Introduction to HER2 in Breast Cancer

The human epidermal growth factor receptor 2 (HER2, also known as ERBB2) gene was discovered by several research groups independently in the 1980s [1,2,3,4]. The role of HER2 in the pathogenesis of human cancer was first revealed in breast cancer [5] and later in ovary, gastric, uterus, and other organs [6, 7]. When HER2 is overexpressed, it will form homodimers or heterodimers by interacting with other HER family members, especially HER3 [8,9,10,11]. Upon the dimerization, the intrinsic kinase activity of HER2 is stimulated, followed by the activation of multiple intracellular signal cascades, and ultimately involves cell proliferation, survival, differentiation, angiogenesis, invasion, and metastasis [12, 13].

HER2 overexpression/amplification is present in ~ 15–20% of breast cancers and it has been recognized as an important prognostic and predictive biomarker. In 1987, Slamon et al. identified the relationship between HER2 and the prognosis in breast cancer patients [5], and thereafter, the literature has consistently demonstrated that HER2 overexpression/amplification in breast cancers is associated with a worse prognosis [14,15,16,17]. The predictive value of HER2 was first revealed in breast cancer with the observation that HER2 status appeared to be associated with sensitivity or resistance to some endocrine and chemotherapy agents [18,19,20]. More importantly, HER2 status in breast cancer patients is now recognized as a critical test for selecting which patients will be appropriate for HER2 targeted therapy. The first HER2-targeted agent, trastuzumab is a monoclonal antibody that binds to the extracellular domain of HER2, suppressing its signaling activity and inducing antibody-dependent cell-mediated cytotoxicity. Clinical trials have demonstrated that a combination of trastuzumab and chemotherapy increase disease-free and overall survival [21,22,23,24]. Trastuzumab was first approved for use in patients with metastatic HER2-positive breast disease in 1998, for the adjuvant treatment of lymph node-positive breast cancer in 2006, and for the adjuvant treatment of lymph node-negative breast cancer in 2008 [25]. Other newly developed HER2 targeted drugs including pertuzumab, trastuzumab emtansine (T-DM1), lapatinib, and neratinib have also been evaluated, either alone or as a combined anti-HER2 approach, and have demonstrated an even more favorable outcome [26,27,28]; however, these treatments are costly with potential serious side effects [29]. Since effective HER2 targeted therapy has only been established in breast cancer patients with HER2 amplification/overexpression, accurate testing for HER2 in breast cancer is critical, and the American Society of Clinical Oncology (ASCO)/College of American Pathology (CAP) developed HER2 testing guideline recommendations.

HER2 Testing Guidelines in Breast Cancer Evolving Over the Years

In 2000, CAP published a consensus statement regarding HER2 testing in breast cancer patients [30]. ASCO subsequently recommended that HER2 needed to be tested in all patients with invasive breast cancer and published the recommendations in 2001 [31]. In 2007, in order to improve the accuracy of HER2 testing through standardization, the two professional societies published the first joint guidelines on HER2 testing in breast cancer [32•]. Based on the extensive literature review and the results of clinical trials, the ASCO/CAP HER2 testing guidelines have evolved through the years with two updates, in 2013 and again in 2018, in the attempt to reliably identify patients who will benefit from HER2 targeted therapy [33•, 34•]. Table 1 summarizes the major changes in the ASCO/CAP HER2 testing guidelines between 2007 and 2018.

Table 1 Summary of the major changes in the ASCO/CAP HER2 testing guidelines between 2007 and 2018
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Main Points in the 2007 Guideline [32•]

The 2007 guidelines focused on the following two clinical questions: (1) what is the optimal testing algorithm for the assessment of HER2 status? And (2) what strategies can help ensure optimal performance, interpretation, and reporting of established assays? The guidelines recommended that HER2 status for all invasive breast cancer should be determined and proposed an algorithmic approach for the evaluation of HER2 by immunohistochemistry (IHC) and by in situ hybridization (ISH). It also specified that important issues be considered in testing protocols such as testing requirements, tissue handling, validation and QA procedures, and others. Importantly, it defined the positive, equivocal, and negative categories for HER2 by IHC and by ISH (Table 1).

