The importance of HER2 as a prognostic marker in invasive breast cancer is well established [1
]. As such, it is critical to validate and standardize testing strategies to make an accurate assessment of HER2 status [3
]. The value of the current study becomes even more relevant with the provocative results from N9831 and NSABP-B31 demonstrating that in patients whose tumors were classified as HER2-normal based on central testing (although originally testing positive in local labs), there appears to be similar hazard ratios for benefit from adjuvant trastuzumab-based therapy when compared to chemotherapy alone [12
]. If patients exist who could benefit from this well-tolerated, effective therapeutic, but who may be misclassified by current HER2 testing (false-negatives), newer methods should be evaluated with attempts to determine better ways of identifying these patients. Similarly, we need to determine if there are patients receiving trastuzumab who are unlikely to benefit [9
]. The main objective of this study is to address critical aspects of HER2 testing through actual collaborative methodologic evaluation rather than a consensus review of literature published by independent groups [9
]. To accomplish this, we conducted a round-robin study among pathologists from three central laboratories utilizing blocks from two HER2-positive adjuvant trastuzumab trials (N9831 and BCIRG-006) and one HER2-normal trial (BCIRG-005) to evaluate current HER2 testing methods and their potential impact on clinical outcomes in tumors from annotated trials.
The pre-round robin discordance rate for HER2 status (both IHC and FISH) in these cases as tested among the three expert pathologists was 8 %. At adjudication, a ≥96 % agreement was observed among these same pathologists, suggesting that interpretation issues and/or HER2 tumor heterogeneity may play a significant role in discordant results. The overall concordance between the adjudicated IHC and FISH results was 92 %.
Similar to the results of an international HER2 proficiency group study performed between five central laboratories [21
], the majority of samples that could not be successfully adjudicated had IHC or FISH equivocal results as defined by the ASCO/CAP guidelines for HER2 positivity [9
]. Of the 14 IHC cases that could not be successfully adjudicated, 64 % were classified as 2+. Circumferential distribution and character (intensity, granularity) of cell staining, rather than quantity of stained cells, was the main reason for discordance among the round-robin pathologists, similar to what was reported in a recent HER2 proficiency testing study [22
]. The 12 non-adjudicated FISH cases had HER2:CEP17 FISH ratios with an average of 1.88 (range 1.13–2.45) and an average HER2 copy number of 4.67 per nucleus across pathologists, both of which are near the FDA-approved cut-offs of 2.0 for ratio and 4.0 for copy number.
Although the absolute HER2 counts were similar across the reading pathologists, small changes in the CEP17 counts (denominator) can and did significantly affect this ratio and changed amplification status of HER2 when using the HER2:CEP17 ratio. As a result, the interpretation of HER2 gene amplification differed in these cases. These data indicate that in equivocal cases, HER2 gene and CEP17 copy numbers should be assessed independently and may have important clinical implications [23
When the HER2 results are in the equivocal range, pathologists should consider consulting with a second pathologist to corroborate or possibly adjudicate the HER2 status. An accompanying explanation/interpretation of the HER2 status from the pathologist(s) is critical to help guide the clinician in making appropriate management decisions [4
]. The patient also needs to be informed of challenges associated with HER2 testing, particularly in cases near the FISH ratio of 2.0 as recommended by the FDA. A trend toward benefit from trastuzumab (adjusted HR = 0.34; p
-0.06) was observed in the small subset of N9831 patients (n
= 53) with disease deemed HER2-normal by central review and confirmed on the limited number of blocks in the round-robin (although initially called HER2-positive locally). While this observation is based on a very small number of events, we recognize that trends toward benefit are important to document, despite failing to reach statistical significance. An alternative and equally plausible explanation for the observation of benefit in HER2-normal cases could be the heterogeneity we observed in both HER2 protein overexpression and gene amplification during assessment of more than one block from the same patient. The current data indicate that 5/22 patients (23 %) that had locally tested as HER2-positive in N9831 and subsequently called IHC-negative/FISH-negative centrally, had a second block from the same primary tumor that we found to be HER2-positive in the round-robin analysis. Accordingly, testing an additional portion of the tumor from “HER2-normal” cases may be advisable to avoid the possibility of not treating patients who might benefit from HER2-targeted therapies. This phenomenon could account for some discrepancies between local and central HER2-testing in N9831 and NSABP B-31. One option to avoid this would be to consider testing a second section from an additional tumor block for each patient whose initial HER2 testing yields normal results.
The incidence of variable IHC staining was higher for overexpression (10 %) compared to heterogeneity of amplification status (5 %). Variable rates of intratumoral HER2 heterogeneity (<1–30 %) of unknown clinical significance have also been reported [15
]. In addition, we occasionally observed distinct intratumoral heterogeneity within the same tumor section (Fig. ). When distinct populations of cells exist in the same section, the HER2 status (by IHC and FISH) should be reported in accordance with established guidelines [9
Approximately 8–10 % of HER2-amplified breast cancers are falsely negative (IHC 0 or 1+) by IHC HER2 testing [14
], and given the high incidence of breast cancer in the US, this rate of false-negativity could negatively and critically impact the care of more than 5,000 patients each year [8
]. Based on the approximate 50 % reduction of relapse events when anti-HER2-targeted therapy is included in the adjuvant setting, thousands of women in the US each year may be experiencing relapses from failed adjuvant regimens that may not have occurred had these women been correctly identified as patients with HER2-positive disease and initially treated with trastuzumab- or lapatinib-based regimens. Improving the accuracy of HER2 testing and reducing the incidence of false-negative results can directly save lives [29