Aromatase [cytochrome P450 19 (CYP19)] is a critical enzyme for estrogen biosynthesis, and aromatase inhibitors are of increasing importance in the treatment of breast cancer. We set out to identify and characterize genetic polymorphisms in the aromatase gene, CYP19, as a step toward pharmacogenomic studies of aromatase inhibitors. Specifically, we ''resequenced'' all coding exons, all upstream untranslated exons plus their presumed core promoter regions, all exonintron splice junctions, and a portion of the 3V-untranslated region of CYP19 using 240 DNA samples from four ethnic groups. Eighty-eight polymorphisms were identified, resulting in 44 haplotypes. Functional genomic studies were done with the four nonsynonymous coding single nucleotide polymorphisms (cSNP) that we observed, two of which were novel. ) displayed a significant change from the WT enzyme in inhibitor constant for the aromatase inhibitors exemestane and letrozole. These observations indicate that genetic variation in CYP19 might contribute to variation in the pathophysiology of estrogendependent disease. (Cancer Res 2005; 65(23): 11071-82)

Purpose: Gemcitabine is a nucleoside analogue with activity against solid tumors. Gemcitabine metabolic inactivation is catalyzed by cytidine deaminase (CDA) or, after phosphorylation, by deoxycytidylate deaminase (DCTD). We set out to study the pharmacogenomics of CDA and DCTD. Experimental Design:The genes encoding CDA and DCTD were resequenced using DNA from 60 African American and 60 Caucasian American subjects. Expression constructs were created for nonsynonymous coding single nucleotide polymorphisms (cSNP) and reporter gene constructs were created for 5V -flanking region polymorphisms. Functional genomic studies were then conducted after the transfection of mammalian cells. Results: CDA resequencing revealed 17 polymorphisms, including one common nonsynonymous cSNP, 79 A>C (Lys27Gln). Recombinant Gln27 CDA had 66 F 5.1% (mean F SE) of the wild-type (WT) activity for gemcitabine but without a significant decrease in level of immunoreactive protein. The apparent K m (397 F 40 Amol/L) for the Gln27 allozyme was significantly higher than that for the WT (289 F 20 Amol/L; P < 0.025). CDA 5V-flanking region reporter gene studies showed significant differences among 5V -flanking region haplotypes in their ability to drive transcription. There were 29 SNPs in DCTD, including one nonsynonymous cSNP, 172 A>G (Asn58Asp), in Caucasian American DNA. Recombinant Asp58 DCTD had 11 F 1.4% of WT activity for gemcitabine monophosphate with a significantly elevated level of immunoreactive protein. No DCTD polymorphisms were observed in the initial 500 bp of the 5V -flanking region. Conclusions: These results suggest that pharmacogenomic variation in the deamination of gemcitabine and its monophosphate might contribute to variation in therapeutic response to this antineoplastic agent.

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. ©2010 AACR.