Major Updates in the 2013 Updated Guideline [33•]

The 2013 ASCO/CAP update committee revised the defining criteria for HER2 categories and recommended that HER2 test must be performed in both all newly diagnosed breast cancer and metastatic disease if tissue sample is available. The fixation time was changed from 6–48 to 6–72 h. The cut off for positivity of HER2 by IHC was decreased from > 30% (2007 guidelines) to > 10% of invasive tumor cells with complete and intense staining by IHC. The HER2/CEP17 ratio for positive ISH result was changed from > 2.2 (2007 guidelines) to ≥ 2.0, with the creation of three positive ISH “groups.” The equivocal and negative categories were also revised accordingly (Table 1). In addition, the testing methodology and quality control were also emphasized.

Major Updates in the Current, 2018 Focused Update Guideline [34•]

The 2018 ASCO/CAP focused update guidelines addressed the following five clinical questions: (1) what is the most appropriate definition for IHC 2+ (IHC equivocal)? (2) Must HER2 testing be repeated on a surgical specimen if initially negative test on core biopsy? (3) Should invasive cancers with an HER2/CEP17 ratio of ≥ 2.0 but an average of HER2 copy number of < 4.0 signals per cell be considered ISH positive? (4) Should invasive cancers with an HER2/CEP17 ratio of < 2.0 but an average of HER2 copy number of ≥ 6.0 signals per cell be considered ISH positive? (5) What is the appropriate diagnostic workup for invasive cancers with an average HER2 copy number of ≥ 4.0 but < 6.0 signals per cell and an HER/CEP17 ratio of < 2.0 and initially deemed to have an equivocal HER2 ISH test result? The 2018 guidelines also re-defined the HER2 IHC equivocal cases as “weak to moderate complete membrane staining observed in > 10% of tumor cells or complete and circumferential membranous that is intense and within ≤ 10% of invasive tumor cells” and clarified the confusion in the 2013 guidelines that defined the equivocal cases as “circumferential membrane staining that is incomplete and/or weak/moderate.” The guidelines also addressed uncommon IHC staining patterns such as moderate to intense but incomplete (basolateral or lateral) staining, or circumferential, intense membrane staining in ≤ 10% of tumor cells (heterogeneous but limited in extent) and recommended that these staining patterns should be considered as equivocal as well. The 2018 guidelines revised the recommendation on repeating HER2 on excision if the prior breast biopsy was HER2 negative, by replacing the word “must” from the 2013 guidelines with “may” in the 2018 guidelines (Table 1).

One of the major updates in the 2018 focused update guidelines was the interpretation of the uncommon ISH HER2 groups 2 (monosomy), 3 (co-amplified/polysomy), and 4 (equivocal). The 2018 guidelines emphasized on coordination between IHC and ISH results and recommended that groups 2 and 3, previously positive by the 2013 recommendations, are no longer positive by default. The 2018 guidelines recommended that the definitive diagnosis for ISH HER2 groups 2 and 3 be rendered based on additional workup. An equivocal result (group 4) is no longer reported, but now rather clarified as positive or negative based on additional workup. Additionally, the 2018 guideline discontinued the option of using alternate probes for solving the group 4 equivocal status. This topic will be elaborated in more detail in the following section.

Applying the Current HER2 Testing Guideline in Breast Cancer

Specimens To Be Tested

The 2007 ASCO/CAP guidelines recommended that all primary breast cancer specimens and metastases should have at least one HER2 test performed [32•]. The majority of earlier studies demonstrated overwhelming consistency of HER2 status in the paired primary and metastatic breast cancers [35,36,37]. However, after the release of 2007 guideline, emerging data suggested that there is up to 42% discordance in HER2 results between primary and metastatic disease, likely due to testing methods, definition of metastatic disease, and the effect of HER2 targeted therapy [38,39,40]. As a result of this discordance, the 2013 and 2018 guidelines recommend that all newly diagnosed patients with breast cancer must have a HER2 test performed and patients who develop metastatic or recurrence disease must also perform HER2 testing in the metastatic site if the tissue sample is available [33•, 34•].

