conducted at an institution where both HER2 IHC and FISH testing were performed at the time of diagnosis for all invasive breast cancers. The rate of true HER2-positive patients was determined, and patient and tumor characteristics were further explored., Results: A total of 1835 invasive breast cancer cases were primarily treated at this institution. A total of 289 cases were HER2 positive on IHC and FISH testing (15.7%). An additional 38 cases were identified as HER2 negative on IHC, but reclassified as HER2 positive on reflex FISH testing. Total HER2 positive cases increased from 289 (15.7%) to 327 cases (17.8%) with reflex FISH testing., Conclusions: The additional HER2-positive cases after completing FISH testing on IHC-negative tumors suggests there may be a role for routine FISH testing in addition to standard IHC staining to determine HER2 status for breast cancer. The ethical, prognostic and even  benefits of a correct diagnosis outweigh the added expense of FISH testing., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

HER2-positive breast cancer accounts for 20 % of all invasive breast cancers. It is associated with increased recurrence, a higher risk of brain metastases and a shorter overall survival than luminal A and B cancers. The presence of ERBB2 gene amplification is predictive of a good response to anti-HER2 therapies. Determining HER2 status is a hot topic, given recent studies on HER2-low subtypes. Although several techniques are available, the latest 2018 update from the American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) recommends a first immunohistochemical study followed by a fluorescence in situ hybridisation study indicated only for equivocal cases. This article summarises the different techniques used to determine HER2 status and provides a detailed review of the literature on pre-analytical factors and intrinsic tumour-specific factors that can distort or complicate the interpretation of results., (Copyright © 2023 Société Française du Cancer. All rights reserved.)

Objective: Currently, human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells are diagnosed under the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2018 guidelines. The guideline combined the results of in situ hybridization (DISH) and immunohistochemistry (IHC) techniques. The IHC technique is easy, cheap, and suitable for developing country. Therefore, in this study, we validated the use of IHC alone compared to the results of HER2 amplification under ASCO/CAP 2018 guidelines in diagnosed HER2 positive breast cancer cells., Methods: A total of 510 breast cancer tissue samples from Rajavithi Hospital in Bangkok, Thailand, from January 1st, 2022, to May 31st, 2023, were analyzed by IHC, followed by dual ISH (DISH). We selected 58 samples of IHC equivocal (score 2+) and 98 samples of IHC positive (score 3+) to analyze the diagnostic values by comparing them to the results of HER2 amplification., Results: The HER2 IHC score was found to agree with HER2 amplification with a sensitivity of 87.96%, a specificity of 93.75%, a positive predictive value of 96.94%, a negative predictive value of 77.59%, a positive likelihood ratio of 14.07, a negative likelihood ratio of 0.13, and an accuracy of 89.74%., Conclusion: The promising outcomes suggest that a positive IHC test result (score 3+) could potentially stand alone for patients with breast cancer undergoing anti-HER2 treatment, even without DISH confirmation.

Context.—: Quantitative imaging is a promising tool that is gaining wide use across several areas of pathology. Although there has been increasing adoption of morphologic and immunohistochemical analysis, the adoption of evaluation of fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded tissue has been limited because of complexity and lack of practice guidelines., Objective.—: To perform human epidermal growth factor receptor 2 (HER2) FISH validation in breast carcinoma in accordance with the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2018 guideline., Design.—: Clinical validation of HER2 FISH was performed using the US Food and Drug Administration-approved dual-probe HER2 IQFISH (Dako, Carpinteria, California) with digital scanning performed on a PathFusion (Applied Spectral Imaging, Carlsbad, California) system. Validation parameters evaluated included z-stacking, classifier, accuracy, precision, software, and hardware settings. Finally, we evaluated the performance of digital enumeration on clinical samples in a real-world setting., Results.—: The accuracy samples showed a final concordance of 95.3% to 100% across HER2 groups 1 to 5. During clinical implementation for HER2 groups 2, 3, and 4, we achieved a final concordance of 76% (95 of 125). Of these cases, only 8% (10 of 125) had discordances with clinical impact that could be identified algorithmically and triaged for manual review., Conclusions.—: Digital FISH enumeration is a useful tool to improve the efficacy of HER2 FISH enumeration and capture genetic heterogeneity across HER2 signals. Excluding cases with high background or poor image quality and manual review of cases with ASCO/CAP group discordances can further improve the efficiency of digital HER2 FISH enumeration., (© 2023 College of American Pathologists.)

Aims: 'HER2-low' breast cancer is an emerging issue as the clinical trials for anti-HER2 antibody-drug conjugates (trastuzumab deruxtecan) are making progress. A reliable method to identify HER2-low cancers is needed. This study aimed to evaluate immunohistochemistry (IHC) and in situ hybridisation (ISH) in detecting HER2-low status., Methods: We evaluated the HER2 ISH data grouped by the IHC consensus in proficiency tests (PTs), and compared the HER2 ISH results between HER2 IHC scored 0 (IHC-0) and IHC scored 1+ (IHC-1+) tumours from in-house tissue microarrays (TMAs). Since benign/normal glands have HER2 expression and ideally should not be affected by targeted therapy, we evaluated HER2 ISH results in peritumoural benign glands of 52 breast cancers as reference values., Results: None of the 565 tissue cores in PTs achieved an 80% participant agreement of IHC-1+. In PTs and in-house TMAs, HER2 signals of the IHC-1+cores (median: 2.6 and 2.0, respectively) were significantly higher than those of IHC-0 cores (median: 2.0 and 1.7, respectively). But the ranges of HER2 signals had a considerable overlap between IHC-1+and IHC-0 cores. The HER2 signals and HER2 :CEP17 ratios of peritumoural benign glands exhibited normal distributions, and their upper bounds of the 95% reference intervals were 2.10 and 1.30, respectively., Conclusions: Current HER2 testing algorithms are unsatisfactory in detecting HER2-low cases. Using ISH to detect tumour with HER2 signals and HER2 :CEP17 ratio higher than the upper bound of the benign glands can be an alternative method., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor that plays a carcinogenic role in breast cancer (BC) through gene amplification, mutation, or overexpression. Traditional methods of HER2 detection were divided into positive (immunohistochemistry (IHC) 3+/fluorescence in situ hybridization (FISH) amplification) and negative (IHC 2+/FISH-, IHC 1+, IHC 0) according to the dichotomy method. Anti-HER2-targeted therapies, such as trastuzumab and pertuzumab, have significantly improved the prognosis of HER2-positive patients. However, up to 75% to 85% of patients remain HER2-negative. In recent years, with the rapid development of molecular biology, gene detection technology, targeted therapy, and immunotherapy, researchers have actively explored the clinicopathological characteristics, molecular biological characteristics, treatment methods, and HER2 detection methods of HER2-low/zero breast cancer. With the clinical efficacy of new anti-HER2 targeted drugs, accurate classification of breast cancer is very important for the treatment choice. Therefore, the following review summarizes the necessity of developing HER2 detection methods, and the clinicopathological and drug treatment characteristics of patients with HER2-low/zero, to light the dawn of the treatment of breast cancer patients with HER2-low/zero expression., (© 2023 UICC.)

