Discussion
Our investigation shows that in HR-positive tumors the response to neoadjuvant treatment with trastuzumab plus anthracyline-taxane chemotherapy is driven by the degree of HER2 mRNA expression. This phenomenon could not be observed in the HR-negative subset. Interestingly, Soon Paik's group has described a similar finding in the adjuvant setting. While their full paper is not published yet, a summary of their results has been included in the recent St Gallen recommendations as follows: 'An interesting STEPP analysis from the adjuvant trastuzumab NSABP B-31 trial examined the degree of HER2 mRNA expression and corresponding trastuzumab benefit separately for patients with estrogen receptor-positive and estrogen receptor-negative disease. The striking finding was that among patients with estrogen receptor-positive disease, trastuzumab benefit in terms of 8-year disease-free survival was entirely confined to those with the higher levels of HER2 mRNA expression' [
15].
Similar to these findings in the adjuvant setting, there is a considerable difference in our neoadjuvant study between ESR1-positive/HER2-positive and ESR1-negative/HER2-positive tumors. For ESR1-negative/HER2-positive tumors the amount of HER2 mRNA is not further relevant for response once a tumor is in the HER2-positive group. mRNA levels of HER2 have a dichotomous distribution and HER2 can be used as a categorical parameter in this group.
For ESR1-positive/HER2-positive tumors the situation is different; HER2 mRNA has a more continuous distribution and the response to neoadjuvant trastuzumab/chemotherapy rises continuously with the amount of HER2 mRNA within the HER2-positive tumor group. This suggests that those luminal tumors with higher activity of the HER2 pathway (measured as increased mRNA levels) are more dependent on this pathway and thus more responsive to trastuzumab targeted therapy. This finding is supported by the STEPP analysis and we observed the same effect with the classical approach of logistic regression, which also showed a significant effect of HER2 mRNA levels (measured as a continuous variable) on pCR only in the ESR1-positive/HER2-positive group. The traditional method of HER2 SISH ratio or copy number was not able to provide a similar result by STEPP or logistic regression analysis, similar to the finding in the adjuvant HERA trial [
27].
The relevance of crosstalk between the ER pathway and the HER2 pathway has been described in several
in vitro cell culture and animal models [
14,
28,
29],
Amplified in breast (
AIB)-
1 [
30,
31] as well as
Paired box gene 2 (
PAX2) [
32] have been identified as relevant mediators of this crosstalk.
The hypothesis derived from those investigations and our results would be that two important growth factor pathways significantly influence ESR1-positive/HER2-positive tumors and either HER2 or ER may be the driver of cell proliferation and survival. With sustained HER2 inhibition ER could function as a key escape or survival pathway, which may result in resistance to trastuzumab. However when HER2 mRNA expression is very high the primary driver of proliferation may still be the HER2 pathway even in the presence of the activated ER pathway. These findings are consistent with two neoadjuvant trials where significantly lower pCR rates were observed in ER-positive/HER-positive tumors compared to ER-negative/HER2-positive disease [
7,
8]. However, in a recently reported neoadjuvant trial, response rates to anti-HER2 treatment with lapatinib and trastuzumab (without chemotherapy) were fairly high (pCR 21%) when combined with endocrine treatment if HRs were present [
33]. As in the adjuvant setting trastuzumab or lapatinib therapy (in contrast to the neoadjuvant approach) is usually combined with endocrine therapy in the HR-positive group; the combined inhibition of both pathways is already used in clinical practice [
34,
35]. It would be interesting to further evaluate the contribution of the endocrine therapy to outcome in ESR1-positive/HER2-positive tumors.
Another finding of our study is the rather high rate of discordance of 27% between central and local evaluation of HER2. We have validated this finding by the use of different methods for central pathology. A similar rate of 20% inaccurate HER2 measurements has been reported before [
9] based on results of the NSABP [
36] and the N9831 study [
37,
38]. Discordance has also been observed between different reference laboratories, in particular for borderline cases [
39]. Interestingly, in our study the discordance was higher in ESR1-positive tumors, which might be partially due to the more continuous distribution of HER2 mRNA in this group. Pinhel
et al. [
40] have also observed this different distribution of HER2 mRNA in ER-positive vs -negative tumors in a recent report.