Tissue Handling, Fixation, and Section

Pre-analytical factors including tissue handling, fixation, and sectioning play a critical role in achieving consistent quality with accurate and reproducible result. The ASCO/CAP guidelines recommend that routinely processed formalin-fixed paraffin-embedded tissue and cell blocks should be used for all HER2 testing [32•, 33•, 34•]. Core biopsy or cytology material should be placed in 10% neutral buffered formalin as soon as possible. Resection specimens should be sliced at 5- to 10-mm intervals after appropriate gross inspection and margin designation and placed in a sufficient volume of 10% neutral buffered formalin within 1 h. The tissue fixation time in formalin for HER2 testing was changed to 6–72 h in the 2013 guidelines [32•] and remained in the 2018 guidelines [33•, 34•]. In addition, it should be noted that after 6 weeks post-tissue sectioning, these tissue sections should not be used for HER2 testing.

Strategies To Ensure Optimal Performance, Interpretation, and Reporting of Established Assays

One of the two main tasks in the 2007 guideline was to ensure that the laboratory had optimal performance, interpretation, and reporting of the established IHC and ISH assays for HER2 testing. These recommendations continued to be emphasized in the revised 2013 and 2018 guidelines. These guidelines provided detailed rejection criteria for IHC and ISH testing, optimal internal validation procedure and monitoring of test concordance between methods, optimal internal QA procedures and external proficiency assessments, as well as optimal laboratory accreditation. The guidelines strongly recommend validation of laboratory assays or modifications, strict adherence to laboratory accreditation standards, proficiency testing, and competency assessment. A recent publication reported that following the ASCO/CAP guidelines resulted in highly accurate and reproducible breast cancer biomarker results, including HER2 [41].

Testing in Central vs Local Laboratory

When evaluating HER2 expression, the issue of local versus central laboratory testing deserves discussion. The initial overall performance in HER2 testing performed by local laboratories was disappointing, with only 74% concordance in HER2 positivity between local and central laboratories [42]. Concordance between central and reference laboratories was found to be higher, suggesting that specifics related to local laboratories may contribute to the discordance between local and central HER2 testing [43].

When To Repeat HER2 Testing

Given the evidence of a high concordance rate (97–99%) for HER2 status between the core biopsy and the excision specimen [44•], the 2018 guidelines recommended that a HER2 negative result diagnosed on core biopsy may be repeated on the resection specimen under the following situations: (1) if the tumor is high grade, (2) if the sample is scarce on core biopsy, (3) if the resection specimen shows new areas of high-grade carcinoma not encountered on the original biopsy or, (4) if the core biopsy result is equivocal after both IHC and FISH testing. A HER2 test should be not ordered on the excision specimen if HER2 was negative on the biopsy specimen when both specimens show grade 1 ductal or lobular carcinoma with ER and PR positivity, tubular, mucinous, cribriform, or adenoid cystic carcinomas. Similarly, if the excision and biopsy specimens both have the same histopathologic findings and a positive initial HER2 result in grade 1 ductal or lobular carcinoma with ER and PR positivity, tubular, mucinous, cribriform, or adenoid cystic carcinoma, a new HER2 test should be ordered. Of note, rare low-grade carcinomas such as approximately 5% of classical lobular carcinoma can be HER2 positive [45]. Our recent findings also support continued HER2 evaluation for all patients with invasive carcinoma, regardless of ductal or lobular morphology [46].