Aims: Accurate assessment of human epidermal growth factor receptor 2 (HER2) expression by HER2 immunohistochemistry and in-situ hybridisation (ISH) is critical for the management of patients with breast cancer. The revised 2018 ASCO/CAP guidelines define 5 groups based on HER2 expression and copy number. Manual pathologist quantification by light microscopy of equivocal and less common HER2 ISH groups (groups 2-4) can be challenging, and there are no data on interobserver variability in reporting of these cases. We sought to determine whether a digital algorithm could improve interobserver variability in the assessment of difficult HER2 ISH cases., Methods and Results: HER2 ISH was evaluated in a cohort enriched for less common HER2 patterns using standard light microscopy versus analysis of whole slide images using the Roche uPath HER2 dual ISH image analysis algorithm. Standard microscopy demonstrated significant interobserver variability with a Fleiss's kappa value of 0.471 (fair-moderate agreement) improving to 0.666 (moderate-good) with the use of the algorithm. For HER2 group designation (groups 1-5), there was poor-moderate reliability between pathologists by microscopy [intraclass correlation coefficient (ICC) = 0.526], improving to moderate-good agreement (ICC = 0.763) with the use of the algorithm. In subgroup analysis, the algorithm improved concordance particularly in groups 2, 4 and 5. Time to enumerate cases was also significantly reduced., Conclusion: This work demonstrates the potential of a digital image analysis algorithm to improve the concordance of pathologist HER2 amplification status reporting in less common HER2 groups. This has the potential to improve therapy selection and outcomes for patients with HER2-low and borderline HER2-amplified breast cancers., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Background: Clear cell carcinoma (CCC) is a rare high-grade adenocarcinoma associated with poor response to platinum-based chemotherapy agents in the female genital tract. Human epidermal growth factor receptor 2 (HER2) overexpression is routinely used as a biomarker for targeted therapy in breast and gastric carcinomas, but its role in CCC remains unclear., Methods: In this study, HER2 overexpression was evaluated by immunohistochemistry (IHC) using College of American Pathologists (CAP) HER2 scoring guidelines for breast and endometrial serous carcinoma (ESC) on tissue microarray blocks. In equivocal and positive cases, fluorescence in situ hybridization (FISH) was performed. IHC score 3, and all amplified cases on FISH test were considered positive., Results: Thirty-six cases of ovarian (OCCC), 36 endometrial (ECCC), and 2 cervical CCC were included. According to ESC and breast scoring guidelines, 20 % and 15.1 % of ECCC and 14.7 % and 6 % of OCCC were HER2 positive, respectively. Both cases of cervical CCC were negative. Scoring based on breast carcinoma guideline showed higher concordance (100 %) with gene amplification results, in comparison with ESC guideline (82.7 %). On multivariate survival analysis, HER2 positive ECCC and OCCC (based on ESC scoring methods) had significantly lower overall and disease-free survivals (OS, DFS) (P < 0.05)., Conclusion: HER2 immunoscoring based on ESC guideline can yield a higher sensitivity with relevant clinical and prognostic features in OCCC and ECCC. HER2 can be considered a potential biomarker for targeted therapy and future clinical trials., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) guidelines are used for human epidermal growth factor receptor 2 (HER2) reporting in breast carcinoma. Cases that demonstrate weak to moderate complete membrane immunohistochemical staining in >10% of the tumor are scored as 2+ (equivocal). This study aimed to determine what proportion of HER2 immunohistochemistry (IHC) score = 2+ breast carcinomas were confirmed to be positive by HER2 fluorescent in situ hybridization (FISH). There were 241 HER2 IHC score = 2+ breast carcinomas included. Most (74.3%) carcinomas were estrogen and progesterone receptor-positive. Invasive breast carcinoma of no special type (89.2%) was the commonest histologic subtype. Most tumors were grade 2 (64.3%). As per the FISH report, at the time of diagnosis, 27 cases (11.2%) were HER2 FISH positive. All HER2 FISH equivocal cases and one FISH positive case assessed using the 2013 ASCO/CAP HER2 criteria were reclassified to HER2 FISH negative when the 2018 criteria were applied. There was a high level of agreement (κ = 0.979) between HER2 FISH results obtained using the 2013 and the 2018 criteria. This study provides insight into the frequency of HER2 FISH positivity (11.2%) among HER2 IHC score = 2+ breast carcinomas and the impact of modifications to the ASCO/CAP HER2 guidelines. Elimination of the HER2 FISH equivocal category by the 2018 guidelines has reduced the need for repeat testing and simplified clinical management. Reclassification of previous HER2 FISH positive to negative has resulted in some patients being ineligible for costly anti-HER therapy., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

The evaluation of the Human Epidermal growth factor Receptor-2 (HER2) expression is an important prognostic biomarker for breast cancer treatment selection. However, HER2 scoring has notoriously high interobserver variability due to stain variations between centers and the need to estimate visually the staining intensity in specific percentages of tumor area. In this paper, focusing on the interpretability of HER2 scoring by a pathologist, we propose a semi-automatic, two-stage deep learning approach that directly evaluates the clinical HER2 guidelines defined by the American Society of Clinical Oncology/ College of American Pathologists (ASCO/CAP). In the first stage, we segment the invasive tumor over the user-indicated Region of Interest (ROI). Then, in the second stage, we classify the tumor tissue into four HER2 classes. For the classification stage, we use weakly supervised, constrained optimization to find a model that classifies cancerous patches such that the tumor surface percentage meets the guidelines specification of each HER2 class. We end the second stage by freezing the model and refining its output logits in a supervised way to all slide labels in the training set. To ensure the quality of our dataset's labels, we conducted a multi-pathologist HER2 scoring consensus. For the assessment of doubtful cases where no consensus was found, our model can help by interpreting its HER2 class percentages output. We achieve a performance of 0.78 in F1-score on the test set while keeping our model interpretable for the pathologist, hopefully contributing to interpretable AI models in digital pathology., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rutger Fick reports financial support was provided by Tribun Health. Manh Dan Pham reports financial support was provided by Tribun Health. Guillaume Balezo reports financial support was provided by Tribun Health. Cyprien Tilmant reports financial support was provided by Tribun Health. Stephanie Pett reports financial support was provided by Tribun Health. Saima Ben Hadj reports financial support was provided by Tribun Health., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose.—: To update the American Society of Clinical Oncology-College of American Pathologists (ASCO-CAP) recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer. An Update Panel is aware that a new generation of antibody-drug conjugates targeting the HER2 protein is active against breast cancers that lack protein overexpression or gene amplification., Methods.—: The Update Panel conducted a systematic literature review to identify signals for updating recommendations., Results.—: The search identified 173 abstracts. Of 5 potential publications reviewed, none constituted a signal for revising existing recommendations., Recommendations.—: The 2018 ASCO-CAP recommendations for HER2 testing are affirmed., Discussion.—: HER2 testing guidelines have focused on identifying HER2 protein overexpression or gene amplification in breast cancer to identify patients for therapies that disrupt HER2 signaling. This update acknowledges a new indication for trastuzumab deruxtecan when HER2 is not overexpressed or amplified but is immunohistochemistry (IHC) 1+ or 2+ without amplification by in situ hybridization. Clinical trial data on tumors that tested IHC 0 are limited (excluded from DESTINY-Breast04), and evidence is lacking that these cancers behave differently or do not respond similarly to newer HER2 antibody-drug conjugates. Although current data do not support a new IHC 0 versus 1+ prognostic or predictive threshold for response to trastuzumab deruxtecan, this threshold is now relevant because of the trial entry criteria that supported its new regulatory approval. Therefore, although it is premature to create new result categories of HER2 expression (eg, HER2-Low, HER2-Ultra-Low), best practices to distinguish IHC 0 from 1+ are now clinically relevant. This update affirms prior HER2 reporting recommendations and offers a new HER2 testing reporting comment to highlight the current relevance of IHC 0 versus 1+ results and best practice recommendations to distinguish these often subtle differences. Additional information is available at www.asco.org/breast-cancer-guidelines., Competing Interests: Authors’ disclosures of potential conflicts of interest are shown after the references., (Copyright 2023 College of American Pathologists and the American Society of Clinical Oncology. This guideline update was developed through collaboration among the College of American Pathologists and the American Society of Clinical Oncology and has been jointly published by invitation and consent in the Archives of Pathology & Laboratory Medicine and the Journal of Clinical Oncology. It has been edited in accordance with style standards established at the Journal of Clinical Oncology. All rights reserved.)