The high level of false-positive cases gave us the possibility to evaluate the response to trastuzumab in HER2-negative tumors. Centrally HER2-negative tumors had a pCR rate of only 20%, which is in the range of the pCR rate of HER2-negative tumors in the GeparTrio trial (17.6%). This validates previous findings that HER2 is the crucial biomarker for trastuzumab-based therapy. However, it differs from an analysis of the NSABP-B31 suggesting a benefit of adjuvant trastuzumab even in cHER2-negative tumors [
41]. There are two main differences between NSABP-B31 and our study: we used the neoadjuvant setting, which allowed us to directly study response in the primary tumor, but we could not evaluate the effects on micro-metastases as well as the contribution of adjuvant endocrine therapy. Furthermore, the rate of cHER2-negative cases in NSABP-B31 was only 9.7%, compared to 27% in our study. It would be very interesting to await the results of the NSABP B47 [
42], which is currently evaluating the benefit of trastuzumab in low HER2-expressing tumors.
As an additional method we have evaluated HER2 mRNA expression by qRT-PCR. Recently, Dabbs
et al. found that HER2 mRNA levels were negative by recurrence score in 10 (42%) of 24 HER2-positive cases [
43]. The same finding had already been shown in 638 samples from the NSABP-B31 with an overlap of HER2 mRNA expression levels between HER2-positive and -negative tumors [
40]. In our study 11 cases were negative for HER2 mRNA despite positivity by SISH, and those cases had a low pCR rate of only 27%. Furthermore seven tumors were mRNA-positive but SISH-negative with a pCR rate of 43%. Therefore, in our small cohort there is no evidence that patients with central IHC-positive results but negative HER2 mRNA have relevant benefit from trastuzumab with regard to pCR, which raises the hypothesis that HER2 mRNA might be more suitable for response prediction than SISH. We would like to emphasize that the low number of cases makes it impossible to fully evaluate this hypothesis in the context of our study, and that currently all indications for HER2-targeted therapies should be based on the established Food and Drug Administration (FDA) criteria.
The differences between HER2 mRNA and the classical methods of IHC and SISH might explain the finding that a different magnitude of benefit according to ER status has not been seen for trastuzumab in any of the adjuvant trastuzumab trials.
There are several limitations of our study; it was retrospective, the analysis could only be performed in a subpopulation, and we used only one pCR definition without separating the group of residual ductal carcinoma in-situ (DCIS). This separation was not possible in our cohort due to the smaller sample size. It should also be noted that some studies suggest that pCR might not be a reliable surrogate for long-term disease outcome in ER+/HER2+ disease [
44]. The advantages of the study are that we used a population from a prospective clinical trial with a standardized assay system, as well as a predefined hypothesis and analysis plan.
Conclusions
In summary, our results provide further evidence for the concept that HER2-positive/non-luminal and ESR1-positive/HERpositive tumors are different biological entities. Several randomized trials have shown that the benefit of adjuvant trastuzumab is significant in the cohort of HER2-positive tumors as well as in subgroups based on HR expression. It would be very interesting to evaluate HER2 mRNA levels in this context, since HER2 mRNA expression may select those ER-positive/HER2-positive tumors with an optimal benefit from trastuzumab. Another important issue would be to evaluate HER2 mRNA levels for response to different types of HER2 targeted agents, for example, lapatinib [
45,
46] or pertuzumab [
47]. Interestingly, a recent analysis in the NSABP B-41 trials has suggested differences between lapatinib, trastuzumab and their combination depending on the protein expression level of HER2 [
48]. Additional evaluations are planned in the GeparQuinto and the GeparSixto trials of the AGO B and the German Breast Group within the European FP7 project, RESPONSIFY.
Competing interests
CD and RK are shareholders of Sividon Diagnostics. CD has received research funding from Siemens Healthcare. CD and JH have received research funding from GSK. All other authors declare no competing interests.
Authors' contributions
CD, JH, SL, RK, GvM and MU have designed the study and participated in data acquisition, analysis and interpretation as well as manuscript writing. KM, SD-E, JCB, BVS and JP participated in the statistical analysis and data acquisition as well as manuscript writing. CS, PAF, BVS, KE, MR, M-LH and HT participated in the acquisition of data as well as writing and revision of the manuscript. All authors have read and approved the manuscript for publication.