Issues Regarding Groups 2, 3, and 4

In the 2013 guidelines [33•], the HER2 ISH categories were modified, creating five different breast cancer ISH groupings, using both the HER2/CEP17 (the centromere enumeration probe for chromosome 17) ratio and the average HER2 copy number per nucleus. Group 2 (monosomy) is defined as a HER2/CEP17 ratio of ≥ 2.0, with an average HER2 copy number of < 4.0 signals/cells. Group 2 result is due to amplification of the HER2 gene and an associated increased HER2 copy number, with a loss of chromosome 17 copy number, resulting in HER2-amplified monosomy of chromosome 17 [47]. In group 2 cases, the CEP17 copy number is so low that even with slight and potentially insignificant elevations of the HER2 copy number, the HER2/CEP17 ratio is ≥ 2.0. The group 2 cases account for approximately 0.4–3.7% of breast cancers [34•, 48•, 49, 50]. Group 3 (co-amplified/polysomy) is defined as a HER2/CEP17 ratio of < 2.0, with an average HER2 copy number ≥ 6.0 signals/cell. Group 3 findings are due to either polysomy of the HER2 gene or more commonly co-amplification of both the HER2 and CEP17 genes [49, 51,52,53]. In these cases, the HER2 copy number is significantly elevated but the CEP17 copy number is also elevated to such a degree that the HER2/CEP17 ratio is < 2.0. The incidence of polysomy/co-amplification is approximately 0.4–3.0% [34•, 48•, 49]. Both groups 2 and 3 were considered as positive in the 2013 guidelines. Group 4 is defined as a HER2/CEP17 ratio of < 2.0, with an average HER2 copy number of ≥ 4.0 and < 6 signals/cell. Group 4 was considered as equivocal (neither amplified nor not-amplified) in the 2013 guidelines. The incidence of group 4 is variable, ranging from 1.9 to 14.2% [34•, 48•, 54, 55, 56•]. Due to the rare incidence of group 2, 3, and 4 cases, and limited data regarding the benefit of HER2 targeted therapy in these three groups, they were very challenging cases for both pathologists and oncologists. In addition, in the 2013 guidelines, since the incidence of true polysomy 17 in breast cancer is rare and the increased CEP17 copy number often results from amplification or copy number gains in the centromeric or pericentromeric regions, the group 4 cases were recommended to be resolved as negative or positive by assessments with alternative, non-centromeric chromosome 17 control probes such as D17S122, Smith–Magenis syndrome (SMS), lissencephaly gene 1 (LIS1), topoisomerase-II-alpha gene (TOP2A), retinoic acid receptor α (RARA), and tumor protein p53 (TP53).

After the release of the 2013 guidelines, new evidence regarding the clinical outcomes of these three groups and whether patients benefited from HER2 targeted therapy has been published. Press et al. [48•] retrospectively analyzed 10,468 breast cancer patients from the Breast Cancer International Research Group (BCIRG)-005, -006, and -007 clinical trials and found that the majority of group 2 patients did not benefit from HER2 targeted therapy. An earlier study also reported that HER2 gene-amplified breast cancers with monosomy of chromosome 17 were poorly responsive to trastuzumab-based treatment [57]. Press et al. [48•] also reported that there was not enough data to determine the benefit of HER2 targeted therapy in group 3 patients and that the outcome of group 4 (equivocal) patients was similar to the outcome of group 5 (classic negative) patients. Furthermore, the 2013 guideline recommendation regarding using alternative chromosome 17 control probes to solve the ISH equivocal cases was also challenged. For example, a recent study showed that the indiscriminate use of alternative control probes to calculate HER2 FISH ratios in HER2-equivocal breast cancers may lead to false-positive interpretations of HER2 status resulting from unrecognized heterozygous deletions of these alternative control genomic sites and incorrect HER2 ratio determinations [58]. Sneige et al. also showed no survival disadvantage between ISH group 4 compared to HER2-negative patients and suggested that alternative chromosome 17 probes may erroneously upgrade the HER2 status, again due to unrecognized reduction in signal numbers resulting from heterozygous deletions [59]. The 2018 guidelines did not recommend using an alternative control probe to resolve ISH equivocal cases.

In the 2018 focused update guideline, the rare “non-classical” HER2 ISH group 2 and 3 cases are no longer considered positive by default, and the ISH group 4 category has been eliminated. Group 2, 3, or 4 cases should be reflexed back to IHC, using the same tissue samples used for ISH. If the reflex IHC is 0 or 1+, the case should be considered negative, and if the reflex IHC is 3+, then the case should be considered positive. If the reflex IHC is 2+, then an observer blinded to the previous results should recount at least 20 invasive tumor cells. If the recount shows a different ISH result, then the new result should be reviewed with reference to the 2018 guidelines and the result should be adjudicated per internal procedures to determine the final category. If the recount shows the same ISH group again, the interpretations among groups 2–4 are slightly different. For group 2 cases, the result should be reported as negative. For group 3 cases, the result should be reported as positive, with an explanatory comment. For group 4 cases, the result should be reported as negative, with an explanatory comment (Fig. 1 and Table 2).

