Ellsworth, Katarzyna A.

Abstract The FKBP5 gene encodes the FKBP51 immunophilin, a negative regulator of AKT. Variability in level of FKBP5 expression is a major factor contributing to variation in response to several classes of chemotherapeutic agents including gemcitabine, first line treatment for pancreatic cancer. Genetic variation of FKBP5 could influence its expression, function, and, ultimately, treatment response of pancreatic cancer. We set out to comprehensively study the role of genetic variation in FKBP5 identified by Next Generation DNA resequencing in response to gemcitabine treatment of pancreatic cancer by utilizing both tumor and germline DNA samples from 43 pancreatic cancer patients, including 19 paired tumor-normal samples. Genotype-phenotype association studies with overall survival (in tumor only) as well as with FKBP5 gene expression were performed using the same samples in which Next Generation DNA resequencing had been performed, followed by functional genomic studies of top selected single nucleotide polymorphisms (SNPs). In-depth resequencing identified 404 FKBP5 SNPs in both normal and tumor DNA patient samples. SNPs with the smallest p-values for association with FKBP5 survival or expression were subjected to functional genomic studies. Electromobility shift assay (EMSA) showed that the rs73748206 “T” allele altered DNA-protein binding patterns, consistent with a significantly increased reporter gene activity. The effect of rs73748206 was also confirmed by mRNA expression studies using lymphoblastoid cell lines (LCL) derived from the same patients from whom DNA used for resequencing had been obtained. This comprehensive FKBP5 resequencing study provides insight into the role of inheritance in variation in response to gemcitabine therapy of pancreatic cancer. Citation Format: Katarzyna A. Ellsworth, Bruce W. Eckloff, Liang Li, Irene Moon, Brooke L. Fridley, Greg D. Jenkins, Erin Carlson, Abra Brisbin, Ryan Abo, William Bamlet, Gloria Petersen, Eric D. Wieben, Liewei Wang. Contribution of FKBP5 genetic variation to gemcitabine treatment and survival in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2209. doi:10.1158/1538-7445.AM2013-2209

Abstract Aromatase (CYP19) is a critical enzyme in estrogen biosynthesis and aromatase inhibitors (AI) are employed widely for endocrine therapy in postmenopausal women with breast cancer. We hypothesized that single nucleotide polymorphisms (SNPs) in the CYP19 gene may alter the effectiveness of AI therapy in the neoadjuvant setting. Genomic DNA was obtained for sequencing from 52 women pre-AI and post-AI treatment in this setting. Additionally, genomic DNA obtained from 82 samples of breast cancer and 19 samples of normal breast tissue was subjected to resequencing. No differences in CYP19 sequence were observed between tumor and germ-line DNA in the same patient. A total of 48 SNPs were identified including 4 novel SNPs when compared with previous resequencing data. For genotype-phenotype association studies, we determined the levels of aromatase activity, estrone, estradiol, and tumor size in patients pre-AI and post-AI treatment. We defined two tightly linked SNPs (rs6493497 and rs7176005 in the 5'-flanking region of CYP19 exon 1.1) that were significantly associated with a greater change in aromatase activity after AI treatment. In a follow-up study of 200 women with early-stage breast cancer who were treated with adjuvant anastrozole, these same two SNPs were also associated with higher plasma estradiol levels in patients pre-AI and post-AI treatment. Electrophoretic mobility shift and reporter gene assays confirmed likely functional effects of these two SNPs on transcription of CYP19. Our findings indicate that two common genetic polymorphisms in the aromatase gene CYP19 vary the response of breast cancer patients to aromatase inhibitors. Cancer Res; 70(1); 319–28