Standardized pathology report for HER2 testing in compliance with 2023 ASCO/CAP updates and 2023 ESMO consensus statements on HER2-low breast cancer

Accurate and reproducible HER2 testing strategies and techniques are crucial for selecting patients who can benefit from novel HER2-ADCs [62]. To minimize the occurrence of false-negative and false-positive results, careful management of preanalytical issues is essential for both biopsy and surgical specimens (Fig. 2) [27, 86]. Various factors, including fixation, antigen retrieval, antibody clones, enzymatic activity, reaction time, temperature, and substrate concentration, can influence HER2 staining intensity and ISH sensitivity [65, 75, 85]. It is recommended to minimize cold ischemia time, which refers to the duration from tissue excision to tissue fixation, ensuring it does not exceed 1 h [78]. After macroscopic examination, the tissue should be fixed in neutral buffered formalin for at least 6 h but less than 72 h [54, 78, 83]. All testing laboratories should employ validated IHC, brightfield ISH, or FISH assays. Discrepancies in the interpretation of IHC HER2 test results can arise due to different antibody clones and antigen retrieval methods. Therefore, the current ASCO/CAP guidelines strongly recommend that laboratories performing HER2 testing participate in regular laboratory inspections and proficiency testing [81]. According to the 2018 ASCO/CAP guidelines, IHC staining should be conducted on freshly cut 2–4-µm-thick sections from representative FFPE blocks. The staining methodology, particularly antigen retrieval, and the availability of diverse antibody clones and staining platforms with varying specificity (such as PATHWAY anti-HER-2/neu (4B5), Ventana Medical Systems, S.A., Illkirch, France and HercepTest™ pharmDx, DakoCytomation, Glostrup, Denmark), can significantly impact result reproducibility and complicate the identification of HER2-low expression [67, 85]. Studies comparing the polyclonal HercepTest and the monoclonal 4B5 have shown acceptable concordance between the two methods for detecting HER2-positive BC [45, 47]. However, recent data suggests that the PATHWAY 4B5 assay may be more sensitive in diagnosing HER2-low diseases [68]. The CE-IVD-marked HercepTest™ monoclonal Ab pharmDx kit (for the Dako Omnis platform) has recently become available and demonstrated the highest overall pass rate (100%) according to the 2021 NordiQC data [57]. In a study comparing the new HercepTest (mAb) with the 4B5 assay using 119 BC samples covering the full range of HER2 IHC expression levels, the former antibody was found to be more sensitive in detecting HER2-low tumors [57]. Regardless of the assay, repeating the test in case of equivocal results may help rule out possible technical issues. Establishing and using positive and negative controls is one of the most important aspects of HER2 quality control [6, 38, 86]. Recommended controls should include low and high expression controls, which are particularly critical for HER2-low detection [37, 73]. Several factors can contribute to false-positive IHC results for HER2-low, including edge artifacts (especially in core biopsies, where cells near the tissue edges may stain more intensely than in the center), cytoplasmic positivity (which may be misinterpreted as membrane staining), and overstaining (potentially due to an inappropriate high antibody concentration) [63]. Causes of false HER2-negative results include prolonged cold ischemia time, intra-tumor heterogeneity (especially in core biopsies), and under-staining (opposite of overstaining, potentially due to excessive antibody dilution). Attention to tissue controls can help reduce both false-negative and false-positive results. Pathologists, biologists, and laboratory technicians must be well-versed in the intricate world of preanalytical issues in predictive pathology [56].