Purpose: To update ASCO-College of American Pathologists (CAP) recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer. The Panel is aware that a new generation of antibody-drug conjugates (ADCs) targeting the HER2 protein is active against breast cancers that lack protein overexpression or gene amplification., Methods: An Update Panel conducted a systematic literature review to identify signals for updating recommendations., Results: The search identified 173 abstracts. Of five potential publications reviewed, none constituted a signal for revising existing recommendations., Recommendations: The 2018 ASCO-CAP recommendations for HER2 testing are affirmed., Discussion: HER2 testing guidelines have focused on identifying HER2 protein overexpression or gene amplification in breast cancer to identify patients for therapies that disrupt HER2 signaling. This update acknowledges a new indication for trastuzumab deruxtecan when HER2 is not overexpressed or amplified but is immunohistochemistry (IHC) 1+ or 2+ without amplification by in situ hybridization. Clinical trial data on tumors that tested IHC 0 are limited (excluded from DESTINY-Breast04), and evidence is lacking that these cancers behave differently or do not respond similarly to newer HER2 ADCs. Although current data do not support a new IHC 0 versus 1+ prognostic or predictive threshold for response to trastuzumab deruxtecan, this threshold is now relevant because of the trial entry criteria that supported its new regulatory approval. Therefore, while it is premature to create new result categories of HER2 expression (eg, HER2-Low, HER2-Ultra-Low), best practices to distinguish IHC 0 from 1+ are now clinically relevant. This Update affirms prior HER2 reporting recommendations and offers a new HER2 testing reporting comment to highlight the current relevance of IHC 0 versus 1+ results and best practice recommendations to distinguish these often subtle differences.Additional information is available at www.asco.org/breast-cancer-guidelines.

The overexpression of the human epidermal growth factor receptor 2 (HER2) is a predictive biomarker in therapeutic effects for metastatic breast cancer. Accurate HER2 testing is critical for determining the most suitable treatment for patients. Fluorescent in situ hybridization (FISH) and dual in situ hybridization (DISH) have been recognized as FDA-approved methods to determine HER2 overexpression. However, analysis of HER2 overexpression is challenging. Firstly, the boundaries of cells are often unclear and blurry, with large variations in cell shapes and signals, making it challenging to identify the precise areas of HER2-related cells. Secondly, the use of sparsely labeled data, where some unlabeled HER2-related cells are classified as background, can significantly confuse fully supervised AI learning and result in unsatisfactory model outcomes. In this study, we present a weakly supervised Cascade R-CNN (W-CRCNN) model to automatically detect HER2 overexpression in HER2 DISH and FISH images acquired from clinical breast cancer samples. The experimental results demonstrate that the proposed W-CRCNN achieves excellent results in identification of HER2 amplification in three datasets, including two DISH datasets and a FISH dataset. For the FISH dataset, the proposed W-CRCNN achieves an accuracy of 0.970±0.022, precision of 0.974±0.028, recall of 0.917±0.065, F1-score of 0.943±0.042 and Jaccard Index of 0.899±0.073. For DISH datasets, the proposed W-CRCNN achieves an accuracy of 0.971±0.024, precision of 0.969±0.015, recall of 0.925±0.020, F1-score of 0.947±0.036 and Jaccard Index of 0.884±0.103 for dataset 1, and an accuracy of 0.978±0.011, precision of 0.975±0.011, recall of 0.918±0.038, F1-score of 0.946±0.030 and Jaccard Index of 0.884±0.052 for dataset 2, respectively. In comparison with the benchmark methods, the proposed W-CRCNN significantly outperforms all the benchmark approaches in identification of HER2 overexpression in FISH and DISH datasets (p<0.05). With the high degree of accuracy, precision and recall , the results show that the proposed method in DISH analysis for assessment of HER2 overexpression in breast cancer patients has significant potential to assist precision medicine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

HER2 (ERBB2) overexpression and/or HER2 gene amplification has been well established in several tumors types and when present HER2 directed therapy may be to be efficacious. While recent findings suggests that HER2 overexpression and HER2 amplification are a relatively common in serous endometrial carcinoma, similar data regarding clear cell endometrial carcinoma (CCC) is difficult to interpret due to issues such as diagnostic criteria, sample type and HER2 interpretation criteria. Our goals were to study HER2 expression and HER2 copy number status in hysterectomy specimens from a large series of patients with pure CCC to determine the frequency of HER2 overexpression and HER2 amplification and evaluate applicability of current HER2 interpretation criteria. Pure CCC specimens derived from hysterectomy specimens from 26 patients were identified. All diagnoses were confirmed by two gynecologic pathologists. Immunohistochemistry for HER2 protein and fluorescence in situ hybridization (FISH) studies for HER2 were performed on whole-slide sections from all cases. Results were interpreted according to the 2018 ASO/CAP HER2 guidelines for breast cancer and International Society of Gynecologic Pathologists (ISGyP) HER2 guidelines for serous endometrial carcinoma. Additional testing was performed when indicated by the guidelines. HER2 expression by immunohistochemistry was 3+ in 4% and 0% of cases, and 2+ in 46% and 52% of cases, by 2018 ASCO/CAP and ISGyP criteria, respectively, while the remaining cases were negative. HER2 testing by FISH showed a positive result in 27% of tumors with 2018 ASCO/CAP guidelines, while 23% were positive with the ISGyP criteria. Our findings indicate that HER2 overexpression and HER2 amplification occur in a subset of CCC. Therefore, additional study into the potential benefit of HER2 targeted therapy in patients with CCC is warranted., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Human epidermal growth factor receptor 2 (HER2) gene amplification helps identify breast cancer patients who may respond to targeted anti-HER2 therapy. This study aims to develop an automated method for quantifying HER2 fluorescence in situ hybridization (FISH) signals and improve the working efficiency of pathologists. An Aitrox artificial intelligence (AI) model based on deep learning was constructed, and a comparison between the AI model and traditional manual counting was performed. In total, 918 FISH images from 320 consecutive invasive breast cancers were analysed and automatically classified into 5 groups according to the 2018 ASCO/CAP guidelines. The overall classification accuracy was 85.33% (157/184) with a mean average precision of 0.735. In Group 5, the most common group, the consistency was as high as 95.90% (117/122), while the consistency was low in the other groups due to the limited number of cases. The causes of this inconsistency, including clustered HER2 signals, coarse CEP17 signals and some section quality problems, were analysed. The developed AI model is a reliable tool for evaluating HER2 amplification statuses, especially for breast cancer in Group 5; additional cases from multiple centres could further improve the accuracy achieved for other groups., (© 2023. The Author(s).)

