Background
Breast cancer treatment is largely determined by hormone receptor and human epidermal growth factor receptor-2 (HER2) expression, but there is significant variability of response and prognosis within the subtypes defined by these markers. Being able to identify characteristics or markers on pretreatment samples that predict a higher likelihood of treatment-refractory disease could spare patients from exposure to ineffective and often toxic therapies, and promote the development of novel treatments that target and neutralize these factors.
In the past, most analyses have focused on identifying markers expressed by the tumor cells themselves. Tumor biopsies and surgical specimens consist of a mixture of cancer cells and surrounding stroma comprised of a variety of cell types, and while the traditional approach to tumor biology disregarded the impact that those other tissues might have on tumor behavior, more recently there has been an increased appreciation of the possibility that the tumor microenvironment and tumor-stromal interactions could play an important role in determining response. These include the abundance and character of tumor-infiltrating lymphocytes (TILs) and levels of expression of proteins such as PD-L1 that can modify immune response to the growing tumor, both of which may have a significant impact on prognosis, especially in more aggressive breast cancer subtypes [
1,
2].
Response rates to NAC vary widely depending on subtype in breast cancer. In HER2+ patients, the addition of the HER2-targeting monoclonal antibody trastuzumab to standard NAC has been shown to improve not only the pathological Complete Response (pCR) rate in the NAC setting, but also recurrence-free and overall survival in the adjuvant setting [
3]. However, despite the addition of trastuzumab or even dual HER2-targeting therapies with trastuzumab and either lapatinib or pertuzumab to NAC (NAC+H), a significant percentage of HER2+ patients do not achieve a pCR or minimal residual disease. In the TRYPHAENA trial, the pCR for ER-/HER2+ is 77 % but only 48 % for ER+/Her2+ with cases from all three arms combined [
4]. In NeoSphere trials, few pathologic complete responses were noted in tumors that are hormonal receptor positive in all four arms [
5]. There was a significant difference in pCR rates between hormone positive and negative tumors; the pCR was 40 % for ER- groups and only 17 % for ER+ groups. This discrepancy may reflect differences in cancer cell biology, related to proliferation or dependence on HER2-mediated signaling; it is also possible that differences in the microenvironment mediate response. At this time, there is no reliable, validated method to distinguish responders from non-responders. Thus, many patients are needlessly treated with toxic chemotherapy with uncertain benefit from the treatment. The aim of this work was to examine gene expression profiles of tumors at the time of the pre-treatment biopsy to identify molecular features that may be associated with chemoresponse. Such markers of NAC response could be useful to reduce chemotherapy-reduced morbidity, and identify new therapeutic approaches to treat ER+/HER2+ breast cancer.
In this study, we chose to focus on the ER+/HER2+ subtype where patients do not respond well to NAC and suffer from overall survival rates comparable to TNBC. The cross-talk between these oncogenic pathways drives cross-resistance to current therapies, leading to the use of cytotoxic chemotherapy to treat this subtype [
6]. We hypothesize that new predictive markers could be developed by identifying candidate genes and pathways from gene expression profiling followed by detailed analysis of candidate markers using immunohistochemistry [
7,
8]. We found that collagens, in particular the expression of the protein product from the ColXA1 gene, were strongly associated with NAC response in ER+/HER2+ breast tumors. The presence of collagen in the surrounding tumor milieu has long been known to influence cancer cells. Collagens can induce epithelial-mesenchymal transitions and related invasive properties of breast cancer cells [
9]. However, the utility of any specific collagen as a prognostic marker remains unclear. Expression of ColXA1 has been included in published stromal expression signatures [
10,
11], but the expression of ColXA1 protein product, colXα1, has not been evaluated. For these reasons, we examined the potential for the expression of the colXα1 protein by immunohistochemistry to predict response to NAC in ER+/HER2+ breast tumors.
Discussion
The diversity of breast carcinoma is increasingly reflected in the spectrum of therapeutic approaches that are based on known biomarkers. ER, PR and HER2 are routinely used in clinical practice as a guide for the selection of therapy for breast cancer patients. In patients with stage II-III breast cancer, achievement of pCR to neoadjuvant chemotherapy correlates with improved long-term outcomes, however the predictive value of the standard clinical biomarkers such as ER, PR and HER2 is limited, motivating this study to identify factors mediating response. We defined the subtype of breast cancer by treatment protocols, and not the molecular markers used to define luminal vs. basal tumors [
23]. We focused on the ER+/HER2+ subtype where less than 50 % of tumors respond to NAC (Table
1), despite combining HER2 targeted therapy, trastuzumab, with taxane and platinum based chemotherapy. To identify new markers, we combined RNA expression profiling with IHC to discover the importance of colXα1 positive stroma. Here, we found that collagens, namely colXα1, are up-regulated in breast tumors that do not respond to therapy in the ER+/HER2+ subtype. We observed an association at the mRNA level by qPCR and microarray and evaluated 50 cases at the protein level by IHC. Together, these RNA and protein data support the conclusion that colXα1 expression is strongly associated with chemotherapy response. We decided to focus on the role of collagens as collagens have been reported in gene expression signatures associated with response in the NAC setting and survival in the adjuvant setting. To our knowledge, this is the first study to evaluate the expression of colXα1 protein in breast tumors.
ColXα1 expression levels in the stroma of ER+/HER2+ tumors have a bimodal distribution, an important characteristic for a biomarker. While some ER+/HER2+ tumors do not express colXα1, those ER+/HER2+ tumors with strong expression (IHC score of 2 or 3) all were resistant to treatment. Thus, in this cohort, colXα1 predicts no false positives, and just 8 false negatives (Table
1).