Fig. 1
figure 1

The ISH workup of an IHC HER2 equivocal (2+) breast carcinoma case using a dual-signal assay

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Table 2 The template for fluorescence in situ hybridization and integrated HER2 status report for cases showing IHC 2+ at the University of Rochester Medical Center (URMC)
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The current 2018 focused update guidelines predominately impact the patients in ISH groups 2, 3, and 4. Gordian-Arroyo et al. found that the 2018 guidelines reclassified the HER2 status of 6% of patients, mostly from HER2 equivocal status to HER2 negative status, and that the HER2 positive rate decreased by 0.4% [60]. Two large Chinese studies also found that using the 2018 guidelines, the HER2 negative rate was increased by 8.1–13.3%, either from an originally equivocal or positive case and that 0.1% of cases were changed from equivocal to positive [61, 62]. Our unpublished data suggests that the 2018 guidelines resulted in a 2.2% decrease in ISH positive patients and an 11.2% increase in ISH-negative patients (unpublished observation).

Issues Not Included in the Current HER2 Testing Guideline

HER2 Testing in Bone Specimen

Bone is the most common site of breast cancer involvement and the bone specimen always requires decalcification. The current ASCO/CAP guidelines do not recommend a specific decalcification process for HER2 testing, although it states that samples with decalcification artifacts should be rejected and that samples that were decalcified in a strong acid solution may be rejected [34•]. Studies have shown that acidification of bone specimen may affect antigenicity of the tumor cells and DNA quality, which can alter the biomarker status of metastatic breast cancer. For example, Maclary et al. demonstrated that most HER2 equivocal cases became negative after 24 h of hydrochloric acid decalcification [63]. Darvishian et al. showed that after 1 h of decalcification in a rapid decalcifying reagent, HER2 IHC showed less intense and more sparse membranous staining with a mean drop of 1 score. Interestingly, FISH analysis on decalcified cases with scores of 3 and 1 by IHC failed to detect any signals [64]. A recent study compared three decalcification solutions including acetic acid, hydrochloric/formic acid, and EDTA and found that bone biopsy specimens can be reliably used for biomarker studies in breast cancer and that EDTA decalcification is the optional method since it minimally affects receptor expression results [65]. At the University of Rochester Medical Center, when a bone biopsy specimen from a patient with history of breast cancer is received, an in-house solution of EDTA with formalin is the preferred method for decalcification and fixation. We recommend that the fresh bone material should be in formalin for at least 1 h before placing in EDTA solution. The decalcification/fixation time depends on the size, thickness, and consistency of the sample, but should be between 6 and 72 h.

Whether to Re-test HER2 Status in the Neoadjuvant Setting

Neoadjuvant chemotherapy (NAC) has been increasingly used for breast cancer in recent years and published literature comparing pre- and post-NAC HER2 results have yielded conflicting information, ranging from no alteration to up to a 43% change. For example, several studies found NAC caused no significant modulation of HER2 status [66,67,68]. In contrast, most studies have demonstrated discordant rates of 4.7–43.0% for HER2 between before-NAC core biopsy and post-NAC resection [69,70,71,72,73,74,75,76,77,78,79]. Studies also found that loss of HER2 amplification in the residual disease was associated with a shorter recurrence-free survival or a higher risk of relapse [72, 74]. Although whether to perform HER2 re-testing on the residual disease after NAC has not been addressed in the ASCO/CAP guidelines, more studies suggest performing HER2 re-testing after NAC, since any significant changes in HER2 status may impact therapeutic management. Interestingly, a few studies have suggested that FISH analysis is preferred to IHC when re-testing, because FISH analysis is more stable than IHC [68, 73].

Challenges Remain for HER2 Testing

Despite the remarkable success realized from targeting HER2-overexpression in a subset of breast cancers over the past two decades, a number of challenges remain, and many of which are directly related to testing.

