Author(s): Susann Busch 1 , Lisa Rydén 2 , Olle Stål 3 , Karin Jirström 4 , Göran Landberg 1 , 4 , 5 , *
The administration of the anti-estrogen tamoxifen is an adjuvant endocrine therapy for patients with ER[alpha]-positive breast cancer. However, many patients do not respond to initial therapy or develop drug resistance and a more patient-tailored therapy approach would be favorable including treatment-predictive markers and alternative treatment options. Therefore, the identification of biomarkers that classify subgroups of breast cancer which will benefit from a particular treatment becomes increasingly relevant .
Recently a vast body of literature has emerged demonstrating the importance of the tumor microenvironment (stroma) on tumor progression , , . Thus, it is evident that exploiting stromal factors will facilitate the discovery of novel biomarkers with prognostic and predictive values , . Cancer-associated fibroblasts (CAFs) may be an attractive target due to their abundance in the tumor. CAFs are also referred to as activated fibroblasts or myofibroblasts, and characterized by the presence of mesenchymal markers such as smooth muscle actin-alpha (SMA[alpha]) and the absence of epithelial and endothelial markers. However, there is yet no marker unique to CAFs  and so far there have been few studies on CAF-specific markers .
Activated (phosphorylated) ERK (pERK) has been reported to be a prognostically relevant tumor-specific biomarker in breast cancer and to date, there is a controversy whether activated ERK signaling in tumor cells is associated with better  or worse ,  relapse-free survival. Previously, our group reported that ERK phosphorylation in tumor cells of invasive breast cancer was correlated to tamoxifen resistance using three different breast cancer cohorts . However, another group has reported that tamoxifen induces sustained activation of ERK in tumor cells leading to rapid cell death indicating an involvement of ERK signaling in the tamoxifen response of ER[alpha]-positive cancer cells . Whether similar effects can be observed in vivo and whether basal ERK phosphorylation levels play a role in tamoxifen response however have not been addressed. Moreover the majority of studies focus on ERK signaling within tumor cells neglecting a possible role of the tumor microenvironment on tumor progression or treatment response.
When analyzing ERK phosphorylation in tumor cells breast cancer tissues, we also observed a distinct staining pattern in the stromal compartment. In order to examine the potential prognostic and treatment-predictive values of stromal ERK phosphorylation we therefore analyzed a unique randomized trial including 564 pre-menopausal breast cancer patients randomized to 2 years of tamoxifen or no adjuvant treatment after surgery, as well as a second cohort of 179 pre- and post-menopausal patients and focused on CAFs. The evaluation of the biomarkers was performed according to the REMARK recommendations in order to provide a more transparent and complete report which may improve ascertaining the relevance of the newly found biomarker (Table S5, Figure S4) .
Materials and Methods
The studies were approved by the Ethics Committee at Universities in Linköping and Lund, Sweden (cohort I SBII:2 and...