Background: Breast cancer with fluorescence in situ hybridization (FISH) group 2 pattern (HER2 <4 and HER2/CEP17 ratio ≥2, a subset of monosomy CEP17) was historically considered HER2-positive, but mostly HER2-negative according to updated 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines unless 3+ by immunohistochemistry (IHC). Therapeutic relevance of this group remained elusive, therefore we assessed if repeat IHC and FISH can assist final HER2 classification., Patient and Methods: We retrospectively reviewed HER2 FISH performed at our institution from 2014 to 2018 and identified 23 of 3554 (0.6%) breast cancer cases with at least one-time measurement of HER2 FISH categorized as group 2. Repeat HER2 tests were performed for cases with available alternative tumor samples and compared with initial testing following 2018 ASCO/CAP guidelines., Results: Only 1 of 23 group 2 cases was HER2-positive, 0/18 in primary and 1/5 in metastatic/recurrent tumors. Of 13 primary tumors with repeat HER2 results; 10 (77%) remained HER2-negative; 3 (23%) changed from HER2-negative (group 2 and IHC 2+) to HER2-positive (group 1 and IHC 2+). Among 8 of these 13 patients receiving neoadjuvant systemic therapy containing anti-HER2 agent, 3 (38%) achieved pathologic complete response (pCR). Two of 3 pCR cases were HER2-positive converters on repeat testing. Three pCR cases were ER-negative or -low positive and Ki67 ≥40%, while 5 partial responders were ER-positive and Ki67 <40% (P < .05)., Conclusion: Breast cancer with HER2 FISH group 2 result may represent heterogeneous populations of tumor cells being originated de novo or preferentially selected secondary to therapy. Repeat HER2 tests on alternative samples may be considered to guide anti-HER2 therapy., Competing Interests: Disclosure All authors declare they have no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The last UK breast cancer (BC) human epidermal growth factor receptor 2 (HER2) testing guideline recommendations were published in 2015. Since then, new data and therapeutic strategies have emerged. The American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) published a focused update in 2018 that reclassified in situ hybridisation (ISH) Group 2 (immunohistochemistry (IHC) score 2+and HER 2/chromosome enumeration probe 17 ( CEP 17) ratio ≥2.0 and HER 2 copy number <4.0 signals/cell), as well as addressed other concerns raised by previous guidelines. The present article further refines UK guidelines, with specific attention to definitions of HER2 status focusing on eight key areas: (1) HER2 equivocal (IHC 2+) and assignment of the ASCO/CAP ISH group 2 tumours; (2) the definition of the group of BCs with low IHC scores for HER2 with emphasis on the distinction between IHC score 1+ (HER2-Low) from HER2 IHC score 0 (HER2 negative); (3) reporting cases showing HER2 heterogeneity; (4) HER2 testing in specific settings, including on cytological material; (5) repeat HER2 testing, (6) HER2 testing turnaround time targets; (7) the potential role of next generation sequencing and other diagnostic molecular assays for routine testing of HER2 status in BC and (8) use of image analysis to score HER2 IHC. The two tiered system of HER2 assessment remains unchanged, with first line IHC and then ISH limited to IHC equivocal cases (IHC score 2+) but emerging data on the relationship between IHC scores and levels of response to anti-HER2 therapy are considered. Here, we present the latest UK recommendations for HER2 status evaluation in BC, and where relevant, the differences from other published guidelines., Competing Interests: Competing interests: The authors attended meetings sponsored by Roche and AstraZeneca on HER2 low and HER2 positive breast cancer. No other relevant conflict of interests., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: T2 gallbladder cancer (GBC) is the only stage showing a survival benefit after complete surgical resection, but recurrence rates remain high. Although human epidermal growth factor receptor 2 (HER2) has emerged as a therapeutic target, its role in T2 GBC remains unclear. This study investigated the status and prognostic impact of HER2 expression on T2 GBC., Materials and Methods: HER2 expression and amplification were detected by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively, in 90 patients with T2 GBC who underwent radical cholecystectomy. We evaluated HER2 status according to the breast and gastric cancer guidelines and analyzed the effect of relevant prognostic factors on survival., Results: HER2 positive status was observed in 11.11% (10/90) and 8.89% (8/90) of cases based on gastric and breast cancer guidelines, respectively. Poor differentiation and a higher level of perineural invasion were independent prognostic factors of disease-free survival (DFS). Old age, male sex, presence of lymph node metastasis, poor differentiation, high levels of perineural invasion, and HER2 positivity based on breast cancer guidelines were identified as independent prognostic factors of overall survival (OS). Patients with HER2-positive T2 GBC according to breast cancer guidelines had worse OS., Conclusions: HER2 positivity based on breast- but not gastric-cancer guidelines was associated with poorer survival. These results provide a criterion for the evaluation of HER2 and a rationale for therapeutic strategies targeting HER2 in T2 GBC., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

