Tamoxifen-predictive value of gene expression signatures in premenopausal breast cancer: data from the randomized SBII:2 trial

Although endocrine therapy with tamoxifen significantly reduces the risk of recurrence in patients with estrogen receptor-positive (ER+) breast cancer, breast cancer recurrence 20 years after diagnosis is not uncommon [1]. Moreover, some patients with ER+ tumors do not benefit from this treatment [2, 3]. Despite this, ER status is the only clinically established predictive marker for tamoxifen response [4], highlighting the need for new predictive tools. In patients treated with five years of adjuvant endocrine therapy, the risk of recurrence is strongly correlated with tumor size, nodal status, and histological grade [1]. Furthermore, the progesterone receptor (PR) has been observed to be prognostic [5], but its independent predictive effect on the response to endocrine therapy has not been established [6]. In recent decades, the clinical use of gene expression (GEX) analysis for prognostication has increased. In addition to providing information on intrinsic subtypes, GEX signatures have been observed to add putative predictive value [7‐11], even for late recurrences [12]. However, the use of risk scores in premenopausal patients is not widely implemented [11, 13].

In addition to routine markers, GEX may provide additional information for predicting the effects of breast cancer drugs [14‐16]. This was exemplified in the FinXX trial using the NanoString Breast Cancer (BC) 360™ panel (BC360 panel), where cytotoxic, endothelial, and Mast cell GEX signatures predicted improved recurrence-free survival, favoring the addition of capecitabine to adjuvant chemotherapy in patients with triple-negative breast cancer (TNBC) [15]. Previously, we demonstrated that PAM50 luminal subtypes are associated with the efficacy of adjuvant tamoxifen in premenopausal patients [9]; however, other gene signatures are currently not used in clinical practice to guide the use of endocrine therapy. The ESR1 gene encodes ER alpha (ERα, denoted as ER in this manuscript), and the GEX of ESR1 and protein expression of ER are strongly correlated [17]. Therefore, high ESR1 GEX levels could indicate responsiveness to tamoxifen therapy, as demonstrated by Chungyeul et al.; however, the same effect was not observed for PGR GEX [16]. Although GEX levels of the androgen receptor (AR) seem to be associated with better outcome [18], and AR overexpression has been reported to induce tamoxifen resistance in a preclinical setting [19], no clear endocrine-predictive effect has been observed [20].

Despite comprehensive studies on GEX signatures in relation to prognosis and prediction of treatment response in primary breast cancer, only a few have been used in the clinical setting. High proliferation scores including Oncotype DX, Prosigna gene assay, and hypoxic GEX signature have been associated with a worse prognosis [21‐24]. In contrast, high expression of the FOXA1 gene seems to be associated with better outcomes in patients with ER+ breast tumors [25, 26].

Previously, we reported the long-term effects of tamoxifen and prognostic value of PAM50 subtypes and the risk of recurrence (ROR) score based on the BC360 panel for premenopausal patients who were randomized between two years of adjuvant tamoxifen and no systemic treatment in the SBII:2pre trial [9]. The primary aim of the present study was to determine the tamoxifen-predictive value of GEX signatures from the BC360 Panel with respect to recurrence-free interval (RFi) and overall survival (OS) in patients with ER+/human epidermal growth factor receptor 2-negative (ER+/HER2 −) tumors. The secondary aim was to decipher the prognostic value of the signatures regardless of molecular subtype.

In the present study, the predictive value of GEX signatures for tamoxifen effect in premenopausal breast cancer patients with early ER+/HER2− tumors was explored. We observed associations between low expression of AR, FOXA1, and surprisingly, ESR1 and improved benefit of tamoxifen. Moreover, in the whole cohort, we found a prognostic effect for each of the GEX signatures BC proliferation, Hypoxia, Mast cells, and the GEX of AR, ESR1, and PGR, even after adjustment for established prognostic factors.

We have previously demonstrated that two years of adjuvant tamoxifen is effective for long-term breast cancer-related survival for patients with ER+ tumors from this trial [29], and that the effect of adjuvant tamoxifen therapy only seemed beneficial in patients with Luminal A tumors, as assessed by PAM50 [9]. ESR1 GEX positively correlated with ER and PR protein levels and the Luminal A subtype. Furthermore, high expression of the BC proliferation and Hypoxia GEX signatures was strongly correlated with high Ki67, high NHG and a Basal-like subtype. This was also reflected in the prognostic analyses, in which these signatures were associated with poor outcomes.

All selected 41 GEX signatures were included in exploratory predictive analyses. The GEX of AR is known to be associated with luminal subtypes and better outcomes [18, 40], and a similar prognostic effect of AR was noted in our study. Interestingly, our results indicate that a high AR GEX level is associated with a negative effect of tamoxifen after ten years, for both RFi and OS. However, no significant AR-by-treatment interactions were observed. Previous preclinical data suggest that AR overexpression might induce tamoxifen resistance; therefore, additional treatments such as AR inhibition may benefit these patients [19, 41]. However, data from clinical trials including patients with ER+ tumors that support the use of AR inhibitors are sparse. Additionally, the results of the study are expected to be influenced by the selection of patients with ER+ and HER2− tumors. However, the selection of patients with a defined phenotype makes clinical interpretation more relevant by reducing tumor heterogeneity in the cohort in which GEX signatures are evaluated.

