Identification of biology-based breast cancer types with distinct predictive and prognostic features: role of steroid hormone and HER2 receptor expression in patients treated with neoadjuvant anthracycline/taxane-based chemotherapy

Neoadjuvant chemotherapy or preoperative systemic therapy is increasingly considered for patients with operable breast cancer [1, 2] as survival rates are similar as in patients receiving standard post-operative chemotherapy and the rate of breast conserving surgery can be significantly increased in patients treated with neoadjuvant chemotherapy [3, 4]. One of the main aims of neoadjuvant chemotherapy is to achieve a pathological complete response (pCR; i.e. absence of malignant cells at the tumor site) because pCR has been found to be associated with longer disease-free and overall survival rates [2, 5‐7]. However, it is not clear if this predictive value is valid for all patients, as a small proportion of patients with pCR still experience distant relapse [8]. In general, pCR rates with classical chemotherapy are rather low and range from 10% to 26% depending on the applied regimes [9]. To date, only a few tumor markers exist for the prediction of pCR, e.g. low tumor differentiation and negative hormone receptor (HR) status [10, 11]. Therefore, reliable predictive and prognostic markers are needed for the optimal selection of patients who might benefit from a neoadjuvant chemotherapy, i.e. who have the chance to achieve a pCR and remain disease-free on the long term.

Studies investigating gene expression profiles in breast cancer have defined different breast cancer subclasses that were based on tumor biology-based characteristics [12‐15]. Luminal cancers were characterized by the expression of HR, the HER2 cluster showed an over-expression of HER2 and associated genes, and basal-like cancers were negative for HR and HER2 ("triple negative") and express basal cytokeratins as well as the proliferative cluster of genes [12]. Despite the fact that these biology-based tumor types are usually seen as different entities, in clinical practice there is a remarkable overlap between HR and HER2 positive cases. As data from preclinical models suggest an interaction between the HER2 and HR pathways [16, 17], we evaluated the hypothesis that these interactions might lead to a different clinical behavior of HR+/HER2+ co-expressing and HR+/HER2- tumors. This might be reflected in a different response to anthracycline/taxane-based neoadjuvant chemotherapy as well as in a different DFS. In addition, it has been suggested that the subgroup of HR-/HER2- (triple negative) carcinomas might constitute a mixture of different biologically and prognostically heterogeneous tumors [18, 19]. Therefore, we evaluated the hypothesis that a subclassification of these carcinomas might be possible using Ki-67 proliferation index, cytokeratin 5/6 (CK5/6), cyclooxygenase-2 (COX-2), as well as Y-box binding protein 1 (YB-1) expression, for the latter two a role in breast cancer progression has been demonstrated previously [20, 21]. We investigated our hypotheses in a cohort of pretherapeutic core biopsies from the neoadjuvant GeparDuo study, in which patients with operable breast cancer have been treated with either dose-dense doxorubicin plus docetaxel (ddADOC) or conventionally-dosed doxorubicin plus cyclophosphamide followed by docetaxel (AC-DOC) [22].

Our study demonstrates that breast cancer subclassification based on HR and HER2 expression as used in standard diagnostics bears potential for the prediction of a pCR in patients with operable breast cancer receiving neoadjuvant chemotherapy with anthracycline and taxane and has a prognostic impact. Interestingly, the coexpression of HER2 and HR was found to be relevant for prediction of therapy response as well as assessment of long term benefit. HR+/HER2+ co-expressing tumors had a high response rate and showed a favorable DFS similarly to HR+/HER2- tumors, which however, responded rarely. A particularly low response rate as well as a poor prognosis was seen for HR-/HER2+ breast cancers. Further on, HR-/HER2- tumors were linked to a higher pCR rate, yet relapsed significantly earlier if they did not achieve a pCR.

Even though the determination of HR and HER2 status is routinely performed in breast cancer diagnostics, the predictive and prognostic value of a classification based explicitly on HR/HER2 expression has been rarely analyzed and in part only been reported in supplemental data. Basically, these studies showed similar results to ours: Guarneri et al. found retrospectively in a cohort of 1,731 breast cancer patients treated with varying neoadjuvant anthracycline-based regimes that ER+/HER2+ carcinomas had a higher pCR rate than ER+/HER2- tumors (15.3% vs. 6%) [30]. The highest pCR rates in this study were observed in ER-/HER2+ (29%) and in ER-/HER2- carcinomas (22.4%). Five-year disease-free survival rate was only slightly lower in ER+/HER2+ than in ER+/HER2- tumors (66.3% vs. 60.2%) and was lowest in ER-/HER2+ tumors (43.7%) just like in our study. Similarly, in a retrospective study including 1,118 patients treated with various neoadjuvant chemotherapeutic regimes Liedke et al. found equally high pCR rates in ER+/HER2+ and ER-/HER2- carcinomas (21% vs. 22%; ER+/HER2-: 5%, ER-/HER2+: 31%). Three-year disease-free survival rate was similar in ER+/HER2+ and in ER+/HER2- carcinomas (70% vs. 73%) and was as low in ER-/HER2+ tumors as in ER-/HER2- tumors (61% and 63%) [31]. In a study setting similar to ours, Carey et al. showed in 107 patients treated with anthracycline-based neoadjuvant chemotherapy that in HR+/HER2+ tumors pCR rate was higher than in HR+/HER2- tumors, and DFS was worse in HR+/HER2+ co-expressing than in HR+/HER2- tumors, yet was still clearly better than in HR-/HER2- or HR-/HER2+ tumors [32]. Regarding these results and our data presented here, it is conceivable that patients with HR and HER2 co-expressing breast carcinomas might constitute a group that particularly benefits from neoadjuvant chemotherapy as demonstrated by high pCR rates and favorable survival times. This is in contrast to HR+/HER2- tumors that rarely respond but nevertheless show favorable survival rates. The results according to the response rate of HR-/HER2+ tumors are conflictive as the studies cited above found high pCR rates in this subgroup, in contrast to our results. Of note, neither in the Geparduo cohort nor in the cohorts described above trastuzumab had been included in the neoadjuvant therapy regime as this was no standard at the time of study execution. As newer studies using trastuzumab in patients with HER2+ tumors have shown almost doubled pCR rates, it will be compelling to elucidate whether the predictive and prognostic effects described above would be altered by the addition of anti-HER2 agents.