The classification of human epidermal growth factor receptor 2 (HER2) expression is optimized to detect HER2-amplified breast cancer (BC). However, novel HER2-targeting agents are also effective for BCs with low levels of HER2. This raises the question whether the current guidelines for HER2 testing are sufficiently reproducible to identify HER2-low BC. The aim of this multicenter international study was to assess the interobserver agreement of specific HER2 immunohistochemistry scores in cases with negative HER2 results (0, 1+, or 2+/in situ hybridization negative) according to the current American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. Furthermore, we evaluated whether the agreement improved by redefining immunohistochemistry (IHC) scoring criteria or by adding fluorescent in situ hybridization (FISH). We conducted a 2-round study of 105 nonamplified BCs. During the first assessment, 16 pathologists used the latest version of the ASCO/CAP guidelines. After a consensus meeting, the same pathologists scored the same digital slides using modified IHC scoring criteria based on the 2007 ASCO/CAP guidelines, and an extra "ultralow" category was added. Overall, the interobserver agreement was limited (4.7% of cases with 100% agreement) in the first round, but this was improved by clustering IHC categories. In the second round, the highest reproducibility was observed when comparing IHC 0 with the ultralow/1+/2+ grouped cluster (74.3% of cases with 100% agreement). The FISH results were not statistically different between HER2-0 and HER2-low cases, regardless of the IHC criteria used. In conclusion, our study suggests that the modified 2007 ASCO/CAP criteria were more reproducible in distinguishing HER2-0 from HER2-low cases than the 2018 ASCO/CAP criteria. However, the reproducibility was still moderate, which was not improved by adding FISH. This could lead to a suboptimal selection of patients eligible for novel HER2-targeting agents. If the threshold between HER2 IHC 0 and 1+ is to be clinically actionable, there is a need for clearer, more reproducible IHC definitions, training, and/or development of more accurate methods to detect this subtle difference in protein expression levels., (Copyright © 2022 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Secretory breast carcinoma (SBC) is a rare malignant breast neoplasm with distinct histological features, including solid, microcystic, tubular, and rarely papillary structures, traditionally characterized by a t (12;15) (p13:q25) translocation, which usually leads to ETV6-NTRK3 fusion, suggesting an early event in tumorigenesis. Due to the rarity of this disease, very few genome sequencing studies have been performed on a series of cases, especially progressive cases., Methods: Seven lesions from 5 patients diagnosed at the Third Affiliated Hospital of Soochow University from 2007 to 2021 were included. Clinicopathological features and prognosis/survival data were collected. Next-generation DNA sequencing was performed on six of the seven lesions., Results: In total, 3/7 (42.9%) lesions demonstrated estrogen receptor (ER) expression, including weak, moderate to strong staining, and no lesion demonstrated progesterone receptor (PR) expression. There were no cases of human epidermal growth factor (HER2) overexpression, and the Ki-67 index was low. S-100 and pan-TRK protein were diffusely positively expressed in all cases. All lesions were characterized by a t(12;15) (p13:q25) translocation, leading to ETV6-NTRK3 fusion confirmed by fluorescence in situ hybridization (FISH). The sequencing results showed that ETV6-NTRK3 fusion was the main driver of early tumorigenesis, while SBC with invasive biological behavior had more complex genomic variation in which TERT promoter mutation was detected., Conclusions: Immunohistochemical staining of a biomarker panel, including ER, PR, HER2, Ki-67, S-100 and pan-TRK, can be used as an auxiliary diagnostic tool, and FISH detection can be used as a diagnostic tool. ETV6-NTRK3 gene fusion involving multiple sites may drive tumorigenesis, while mutations in the TERT promoter region may be a factor driving tumor progression., (© 2022. The Author(s).)

Performance of the new CE-IVD-marked HercepTest™ mAb pharmDx (Dako Omnis) assay (HercepTest (mAb)) was compared against the PATHWAY® anti-HER-2/neu (4B5) (PATHWAY 4B5) assay using 119 pre-selected breast cancer samples covering the entire range of HER2 immunohistochemistry (IHC) expression scores (0, 1 + , 2 + , 3 +). The sensitivity and specificity of both assays were assessed based on consensus IHC scores and amplification status, as determined by fluorescence in situ hybridization (FISH) according to 2018 ASCO/CAP testing guidelines. There was a high concordance between results from the HercepTest (mAb) and PATHWAY 4B5 assays for HER2-negative (IHC 0, 1 + , 2 + and FISH negative) and HER2-positive (IHC 3 + , 2 + and FISH positive) breast carcinomas (98.2%). Regarding individual IHC scores, complete agreement was achieved in 69.7% (83/119) of cases, and all but one of the discordant cases were due to higher HER2-status scoring using the HercepTest (mAb). Thus, more tumors were overscored as IHC 2 + by HercepTest (mAb) (27 versus 15) as evidenced by their lower FISH positivity rate (48.1% versus 80%). However, two amplified tumors identified as IHC 2 + by HercepTest (mAb) were missed by PATHWAY 4B5 (IHC 1 +). Four additional cases identified as IHC 2 + by HercepTest (mAb), with FISH ratio < 2 but elevated gene counts (≥ 4 to < 6), were recorded negative by PATHWAY 4B5. The HercepTest (mAb) detects HER2 expression with higher sensitivity in tumors with gene amplification (ISH group 1) and increased gene counts (ISH group 4) as well as in HER2-low tumors (HER2 IHC2 + /FISH negative or IHC 1 +). Future studies will demonstrate whether this translates into improved patient selection especially for new HER2-directed therapies., (© 2022. The Author(s).)

Invasive breast carcinomas are routinely tested for HER2 using immunohistochemistry (IHC), with reflex in situ hybridization (ISH) for those scored as equivocal (2+). ISH testing is expensive, time-consuming, and not universally available. In this study, we trained a deep learning algorithm to directly predict HER2 gene amplification status from HER2 2+ IHC slides. Data included 115 consecutive cases of invasive breast carcinoma scored as 2+ by IHC that had follow-up HER2 ISH testing. An external validation data set was created from 36 HER2 IHC slides prepared at an outside institution. All internal IHC slides were digitized and divided into training (80%), and test (20%) sets with 5-fold cross-validation. Small patches (256×256 pixels) were randomly extracted and used to train convolutional neural networks with EfficientNet B0 architecture using a transfer learning approach. Predictions for slides in the test set were made on individual patches, and these predictions were aggregated to generate an overall prediction for each slide. This resulted in a receiver operating characteristic area under the curve of 0.83 with an overall accuracy of 79% (sensitivity=0.70, specificity=0.82). Analysis of external validation slides resulted in a receiver operating characteristic area under the curve of 0.79 with an overall accuracy of 81% (sensitivity=0.50, specificity=0.82). Although the sensitivity and specificity are not high enough to negate the need for reflexive ISH testing entirely, this approach may be useful for triaging cases more likely to be HER2 positive and initiating treatment planning in centers where HER2 ISH testing is not readily available., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background and Aims: Chromosome 17 alterations affect the assessment of HER2 gene amplification in breast cancer (BC), but its clinical significance remains unclear. This study aimed to identify the prevalence of centromere enumeration probe 17 (CEP17) alterations, and its correlation with response to neoadjuvant therapy (NAT) in BC patients with human epidermal growth factor receptor 2 (HER2) immunohistochemistry-equivocal score., Methods and Results: A large BC cohort (n = 6049) with HER2 immunohistochemistry score 2+ and florescent in-situ hybridisation (FISH) results was included to assess the prevalence of CEP17 alterations. Another cohort (n = 885) with available clinicopathological data was used to evaluate the effect of CEP17 in the setting of NAT. HER2-amplified tumours with monosomy 17 (CEP17 copy number < 1.5 per nucleus), normal 17 (CEP17 1.5-< 3.0) and polysomy 17 (CEP17 ≥ 3.0) were observed in 16, 59 and 25%, respectively, compared with 3, 74 and 23%, respectively, in HER2-non-amplified tumours. There was no significant relationship between CEP17 alterations and pathological complete response (pCR) rate in both HER2-amplified and HER2-non-amplified tumours. The independent predictors of pCR were oestrogen (ER) negativity in HER2-amplified tumours [ER negative versus positive; odds ratio (OR) = 11.80; 95% confidence interval (CI) = 1.37-102.00; P = 0.02], and histological grade 3 in HER2 non-amplified tumours (3 versus 1, 2; OR = 5.54; 95% CI = 1.61-19.00; P = 0.007)., Conclusion: The impacts of CEP17 alterations are not as strong as those of HER2/CEP17 ratio and HER2 copy number. The hormonal receptors status and tumour histological grade are more useful to identify BC patients with a HER2 immunohistochemistry-equivocal score who would benefit from NAT., (© 2022 The Authors. Histopathology published by John Wiley & Sons Ltd.)