In line with previous studies, we found that patients with high ESR1 GEX had better outcome [17]. Since ER protein expression is associated with a better response to endocrine therapy, and ESR1 GEX is positively correlated with ER status, an expression-dependent relationship between ESR1 expression and tamoxifen benefits [42] may be anticipated. In contrast to our results, a high ESR1 expression was a strong predictor of tamoxifen benefits in ER+ breast cancer in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 trial [16]. Kuske et al. stated that endocrine resistance to aromatase inhibitors can be linked to high ER expression and reduced ER phosphorylation [43], and other mechanisms of ER resistance have been proposed based on results in the metastatic setting, including mutations in ESR1 [44]. Although we observed PGR as strong prognostic factor in this cohort, no predictive effect of tamoxifen was found, as reported in the NSABP B-14 study [16].

FOXA1 plays a critical role in the regulation of ER function and may contribute to endocrine resistance in breast cancer [45‐47]. Clinically, FOXA1 protein expression has been associated with a luminal phenotype, including increased hormone receptor expression and improved outcomes [25, 26, 48, 49]. One study indicated that FOXA1 IHC staining decreased after neoadjuvant endocrine treatment, but the staining intensity (%) was not linked to treatment benefits [50]. To the best of our knowledge, no clear clinical evidence has been provided regarding the predictive effect of FOXA1 GEX in breast cancer. In this study, we showed that the benefit of tamoxifen decreased with increasing GEX of FOXA1, revealing a group of patients with ER+/HER2− tumors and low expression of FOXA1 who had an excellent response to tamoxifen treatment. In line with our results, previous studies have suggested that overexpression and mutation of FOXA1 could be underlying factors in endocrine resistance [46, 51]. In contrast to the observation that high FOXA1 reduces the benefit of tamoxifen in the ER+/HER2− subgroup, we observed high FOXA1 GEX to be prognostically favorable in the whole cohort, although no clear dose–response relationship was observed. A possible explanation for this may be the association between FOXA1 expression and luminal traits. In a subgroup analysis including only ER+/HER2− tumors, to mitigate this possible confounder, high FOXA1 GEX was a negative prognostic factor for both RFi (Additional file 4 g) and OS (Additional file 5 g). Interestingly, high FOXA1 was strongly associated with inferior outcome in the ER+/HER2− subgroup of patients allocated to tamoxifen, which was not true for the corresponding patients in the control arm. Together, these results strongly support that FOXA1 is a putative tamoxifen-predictive factor in patients with ER+/HER2− tumors.

Previously, we reported PAM50 subtypes to have prognostic relevance in this premenopausal cohort [9]. Although we identified four GEX clusters with prognostic effects in this cohort, these did not outperform PAM50 (Additional file 6). Focusing on the respective GEX signatures of BC360, those related to proliferation, hypoxia, immunology, and hormone receptors were associated with long-term prognosis in this cohort. High expression of BC proliferation and hypoxia gene signatures was associated with worse RFi and OS outcomes. An association between BC proliferation and poor outcome was expected, because MKI67, which encodes Ki67, is included in this signature. Ingebriktsen et al. demonstrated that a 6 Gene Proliferation Score (6GPS) incorporating proliferation in young breast cancer patients (< 40 years) is of prognostic significance [21]. Oncotype DX includes 5 of the 16 genes of the BC proliferation GEX signature, further illustrating how proliferation markers at the RNA level can be of clinical interest [22]. Several research groups have also shown that hypoxia-related GEX profiles have prognostic value in breast cancer, which supports our results [23, 52, 53].

We have previously shown that TILs are independently associated with prognosis in premenopausal patients [27]. Mast cells are a part of the innate immune system and are more frequent in hormone receptor-positive breast cancers [54]. The Mast cell GEX signature incorporated multiple genes (Additional file 2), and we demonstrated a possible association between high expression of this signature and better prognosis. Another Mast cell gene signature (MCS) has been shown to be prognostic and suggested as a potential indicator of immunotherapy response for patients with head and neck squamous cell carcinoma [55]. In early TNBC, the benefit from capecitabine has been demonstrated to be linked to the Mast cell signature used in our study [15]. Data on the endocrine therapy-predictive effects of this signature in early breast cancer are lacking, and predictive effects were not observed in our cohort.

The strengths of this study include its pure premenopausal cohort, long-term follow-up, and randomized design. Furthermore, the tumor material in this cohort was treatment-naïve, making the GEX readings representative of newly diagnosed tumors. We illustrated the predictive results in terms of quartiles to visualize any dose–response relationship with tamoxifen. However, the cutoffs of GEX signatures have not been settled for clinical use, and more data are needed to further explore this. The limitations of this study are the limited cohort size and, hence, low power, especially for the detection of interaction effects. Moreover, the treatment of this cohort today would differ in terms of systemic therapy from the guidelines of that time. A data-driven selection of signatures was used for some analyses, which increased the risk of false positives. However, we prespecified the evaluation of biologically important signatures such as ESR1 and PGR, and the analyses were adjusted for multiple testing. Regarding the endpoints, we chose RFi rather than the breast cancer-free interval (BCFi). The difference lies in the inclusion of contralateral breast cancer (CBC; invasive and/or in situ) in the latter definition. The inclusion of the CBC would have resulted in more events; however, as in other randomized studies, including those evaluating the clinical utility of GEX assays, the CBC is often considered a censoring event. In addition, we focused on the potential effect of tamoxifen in reducing breast cancer recurrence, not as chemoprevention.

In summary, this study showed an association between low gene expression of FOXA1 and tamoxifen benefit in premenopausal patients with ER+/HER2− tumors. In addition, the findings confirmed that BC proliferation and Hypoxia gene expression signatures identify patients with a dismal prognosis. The gene expression of ESR1, PGR, and the Mast cells gene expression signature were observed to be associated with improved outcomes. The results warrant future validation in independent cohort studies.