The importance of the tumor microenvironment in influencing chemosensitivity is becoming increasingly clear [
23]. The amount of stroma has been associated with chemosensitivity in many studies [
24,
25]. Various stromal markers including tenascin, fibronectin and collagen type IV have been correlated with more aggressive behavior in breast cancer [
26]. Although the quantity of stroma is correlated with pCR in this study, it is also clear that there are different types of stroma [
27]. A variety of collagens are highly expressed in breast tumors contributing to its dense structure [
9]. Collagens have long been known to be critical players in the extracellular matrix of breast tumors [
9,
27], including mediating drug resistance [
28], and alignment of collagens has been proposed to indicate progression in breast tumors [
29,
30]. Collagen alignment was reported to correlate with expression of syndecan-1, but this gene was not significantly differentially expressed in this study (Fc = 0.15). However, there are limited data on the expression and function of many specific collagen subtypes by IHC in breast cancer patients. Collagen is associated with the major ECM transformations and the collagen subtype COLl11A1 has been associated with metastasis and disease progression in breast cancer [
9]. In this study, COL3A1 and COL14A1 are among the most significantly differentially expressed transcripts (see Additional file
4: Table S3) and are good candidates for further evaluation, as they are not one of the dominant forms of collagen in normal breast tissue. Further studies are warranted to test which collagens, and if a combination of specific collagens are good predictors of response at the RNA and protein levels.
Col10A1 mRNA expression is up-regulated in a variety of human malignancies compared to normal tissue, including breast tumors [
8]. Increased expression of Col10A1 has been a part of breast cancer signatures, including a CD10+ signature to discriminate in situ from invasive breast cancer [
11] and a stroma expression signature to predict resistance to neoadjuvant chemotherapy in breast cancer [
10], though the specific ER+/HER2+ subtype was not specifically evaluated. In normal tissue, colXα1 expression is distinct among all collagens as it is only expressed in hypertrophic chondrocytes [
7]. These observations and our data highlight how colXα1 can be an excellent biomarker for chemoresistance and is a candidate target for specific delivery to ER+/HER2+ breast tumors. It is not clear why expression of the COL10A1 gene is such a good marker. In pre-clinical models, collagens increase multiple tumor properties including growth, tumorigenicity, invasion [
9,
31], the drug resistance, and the mesenchymal state [
32,
33]. Here, many related genes and pathways involved in chemoresistance were correlated with COL10A1 mRNA expression. These observations further support the development and testing of colXa1 protein expression as a biomarker, perhaps in combination with other pathways such as sTILs, EMT markers. Further study is warranted to determine the function of colXα1 in mediating resistance.
We were motivated to look at TILs because of the enrichment of immune pathways, including genes indicative of T cells, in the gene expression data. Several clinical studies have evaluated TILs as a positive prognostic biomarker in TNBC [
2,
34‐
42]. However, TILs were not a positive biomarker for luminal subtypes [
43]. In this study, TILs were a strong independent predictor in in ER+/HER2+ subtypes (Table
2, see Additional file
7: Table S5). In the ER+/HER2+ subtype, TILs and colXα1 both contribute to predict chemosensitivity (Fig.
4, Tables
1 and
2), suggesting that the combination of TILs and colXα1 IHC score is a strong predictor for ER+/HER+ breast cancers.
This study focused on patients with ER+/HER2+ breast cancer treated with neoadjuvant chemotherapy and HER2-targeted therapy and was designed to identify novel biomarkers predictive of pathologic response. This definition may have impacted our ability to detect Col10A1 and TIL association not previously observed in luminal tumors. The HER2-positive patients generally have a significantly higher pCR rate in response to NAC+H. However, within the HER2-positive population, pCR was more common for ER-negative tumors than for ER- positive tumors [
4,
5,
44]. This suggests that for a subset of HER2+ tumors, ER or a more complex molecular pathway drives response, and ER+/HER2+ tumors are biologically different than ER-/HER2+ tumors [
45,
46].
Our data represent initial, preliminary evidence that colXα1 may be a marker for NAC response. The present study has some limitations, for example, the modest number of cases could be affected by unknown sample biases. We have selected cases with similar, but not identical treatment schedules. Even with a variety of treatment schedules, TILs and colXα1 have strong predictive power for chemoresponse suggesting that they are general factors for NAC responsiveness in ER+/HER2+ breast tumors and further study of colXα1 and other collagens as predictive markers is warranted.
Acknowledgements
Funding is provided by the Department of Pathology at Rhodes Island Hospital (Y.W.), Brown University (ASB) and the Mary Kay Foundation (ASB). The Brown University Genomics Core Facility has received partial support from the National Institutes of Health (NIGMS grant Number P30GM103410, NCRR grant Numbers P30RR031153, P20RR018728 and S10RR02763), National Science Foundation (EPSCoR grant No 0554548), Lifespan Rhode Island Hospital, and the Division of Biology and Medicine, Brown University.
The Molecular Pathology Core of the COBRE Center for Cancer Research Development is funded by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM103421.
Competing interests
The authors declare that a patent application has been filed.
Authors’ contributions
ASB, WMS, MBR and YW conceived of the study, participated in its design, and prepared the manuscript. ASB performed the statistical analyses. YW and JX collected, reviewed and scored all the cases in the study including morphological and immunohistochemical evaluation. DY and CS performed the molecular experiments. ASB and YW wrote the manuscript. MAF and TAG reviewed the data and participated in revising the manuscript. All authors read and approved of the final manuscript.