BACKGROUND Secretory breast carcinoma (SBC), an extremely rare malignancy, is related to a chromosomal translocation which leads to an ETV6-NTRK3 fusion mutation. SBC is characterized by eosinophilic secretions and is usually triple-negative, with a small number of patients demonstrating ER-positivity of the tumors. Diagnosis can be challenging and requires genomic testing for confirmation. CASE REPORT A 40-year-old woman presented with a breast mass found on mammography. She underwent an ultrasound-guided biopsy of the tumor. Initial pathology evaluation revealed features consistent with invasive ductal carcinoma. The immunochemistry report described an ER-positive, PR-negative, and HER2-negative tumor. The specimen was sent for oncotype scoring, which was not performed due to the specimen not meeting the criteria for invasive ductal carcinoma and displaying pathological features of SBC. A fluorescent in situ hybridization (FISH) study revealed ETV6 translocation, consistent with the diagnosis of SBC. The patient underwent lumpectomy followed by adjuvant radiotherapy and endocrine therapy. She remains in complete remission 3 years after treatment. CONCLUSIONS Accurately diagnosing SBC is of extreme importance as it has an indolent clinical course, but has a favorable prognosis if detected early. Due to nonspecific imaging findings, pathology evaluation with immunohistochemical staining followed by genomic testing is required. Our case highlights the challenges associated with SBC diagnosis requiring genomic testing due to equivocal pathological findings, along with increasing incidence of SBT in adults. There are no established guidelines for SBC management. The mainstay of treatment is partial or total mastectomy. Data on the benefits of adjuvant endocrine therapy, chemotherapy, and radiotherapy are inconclusive.

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) GH correlated 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 15 cases (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 6 patients (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., (Copyright © 2022. Published by Elsevier Inc.)

Novel human epidermal growth factor receptor 2 (HER2)-directed antibody-drug conjugates have demonstrated efficacy in HER2-low expressing breast cancers, which are currently defined as those with immunohistochemistry (IHC) scores of 1+ or 2+ with a negative in situ hybridization assay. However, current HER2 testing methods are designed to identify HER2-amplified tumors with high expression levels. The true definition of HER2-low expressing breast cancers remains controversial. Using quantitative molecular analysis of breast cancers based on RNA expression, the dynamic range of HER2 expression exceeds that detected by in situ IHC approaches. Erb-B2 receptor tyrosine kinase 2 (ERBB2) mRNA expression levels across IHC groups using patient samples derived from the Tamoxifen Exemestane Adjuvant Multicenter Trial were investigated. The standardized mean differences in ERBB2 mRNA scores in log base 2 are 0.47 (95% CI, 0.36-0.57), 0.58 (95% CI, 0.26-0.70), and 0.32 (95% CI, -0.12 to 0.75) when comparing IHC 0+ without staining versus IHC 0+ with some staining, IHC 0+ with some staining versus IHC 1+, and IHC 1+ versus IHC 2+/fluorescence in situ hybridization-negative, respectively. The results showed immunohistochemical methods have a comparatively limited dynamic range for measuring HER2 protein expression. The range of expression based on RNA abundance suggests a molecular method defining HER2-low cancers may better serve the treatment decision needs of this group. Indeed, the validity of RNA abundance to identify HER2-low cancers and predict treatment response needs to be further evaluated by prospective clinical trials., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Context.—: 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., Objective.—: To evaluate the characteristics of HER2 amplification in ESC in the context of breast and gastric HER2 fluorescence in situ hybridization (FISH) guidelines., Design.—: 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., Results.—: 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., Conclusions.—: 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., (© 2022 College of American Pathologists.)

Context.—: 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+., Objective.—: To assess the clinical, pathologic, and treatment aspects of patients with ISH Group 2 results., Design.—: 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., 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)., Conclusions.—: 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.

Background: In breast cancer (BC), overexpression of HER2 on the primary tumor (PT) is determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) to stratify samples as negative, equivocal and positive to identify patients (pts) for anti-HER2 therapy. CAP/ASCO guidelines recommend FISH for analyzing HER2/neu (ERBB2) gene amplification and for resolving equivocal HER2 IHC results. However, pre-analytical and analytical aspects are often confounded by sample related limitations and tumor heterogeneity and HER2 expression may differ between the PT and circulating tumor cells (CTCs), the precursors of metastasis. We used a validation cohort of BC patients to establish a new DEPArray™-PT-HER2-FISH workflow for further application in a development cohort, characterized as PT-HER2-negative but CTC-HER2/neu-positive, to identify patients with PT-HER2 amplified cells not detected by routine pathology., Methods: 50 µm FFPE tumor curls from the validation cohort (n = 49) and the development cohort (n = 25) underwent cutting, deparaffinization and antigen retrieval followed by dissociation into a single-cell suspension. After staining for cytokeratin, vimentin, DAPI and separation via DEPArray™, single cells were processed for HER2-FISH analysis to assess the number of chromosome 17 and HER2 loci signals for comparison, either with available IHC or conventional tissue section FISH. CTC-HER2/neu status was determined using the AdnaTest BreastCancer (QIAGEN, Hilden, Germany)., Results: Applying CAP/ASCO guidelines for HER2 evaluation of single PT cells, the comparison of routine pathology and DEPArray™-HER2-FISH analysis resulted in a concordance rate of 81.6% (40/49 pts) in the validation cohort and 84% (21/25 pts) in the development cohort, respectively. In the latter one, 4/25 patients had single HER2-positive tumor cells with 2/25 BC patients proven to be HER2-positive, despite being HER2-negative in routine pathology. The two other patients showed an equivocal HER2 status in the DEPArray™-HER2-FISH workflow but a negative result in routine pathology. Whereas all four patients with discordant HER2 results had already died, 17/21 patients with concordant HER2 results are still alive., Conclusions: The DEPArray™ system allows pure tumor cell recovery for subsequent HER2/neu FISH analysis and is highly concordant with conventional pathology. For PT-HER2-negative patients, harboring HER2/neu-positive CTCs, this approach might allow caregivers to more effectively offer anti-HER2 treatment., (© 2022. The Author(s).)