Quantitative Measurement of HER2

While the IHC and ISH methodologies for assessing HER2 in breast cancer have undergone extensive analytic and clinical validation over the years to demonstrate clinical utility, both of these methodologies have limitations [32•, 33•]. IHC utilizes chromogenic dyes that enable semi-quantitative measurement of HER2 receptor overexpression; however, this detection methodology has a limited dynamic range where the test is linear, restricting the capability to obtain accurate quantitative results [80]. Furthermore, the intensity of staining is dependent on the enzymatic activity of the detection system being employed, the reaction time, and temperature as well as substrate concentration, thus limiting the quantitative sensitivity of IHC [81]. A number of studies have established a correlation between the level of HER2 protein expression and breast cancer dependence on HER2 signaling, which in turn would be predictive of sensitivity to HER2-targeted therapy [82]. Given the limitation of IHC for accurate quantitative assessment of HER2 protein levels, more reliable methods to quantify HER2 protein expression have been proposed for more accurate prediction of therapeutic efficacy. The development of quantitative methodologies for the measurement of a predictive biomarker such as HER2 has in fact provided compelling evidence to suggest that this approach can improve the prediction of the response to HER2-targeted therapies in breast cancer [83, 84]. The measurement of HER2 protein by a selected reaction monitoring mass spectrometry has been shown to significantly correlate with both IHC and ISH, and these quantitative measurements were superior to IHC and ISH for predicting outcome after HER2-targeted therapy in the metastatic and adjuvant settings [85]. In addition, a number of studies have shown that compared to IHC and ISH, the quantitative measurement of HER2 protein, regardless of the methodology, is associated with a significantly higher pathologic complete response rate (a surrogate for patient outcome) following NAC that included HER2-targeted therapy [84, 86,87,88]. As with any other predictive biomarkers or companion diagnostics, these new methodologies will need to undergo rigorous analytic and clinical validation to demonstrate level-1 evidence of clinical utility before entering clinical practice [89].

Expansion of HER2 Indications and the Need for Testing in Other Solid Tumors

The remarkable clinical benefit achieved by targeting the HER2-pathway in HER2-positive breast cancer has generated intense interest in expanding the clinical indications for this treatment approach to other solid tumors [90]. With increased genomic profiling of many tumor types, there is an emerging recognition that HER2 gene amplification and activating mutations can occur in a number of different solid tumor types including lung, colon, bladder, salivary gland, ovary, and uterine serous carcinomas, as well as adenocarcinomas of the upper gastrointestinal tract [91,92,93,94]. Clinical trials, such as the MY PATHWAY basket trial have shown objective responses across a variety of solid tumors with HER2 overexpression and/or amplification in 30 of 114 treatment refractory patients (26%, 95% confidence interval 19–35%), with the highest response rates seen in patients with colorectal carcinoma [95]. The data from this and other trials support the notion that an activated HER2 pathway may be a potential therapeutic target in a number of different solid tumors and has led to a number of trials exploring the clinical efficacy of this approach. The Trastuzumab for Gastric cancer trial (ToGA) was the first phase III randomized, controlled study that showed a survival benefit for patients with metastatic HER2-positive gastric cancer, which led to the approval of trastuzumab for these patients in both the USA and Europe in 2010 [96]. Before the initiation of the ToGA trial, the investigators needed to validate which analytic method should be used for HER2 assessment in determining the most appropriate scoring criterion for HER2 analysis, so that an appropriate inclusion algorithm could be used [97]. This HER2 gastric validation study led to the development of a modified scoring system that was distinctive for gastric cancer, incorporating pathologic and biologic features unique to upper gastrointestinal tract tumors [97, 98]. Going forward, similar rigorous validation studies will need to be performed to help determine the most appropriate assessment methodology and HER2 scoring criterion for each new emerging HER2 indication.

Conclusion

Accurate assessment of HER2 status in breast cancer is critically important and clinically relevant. The ASCO/CAP current guidelines provide very valuable recommendations for the accurate evaluation of HER2 status, based on the results of clinical trials and an extensive literature review. Additional studies, especially those with long-term outcome data are needed to confirm the clinical validity of the 2018 ASCO/CAP guideline recommendations, in order to evaluate the clinical utility of the new criteria and to help with the development of new recommendations. The expansion of HER2 indications in other solid tumors also emphasizes the need for the most appropriate assessment methodology and HER2 scoring criterion for each new emerging HER2 indication.