Some interesting facts about the molecular interaction of hormone receptors (particularly ER) and HER2 have been reported to date: estrogen generally downregulates HER2 expression [33, 34], a mechanism that does not seem to be relevant in HR/HER2 co-expressing carcinomas from hitherto unknown reasons [16]. Moreover, ER can activate HER2 by membrane non-genomic estrogen signaling, while HER2 activates ligand-independently ER by mitogen-activated protein kinase (MAPK)-/protein kinase B (AKT)-mediated phosphorylation [35]. These interactions have been supposed to be underlying the relative resistance and worse prognosis of breast cancers that co-express ER and HER2 and that have been treated with tamoxifen [36]. However, the situation in patients that are treated with primary chemotherapy is presumably quite different and there are no functional data that explicitly refer to this group of tumors. In the adjuvant setting HER2+ tumors respond well to an anthracycline-based therapy [37] and it is conceivable that this might be also the case in HR+/HER2+ tumors in the neoadjuvant setting as reflected by high pCR rates. For long-term prognosis the phenotype of HR positive tumors cells (higher differentiation, slower proliferation, etc.) seems to be more relevant in receptor co-expressing tumors and may even be amplified by HER2-mediated ER activation. Yet, the exact mechanisms remain to be elucidated in future functional studies.

The other biology-based tumor type for which we found a particular behavior as to response and survival is the HR-/HER2- (triple negative) subgroup. In line with previous reports in the neoadjuvant and adjuvant setting [30‐32, 38‐40], we observed a relatively short survival time in spite of a high response rate. Carey and Liedke explained this with the exceptionally poor prognosis of patients with HR-/HER2- tumors not achieving a pCR [31, 32]. We also saw a higher rate of disease-relapses in HR-/HER2- tumors without pCR than with pCR (42.1% vs. 20%). A predictive factor for neoadjuvant chemotherapy response might thus also be a reliable prognostic factor in the HR-/HER2- subtype. However, in our group highly proliferating tumors were more likely to respond, which may be explained by the fact that actively dividing cells are the target of cytotoxic drugs, but a high Ki67 labeling index was not linked to a better prognosis. Independent of the biology-based tumor type, Ki67 staining per se was an independent predictive but no prognostic factor in the whole study group, indicating that as to long-term survival proliferation is not as relevant as HR or HER2 expression. Yet, our findings support the concept that HR-/HER2- carcinomas are a heterogeneous groups of tumors which should be subdivided further [18, 19]. The expression of basal cytokeratins has been reported as one distinctive feature of the so-called basal-like carcinomas, a highly aggressive breast cancer subtype according to the concept of intrinsic breast cancer subtypes, which have been defined by gene expression analysis [12]. In our whole study group CK5/6 expression was a predictive factor, but within the HR-/HER2- group the addition of CK5/6 expression did not add predictive or prognostic information to the determination of ER, PgR, and HER2, which however might have been a sample size problem. The problem with the poor-prognosis HR-/HER2- tumors is that no specific targeted therapy exists to date, in contrast to endocrine therapy in HR+ and trastuzumab or lapatinib in HER2+ breast cancers. Our hypothesis of a special predictive and prognostic role of COX-2 or YB-1-expression (for both molecules targeted therapies are available or are in development) [41, 42] in HR-/HER2- carcinomas could not be proven in our study.

Certain limitations of our study should be stated: Due to the retrospective evaluation and the limited sample size it is primarily a hypothesis-generating study, and results remain to be investigated further in larger cohorts, preferentially in prospective trials [43]. However, the setting of a clinical trial ensures a clearly described population, homogenous treatment as well as well-documented and monitored data.

In summary, our results demonstrate that a breast cancer classification, simply based on the expression of the standard markers ER, PgR, and HER2 bears independent predictive and prognostic potential. Patients with HR-/HER2- tumors, particularly those without achievement of a pCR, are in need for further treatment options. Patients with HR-/HER+ tumors had an unfavorable prognosis, but can now be treated with anti-HER2 agents. In contrast, for HR+/HER2- carcinomas pCR was not relevant for prognosis, as DFS was long in spite of a low response rate. The HR+/HER2+ co-expressing carcinomas, so far insufficiently investigated, emerged as a group of tumors with a good response rate to neoadjuvant anthracycline/taxane chemotherapy and a favorable prognosis. This interesting group of tumors should be further investigated in prospective clinical trials and in functional studies.