The successful experiences of HER2 inhibitors in patients with HER2 ( +) breast cancer (BC) and advanced gastroesophageal adenocarcinoma (GEA) have encouraged us to continuously explore the HER2 status and its potential as a therapeutic target in primary mucinous ovarian carcinoma (mOC). Using 49 primary mOC samples, we compared the assay characteristics of HER2 status between both 2017 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) for GEA and 2018 ASCO/CAP for BC guideline recommendations. We demonstrated moderate to strong agreement between their HER2 IHC results (Weighted Kappa = 0.78) and perfect agreement between their HER2 FISH results (Kappa = 1.00). The overall concordance of non-equivocal HER2 IHC and HER2 FISH results was 97.56% (kappa = 0.93) by 2017 ASCO/CAP for GEA criteria and 100% (kappa = 1.00) by 2018 ASCO/CAP for BC criteria. The number (n = 8; 16.32%) of HER2 IHC equivocal (score + 2) by 2017 ASCO/CAP for GEA criteria was twofold higher than that (n = 4; 8.16%) by 2018 ASCO/CAP for BC criteria. Additionally, we identified one false-positive (FP) case (n = 1; 2.04%) that was HER2 IHC positive (score + 3), but HER2 FISH non-amplified result by the 2017 ASCO/CAP for GEA criteria. In conclusion, owing to the absence of FP/ FN and fewer equivocal cases of HER2 IHC, we recommend that the 2018 ASCO/CAP for BC are more appropriate than 2017 ASCO/CAP for GEA criteria in appraising the HER2 status in mOC and justifying the inclusion of eligible subjects for basket clinical trials of the newly developmental anti-HER2 treatments., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 (R 2  = 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., (© 2021. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Objectives: Recent clinical trials have demonstrated significant clinical benefits from novel therapeutic compounds in breast cancer patient with human epidermal growth factor receptor 2 (HER2) immunohistochemical (IHC) score of 1+ or 2+ and negative in situ hybridization (ISH) result. A new concept of "HER2-low" breast cancer has been proposed and applied in the recent and ongoing clinical trials. In this article, we review the literature on the topic of HER2-low breast cancer., Methods: A literature search in PubMed was performed using key words related to HER2-low breast cancer. Major relevant studies that were presented in international breast cancer conferences were also included., Results: HER2-low breast cancer is currently defined as breast cancer with HER2 IHC score of 1+ or 2+ and negative ISH result. It likely represents a group of tumors with significant biological heterogeneity. Reports of clinical activity using the next generation of HER2-targeting antibody-drug conjugates in HER2-low breast cancers suggest that some strategies of targeting HER2 might be effective in this patient population while raising considerable concerns over limitations in our current testing methodologies and our ability to accurately identify such patients., Conclusions: The promising efficacy of novel HER2-targeted therapy in advanced HER2-low breast cancers has raised the possibility for changing the clinical interpretation of HER2 status in breast cancer to include a HER2-low category; however, the definition of HER2-low breast cancer, the corresponding reliable and accurate quantitative HER2 testing methodology, and the biology of HER2-low breast cancer remain poorly defined., (© American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Human epidermal growth factor 2 (HER2) amplification and/or overexpression occurs in 12% to 25% of breast cancers. Accurate detection of HER2 is critical in predicting response to HER2-targeted therapy. Both immunohistochemistry (IHC) and in situ hybridization (ISH) are FDA-approved methods for detecting HER2 status because its protein overexpression is largely attributable to gene amplification. However, variable discordant results between IHC and ISH have been reported., Methods: We determined the frequency of HER2 IHC/ISH discordance in these patients and also performed a pooled literature review analysis., Results: Of the 1125 consecutive primary or metastatic breast cancers with HER2 IHC and ISH performed simultaneously between 2015 and 2020, 84.6% had an unequivocal HER2 status. Discordance was found in 30 cases from 26 patients, including 13 IHC - /ISH + and 17 IHC + /ISH - , representing 1.6% and 11.9% of IHC - and IHC + cases, respectively. Review of the literature between 2001 and 2020 identified 46 relevant studies, with a total of 43,468 cases with IHC and ISH performed. The IHC - /ISH+ and IHC + /ISH - discordances were seen in all antibody clones and ISH methods used. The IHC + /ISH - discordance was significantly higher than IHC - /ISH + (13.8% vs. 3%, P < .0001). The overall discordance constituted 4% of all cases and 5.4% of those with an unequivocal IHC status. Significantly lower incongruities for both IHC - /ISH + and IHC + /ISH - were found in those published after 2018. The discordances probably reflect altered biology of HER2 oncogene/oncoprotein. Routinely performing both IHC and ISH may uncover such cases to prevent denial of potentially beneficial targeted therapy., (Copyright © 2021. Published by Elsevier Inc.)

Molecular testing in breast cancer is a rapidly developing field that is becoming increasingly integral to patient care. This article provides an overview of currently available molecular assays and testing modalities that have prognostic, predictive, and therapeutic value. These include multigene assays for invasive breast cancer (Oncotype DX, MammaPrint, Prosigna, and Breast Cancer Index) and ductal carcinoma in situ (Oncotype DX DCIS and DCISionRT) and companion tests to detect PIK3CA mutations and NTRK fusions. The various assays related to immune checkpoint inhibitors, consisting of immunohistochemistry with anti-programmed death-ligand 1 antibodies SP142 and 22C3 and detection of microsatellite instability, mismatch repair deficiency, and tumor mutational burden are also discussed. Finally, the practical utility and hopeful promise of next-generation sequencing panels and circulating tumor (cell-free) DNA assays are evaluated. This review should serve as a useful and practical reference for practicing pathologists, molecular pathologists, clinicians, and researchers., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Bone is the most common site of metastasis in breast carcinoma (BC). Treatments for metastatic BC depend on various factors, including the tumor's estrogen receptor (ER), progesterone receptor (PR), and HER2 status. Bone biopsies require decalcification which may affect the accuracy of ER and PR immunohistochemistry (IHC) and HER2 situ hybridization (FISH) studies. Ethylenediaminetetraacetic acid (EDTA) decalcifying solutions have been theorized to have no significant impact on ER and PR IHC or HER2 FISH analyses. We completed a prospective study of the effect of EDTA decalcification on ER and PR IHC and HER2 FISH in 29 cases of BC. Samples from 29 BC resections were collected and formalin fixed between 12 and 24 h. Control samples were routinely processed, whereas test samples were placed in EDTA for 48 h. ER and PR slides were blinded, randomized, and evaluated. Blinded samples underwent HER2 FISH assays where an average HER2 copy number and HER2/CEP17 ratio were calculated. Paired differences between EDTA and control samples were compared for ER and PR positivity, average HER2 copy number, and HER2/CEP17 ratios using paired-samples t-tests (PST) and Wilcoxon signed-rank test (WSR). PST and WSR tests yielded no significant difference between EDTA and control tissue for ER% (PST: P = 1; WSR: P = 0.916), PR% (PST: P = 0.973; WSR: P = 0.984), HER2 copy number (PST: P = 0.124; WSR: P = 0.103), and HER2/CEP17 ratio (PST: P = 0.25; WSR: P = 0.105). The use of EDTA in bony tissue is therefore a valid decalcification method to ensure accurate assessment of ER and PR IHC and HER2 FISH in metastatic BC., (Copyright © 2021. Published by Elsevier Inc.)