With the advent of HER2-ADCs, the distinction between HER2 score 0 and score 1 + BCs has become clinically relevant, significantly impacting the routine of pathologists in biomarker testing. However, the currently employed HER2 assays were primarily designed to identify BC exhibiting HER2 overexpression, lacking specific validation for detecting HER2 low expression. A comprehensive assessment of inter-observer reproducibility for HER2-low status was conducted, encompassing a vast CAP survey involving over 1400 pathology laboratories, along with a Yale University study examining the concordance of 18 pathologists analyzing 170 BC biopsies [21]. The findings revealed a low scoring accuracy (26%) for HER2 IHC in the low expression range (score 0 vs. score 1 +), raising concerns about the potential misclassification of numerous patients for HER2-ADCs treatment in clinical practice. Notably, the level of agreement was significantly poorer compared to that observed between score 2 + and score 3 + . Due to inconsistent findings concerning the prognosis of HER2-low BC, a comprehensive retrospective cohort study was undertaken utilizing the National Cancer Database [55]. This study encompassed 1,136,016 US patients diagnosed with invasive BC between January 1, 2010, and December 31, 2019, who exhibited ERBB2-negative disease and had accessible immunohistochemistry results [55]. The investigation compared the characteristics of HER2-low and HER2-zero BC cases and revealed minimal prognostic disparities, implying that the effectiveness of HER2-directed therapies will play a crucial role in shaping outcomes, rather than inherent biological differences associated with low levels of HER2 expression. Nonetheless, encouraging outcomes surfaced from the preliminary results of a multinational, multicenter, non-interventional, retrospective study on HER2 retesting (NCT04807595) [77]. This study included 798 patients diagnosed with locally advanced or metastatic BC between 2015 and 2017, displaying canonical HER2-negative status (IHC score 0, 1 + , or 2 + without gene amplification). Following a web-based training session for pathologists involved in scoring low-end HER2 expression, who were blinded to historical HER2 scores, the original HER2 IHC-stained slides (mainly employing the Ventana 4B5 assay) were reassessed and reclassified as either HER2-low or HER2-zero. HER2 rescores were successfully obtained for 786 patients, demonstrating an 81.3% concordance between historical HER2 scores and rescores. It should be noted, however, that in the study conducted by Fernandez et al. [21], the participating pathologists were unaware that the interobserver agreement between the IHC score 0 and 1 + would be evaluated. In contrast, the observers in the NCT04807595 trial were fully aware of the investigation’s scope and, more importantly, the clinical implications of the new HER2-low category. The interpretation challenges are further complicated by the heterogeneity of HER2 expression [15, 22, 24, 43, 46]. This phenomenon is considered an independent risk factor for decreased disease-free survival and poses difficulties in selecting appropriate treatments for such patients [5, 54, 64]. In this context, the patterns of protein distribution hold significant relevance [30]. Geographical variations in HER2 staining within the same tumor, characterized by distinct patterns such as “clustered,” “mosaic,” and “scattered,” can significantly influence the identification of HER2-low BC, particularly in cases classified as HER2 “equivocal” [46, 84]. The concept of HER2 heterogeneity encompasses not only variations in HER2 expression levels but also the phenomenon of HER2 status switching and loss of HER2 expression, which can occur as a consequence of HER2-targeted therapy or following neoadjuvant treatment [23, 34, 49, 70]. In these settings, we encourage HER2 retesting [3, 10]. Recently, the change of HER2-low status from primary tumors to metastatic sites was investigated through a retrospective analysis of 554 BC [70]. Overall, HER2 discordance between primary and metastatic disease occurred in half of the cases [70]. Similarly, Miglietta et al., who assessed HER2 status in 547 patients with matched primary and recurrent samples, observed an overall HER2 discordance rate of 38%, primarily characterized by HER2-zero switching to HER2-low (15%) and HER2-low switching to HER2-zero (14%) [49]. These findings may indicate a genuine possibility that the conditions of the tissue, the test itself, or the interpretation of the test are not adequately sensitive in detecting low levels of HER2 protein expression. Considering the instability of HER2-low expression during disease progression, it is recommended to perform a biopsy of recurrent lesions if the primary tumor was previously scored as 0, whenever feasible from a clinical standpoint. Conversely, when conducting a biopsy of metastatic lesions and obtaining a score of 0, it is advisable to take into account the initial HER2 testing result of the primary tumor and reassess it if it was initially diagnosed as HER2-zero. Eligibility for T-DXd treatment is granted to patients if at least one tumor sample demonstrates HER2-low status, regardless of when the sample was obtained.

Timely release of a clear, concise, and comprehensive pathology report plays a vital role in facilitating clinical decision-making [26, 40]. To maintain quality assurance, strict adherence to standard operating procedures (SOPs) is imperative, outlining the diagnostic workflow from specimen excision to the final HER2 report [8, 14]. Current ASCO/CAP guidelines recommend the use of Food and Drug Administration (FDA)-approved IHC/ISH assays and periodic inspections of testing laboratories [80]. Efficient organization of sample storage and laboratory documentation is crucial to facilitate retesting if necessary [7, 12, 27, 37, 41, 49]. The quality of HER2 testing results and adherence to standard operating procedures rely directly on the laboratory personnel, encompassing technicians, molecular biologists, and pathologists [4, 31, 56]. Evaluation of HER2 status should align with the current ASCO/CAP guidelines, encompassing the IHC score ranging from 0 to 3 + , intensity and pattern of staining, and the percentage of positive cells with the highest staining pattern seen in > 10% of invasive tumor cells. Moreover, although not currently recommended for routine practice outside of clinical trials, providing the percentage (10% or less) of immunostained cells in samples with an IHC score of 0 could hold future value. This approach may prove beneficial for investigating the potential advantages of new ADCs in the context of HER2-ultra low BC (defined as a score of 0 with incomplete and faint staining in  >0 and ≤ 10% of tumor cells) in forthcoming studies [61]. Methodological information, including the primary antibody used (e.g., 4B5, HercepTest GE001), should be provided, along with a statement confirming adherence to the ASCO/CAP guidelines. The ISH test should indicate the positivity or negativity of the test, specify the number of observers and invasive tumor cell counts, and offer additional details regarding aneusomy, signal heterogeneity, and the percentage of cells with amplified HER2 signals [78]. In Fig. 3, we present a proposed optimal report for HER2 testing, tailored specifically for HER2-low BC. This report aims to provide a standardized framework that promotes consistent and informative documentation of HER2 testing results.