Folate Receptor-α (FOLR1) Expression and Function in Triple Negative Tumors

March Folate Receptor- (FOLR1) Expression and Function in Triple Negative Tumors Brian M. Necela 0 1 Jennifer A. Crozier 0 1 Cathy A. Andorfer 0 1 Laura Lewis-Tuffin 0 1 Jennifer M. Kachergus 0 1 Xochiquetzal J. Geiger 0 1 Krishna R. Kalari 0 1 Daniel J. Serie 0 1 Zhifu Sun 0 1 Alvaro Moreno Aspita 0 1 Daniel J. O'Shannessy 0 1 Julia D. Maltzman 0 1 Ann E. McCullough 0 1 Barbara A. Pockaj 0 1 Heather E. Cunliffe 0 1 Karla V. Ballman 0 1 E. Aubrey Thompson 0 1 Edith A. Perez 0 1 0 1 Department of Cancer Biology, Mayo Clinic , Jacksonville, Florida , United Sates of America, 2 Department of Hematology and Oncology, Mayo Clinic , Jacksonville , Florida, United States of America, 3 Department of Pathology and Laboratory Medicine, Mayo Clinic , Jacksonville , Florida, United States of America, 4 Department of Health Sciences Research, Mayo Clinic , Rochester , Minnesota, United States of America, 5 Department of Health Sciences Research, Mayo Clinic , Jacksonville , Florida United States of America, 6 Department of Biomedical Statistics and Informatics, Mayo Clinic , Rochester , Minnesota, United States of America, 7 Department of Translational Medicine and Diagnostics, Morphotek, Exton, Pennsylvania, United States of America, 8 Department of Clinical Development, Morphotek, Exton, Pennsylvania, United States of America, 9 Department of Laboratory Medicine and Pathology, Mayo Clinic , Scottsdale , Arizona, United States of America, 10 Department of Laboratory Medicine and Pathology, Mayo Clinic , Scottsdale , Arizona, United States of America, 11 Department of Pathology, Dunedin School of Medicine, University of Otago , Dunedin , New Zealand 1 Funding: This work was supported in part by grants from the Breast Cancer Research Foundation, Mayo Clinic Cancer Center (P30 CA15083) and the Florida Bankhead-Coley Research Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Folate receptor alpha (FOLR1) has been identified as a potential prognostic and therapeutic target in a number of cancers. A correlation has been shown between intense overexpression of FOLR1 in breast tumors and poor prognosis, yet there is limited examination of the distribution of FOLR1 across clinically relevant breast cancer subtypes. To explore this further, we used RNA-seq data from multiple patient cohorts to analyze the distribution of FOLR1 mRNA across breast cancer subtypes comprised of estrogen receptor positive (ER+), human epidermal growth factor receptor positive (HER2+), and triple negative (TNBC) tumors. FOLR1 expression varied within breast tumor subtypes; triple negative/ basal tumors were significantly associated with increased expression of FOLR1 mRNA, compared to ER+ and HER2+ tumors. However, subsets of high level FOLR1 expressing tumors were observed in all clinical subtypes. These observations were supported by immunohistochemical analysis of tissue microarrays, with the largest number of 3+ positive tumors and highest H-scores of any subtype represented by triple negatives, and lowest by ER+ tumors. FOLR1 expression did not correlate to common clinicopathological parameters such as tumor stage and nodal status. To delineate the importance of FOLR1 overexpression in triple negative cancers, RNA-interference was used to deplete FOLR1 in overexpressing triple negative cell breast lines. Loss of FOLR1 resulted in growth inhibition, whereas FOLR1 overexpression promoted folate uptake and growth advantage in low folate conditions. Taken together, our data suggests patients with triple negative cancers expressing high FOLR1 expression represent an important population of patients that may benefit - Competing Interests: The authors confirm that Daniel J. OShannessy and Julia D. Maltzman are employees of Morphotek, developers of the FOLR1 antibody farletuzumab and as such have received support in the form of salaries from Morphotek. Daniel J. OShannessy and Julia D. Maltzman did not from targeted anti-FOLR1 therapy. This may prove particularly helpful for a large number of patients who would typically be classified as triple negative and who to this point have been left without any targeted treatment options. Every year, worldwide, more than a million breast cancer cases are diagnosed [1]. Deaths have been declining due to better early detection strategies and improved therapies, particularly those targeted to specific biomarkers. Despite these improvements, there are approximately 230,000 deaths per year worldwide due to breast cancer, including about 40,000 deaths in the United States [1]. Breast cancer is currently grouped into three main clinically relevant molecular subtypes: hormone receptor positive [estrogen receptor (ER+) and/or progesterone receptor (PR+)], human epidermal growth factor receptor positive (HER2+), and triple negative (TNBC) which is ER-, PR-, and HER2- [2]. ER+ tumors account for nearly 70% of invasive breast cancers diagnosed, and the widespread use of ER antagonists in this subset of patients account for the bulk of the treatment-related decreases in mortality. The remaining 30% of breast cancers are closely split between HER2+ breast cancers and triple negative breast cancers, both of which have a worse prognosis compared to ER+ disease. Use of trastuzumab (a humanized monoclonal antibody against HER2) has improved survival in women with HER2+ breast cancer [35]. There is currently no known specific targeted therapy for triple negative breast cancers that significantly affects survival [6] although there is emerging evidence that this subgroup of breast cancers is heterogeneous [79]. A biomarker of recent interest in the cancer field is folate receptor alpha (FOLR1), a membrane-bound protein with high affinity for binding and transporting folate into cells. Folate is a necessary component of cell metabolism. Overexpression of FOLR1 may confer a growth advantage to tumors by increasing folate uptake and/or may affect cell proliferation via alternative cell signaling pathways [1012]. FOLR1 levels have been found to be elevated in tumors of epithelial origin compared to normal tissue, including ovarian, breast, brain, lung and colorectal cancers [1317]. The tumor specificity of FOLR1 makes it a promising target for diagnosis and treatment strategies. Several types of folate receptor targeted therapies, such as antibodies and folic acid-drug conjugates, have been developed and are in various phases of clinical trials for treatment of ovarian and lung cancer [18]. Farletuzumab (MORAb-003) is a monoclonal anti-FOLR1 antibody that elicits antibody dependent cellular cytotoxicity (ADCC) [19]. Vintafolide (EC145) is a folate-conjugated small molecule designed to deliver the chemotherapeutic drug vinblastine selectively to cells expressing the folate receptor [20]. Moreover, diagnostic FR-targeted imaging agents have become available to help select patients with FR-expressing tumors lik (...truncated)