Objectives: We investigated the impact of our laboratory's reflex testing process for resolving ERBB2 (HER2) status on breast cancer samples that require additional workup after fluorescence in situ hybridization (FISH), per guideline recommendations published in 2018 by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP)., Methods: In total, 500 breast cancer specimens with ERBB2 FISH results in groups 2 through 4 (all reported as immunohistochemistry [IHC] equivocal [2+] at external laboratories) were resubmitted for IHC testing in our laboratory. Per the ASCO/CAP guideline, FISH was rescored when internal IHC was also equivocal (2+), targeted to tumor areas demonstrating more intense IHC staining, if observed., Results: Reflex IHC/FISH testing changed the final reported ERBB2 status in 185 of 500 (37.0%) samples. Result changes included discordant IHC (n = 4 score 0, n = 132 score 1+, and n = 16 score 3+) and discordant FISH (n = 33). Numerical differences in FISH scores were comparable for targeted vs nontargeted FISH rescoring (P = .086 for ERBB2 copy number; P = .49 for ERBB2 ratio). Two cases showed larger differences in FISH scores, suggesting heterogeneity., Conclusions: Retesting of breast cancer samples with equivocal IHC frequently changes IHC results, but targeted reanalysis of borderline FISH results rarely identifies significant differences in ERBB2 copy number or ratio., (© American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The diagnostic accuracy of the multigene panel test (MPT) and OncoScan™ in the determination of HER2 amplification in breast tumors remains controversial. In the present study, HER2 copy number was analyzed using both MPT and OncoScan™ in 45 breast tumors and was compared with that in fluorescent in situ hybridization (FISH) analysis. Tumors with low cellularity were examined using tumor cell enrichment and fluorescence‑activated cell sorting. Both MPT and OncoScan™ exhibited significant correlations with FISH with respect to the determination of HER2 amplification in breast tumors. However, the correlation coefficient was significantly higher for the comparison of MPT and FISH (r=0.770) compared with that between OncoScan™ and FISH (r=0.564). The accuracy of MPT (93.3%) was slightly higher compared with that in OncoScan™ (84.4%) in determining the HER2 status, which was mostly explained by the higher sensitivity of MPT in tumors with low cellularity (83.3 vs. 33.3%), but not in those with high cellularity (81.8 vs. 72.7%). The specificity was 100% for both tests. The MPT exhibited higher sensitivity in the determination of the amplification of other genes, including MYC , fibroblast growth factor receptor 1 and GATA binding protein 3 in tumors with low cellularity compared with that in tumors with high cellularity. OncoScan™ exhibited low sensitivity without tumor cell enrichment. The results suggested that MPT could be a promising method to determine HER2 status in breast tumors and that it could exhibit improved accuracy compared with that in OncoScan™ in tumors with low cellularity.

There are contradictory data regarding the correlation between HER2 amplification level determined by in situ hybridization and evolution after treatment with anti-HER2 therapies. The aim of this study was to correlate quantitative results of FISH (ratio HER2/CEP17 and number of HER2 signals/nucleus) with pathological response achieved after neoadjuvant treatment with trastuzumab and chemotherapy. For this purpose, we analysed 100 consecutive HER2-positive cases of breast carcinoma treated with neoadjuvant therapy. HER2 amplification determined by FISH was found in 92 of the 100 cases studied. pCR was obtained in 58% of the patients whose tumours presented amplification. In contrast, no pCR was obtained in the 8 patients with non-amplified tumours. A significant direct correlation between HER2 high amplification (HER2/CEP17 ratio > 5 or HER2 signals/nucleus > 10) and pCR was found. In conclusion, HER2 amplification levels are clinically relevant because they provide oncologists with valuable information on the possibilities of achieving pCR after neoadjuvant treatment., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Decades of quality control efforts have raised the standards of immunohistochemistry (IHC), the principle method used for biomarker testing in breast cancer; however, computational pathology and reverse transcription quantitative PCR (RT-qPCR) may also hold promise for additional substantial improvements., Methods: Herein, we investigated discrepancies in the assessment of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and marker of proliferation Ki67 comparing routinely obtained IHC (and FISH) data (ORI) with the results of manual (REV) and semi-automated (DIA) re-evaluation of the original IHC slides and then with RNA expression data from the same tissue block using the MammaTyper® (MT) gene expression assay., Results: Correlation for ER and PR was high between ORI IHC and the other three study methods (REV, DIA and RT-qPCR). For HER2, 10 out of 96 discrepant cases can be detected between ORI and REV that involved at least one call in the equivocal category (except for one case). For Ki67, 22 (29.1%) cases were categorized differently by either REV alone (n = 17), DIA alone (n = 15) or both (n = 10) and 28 cases (29.2%) for RT-qPCR. Most of the discrepant Ki67 cases changed from low to high between the original and following assessment and belonged to the intermediate Ki67 expression range (between 9 and 30%)., Conclusions: Determination of the breast cancer biomarkers ER, PR, HER2 and Ki67 at the mRNA level shows high degree of correlation with IHC and compares well with correlations between original with subsequent independent manual or semi-automated IHC assessments. The use of methods with wider dynamic range and higher reproducibility such as RT-qPCR may offer more precise assessment of endocrine responsiveness, improve Ki67 standardization and help resolve HER2 cases that remain equivocal or ambiguous by IHC/FISH. In summary, our findings seem to configure RT-qPCR as a complementary method to be used in cases of either equivocal results or presenting, at the traditional determination assays, biomarkers expressions close to the cut-off values., Competing Interests: Kerstin Hartmann, Mark Laible, Ugur Sahin: Salary and stock ownership BioNTech Diagnostics GmbH / BioNTech AG. These commercial affiliations do not alter our adherence to PLOS ONE policies on sharing data and materials. Mark Laible: Patent ownership of WO 2015/024942, Commercialized as MammaTyper (TM) Kit. The other authors declare that they have no conflict of interest.

Uterine carcinosarcoma (UCS) is an uncommon and highly aggressive tumor. There is no HER2 testing protocol for UCS despite the development of HER2 antibody conjugates. We aimed to elucidate histopathological HER2 expression details in UCS, to compare HER2 scores between ASCO/CAP criteria for gastric and breast cancer, and to propose requirements for HER2 testing for UCS. Eighty-nine specimens from 84 patients with metastatic/recurrent UCS were prospectively collected from May 2018 to July 2020. We performed HER2 immunohistochemistry (IHC) for 89 specimens and FISH for 44 specimens. HER2 expression details and HER2 score were evaluated according to the latest ASCO/CAP criteria for gastric (2016) and breast cancer (2018). HER2 IHC scores according to the gastric cancer criteria were 0 in 31 cases (35%), 1+ in 26 (29%), 2+ in 22 (25%), and 3+ in 10 cases (11%) of the 89 specimens. A lateral/basolateral membranous staining pattern was observed in 28/32 (88%) specimens with HER2 scores of 2+/3+. HER2 intratumoral heterogeneity was identified in 28/32 (88%) of the specimens with HER2 scores of 2+/3+. The overall concordance rate of HER2 score was 70% between the gastric and breast criteria. FISH revealed HER2 gene amplification in 10/44 (23%) specimens containing only lateral/basolateral membranous staining pattern. Based on the histopathological features of HER2 expression in UCS, a scoring system that accepts lateral/basolateral staining patterns should be applied. Furthermore, we proposed specific requirements for UCS testing, including specimen selection, scoring system, and calculating the proportion of HER2-positive cells.