1226 Vol. 11, 1226–1236, February 1, 2005 Clinical Cancer Research UGT1A7 and UGT1A9 Polymorphisms Predict Response and Toxicity in Colorectal Cancer Patients Treated with Capecitabine/Irinotecan Leslie E. Carlini,1 Neal J. Meropol,1 John Bever,2 irinotecan. Specifically, patients with genotypes conferring Michael L. Andria,2 Todd Hill,2 Philip Gold,3 low UGT1A7 activity and/or the UGT1A9 (dT)9/9 genotype may be particularly likely to exhibit greater antitumor Andre Rogatko,1 Hao Wang,1 and response with little toxicity. Rebecca L. Blanchard1 1 2 Fox Chase Cancer Center, Philadelphia, Pennsylvania; Roche INTRODUCTION Laboratories, Inc., Nutley, New Jersey; and 3Swedish Cancer Institute, Seattle, Washington Colorectal cancer (CRC) is the third most common cancer in both men and women and the third most prevalent cause of cancer-related death (1). A common approach to the systemic ABSTRACT management of metastatic colorectal cancer is a combination Purpose: Capecitabine and irinotecan are commonly of fluoropyrimidine (e.g., 5-fluorouracil or capecitabine) used in the treatment of metastatic colorectal cancer (CRC). and irinotecan (reviewed in ref. 2). Fluoropyrimidines act We hypothesized that germline polymorphisms within genes primarily through the inhibition of thymidylate synthase (TS), related to drug target (thymidylate synthase) or metabolizing resulting in impaired DNA synthesis and cell death. Irinotecan is enzymes (UDP-glucuronosyltransferase, UGT) would impact a camptothecin compound that inhibits DNA topoisomerase I, response and toxicity to the combination of capecitabine plus resulting in an accumulation of DNA damage and cell death. irinotecan (CPT-11). Irinotecan is a prodrug that undergoes conversion by liver Experimental Design: Sixty-seven patients with measur- carboxylesterases to form the active compound SN-38, 7-ethyl- able CRC were treated with irinotecan i.v. (100 or 125 mg/m2) 10-hydroxy-camptothecin (2, 3). SN-38 is largely metabolized on days 1 and 8 and capecitabine orally (900 or 1,000 mg/m2, to the inactive glucuronide, SN-38G, via the action of several twice daily) on days 2 through 15 of each 3-week cycle. UDP-glucuronosyltransferase (UGT) including hepatic Genomic DNA was extracted from peripheral blood and ge- UGT1A1, UGT1A6, and UGT1A9 and extrahepatic UGT1A7 notyped using Pyrosequencing, GeneScan, and direct se- (4–7). quencing (Big Dye terminator) technologies. The existence of significant patient variability in response Results: The overall objective response rate was 45% to fluoropyrimidines and irinotecan, coupled with knowledge with 21 patients (31%) exhibiting grade 3 or 4 diarrhea and of common genetic polymorphisms within genes important to 3 patients (4.5%) demonstrating grade 3 or 4 neutropenia in the pharmacology of these drugs, suggests that pharmacogenetic the first two cycles. Low enzyme activity UGT1A7 genotypes, studies could identify individuals likely to benefit from treatment UGT1A7*2/*2 (six patients) and UGT1A7*3/*3 (seven or develop severe toxicity. Genetic variation within the promoter patients), were significantly associated with antitumor and the 3V-untranslated region (UTR) of the human TS gene has response ( p = 0.013) and lack of severe gastrointestinal been previously linked to the efficacy of 5-fluorouracil-related toxicity ( p = 0.003). In addition, the UGT1A9 À118 (dT)9/9 drugs (reviewed in ref. 8). The polymorphic UGT1A family genotype was significantly associated with reduced toxicity members may govern variability in patient response to irinotecan ( p = 0.002) and increased response ( p = 0.047). There were (reviewed in ref. 9). A common TA repeat within the promoter of no statistically significant associations between UGT1A1, the human UGT1A1 gene has been associated with CPT-11- UGT1A6, or thymidylate synthase genotypes and toxicity or related toxicity, most predominantly neutropenia (10–12). tumor response. Functionally significant genetic variation has also been described Conclusions: These data strongly suggest that UGT1A7 for human UGT1A6, UGT1A7, and UGT1A9 (6, 13–17). In this and/or UGT1A9 genotypes may be predictors of response study, we correlate the efficacy and toxicity of combined and toxicity in CRC patients treated with capecitabine plus capecitabine/CPT-11 therapy with genetic variation in genes important in the metabolism of CPT-11 (UGT1A1, UGT1A6, UGT1A7, and UGT1A9). Given the narrow therapeutic index, Received 9/2/04; revised 11/3/04; accepted 11/9/04. frequent resistance, and expanding options for treatment of CRC, Grant support: Roche Laboratories, Inc. the need for predictive markers of response and toxicity has The costs of publication of this article were defrayed in part by the assumed increased importance. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Presented in part at the 40th Annual Meeting of the American MATERIALS AND METHODS Society of Clinical Oncology, June 5-8, 2004, New Orleans, LA. Patient Eligibility. This pharmacogenetic analysis was a Request for reprints: Rebecca L. Blanchard, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497. Phone: 215-728- secondary objective of a multicenter phase II trial of capecitabine/ 3141; Fax: 215-728-4333; E-mail: [email protected]. CPT-11 combination therapy in patients with metastatic CRC. #2005 American Association for Cancer Research. The primary clinical objective was determination of objective Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Clinical Cancer Research 1227 response. Eligible patients were at least 18 years old with PCR reaction mixture included 20 mmol/L Tris-HCl (pH 8.3), histologically confirmed metastatic colorectal adenocarcinoma. 50 mmol/L KCl, 1.5 mmol/L MgCl2,0.2mmol/Leach Previous cytotoxic chemotherapy was not permitted except for deoxynucleotide triphosphate (dNTP), 0.2 Amol/L each primer, neoadjuvant or adjuvant treatment completed 12 months before 1.25 units Platinum Taq polymerase (Invitrogen, Carlsbad, CA), study enrollment. Patients were required to be ambulatory with a and 20 ng genomic DNA in a 50 AL reaction volume. The PCR Karnofsky performance status of z70%. Required laboratory profile included 1-minute denaturation at 94jC, 34 cycles of 30 9 values for inclusion included neutrophil count z1.5 Â 10 , seconds at 94jC, 30 seconds at 52jC, and 30 seconds at 72jC, 9 platelet count z100 Â 10 /L, serum creatinine V1.5 Â upper limit with a final 5-minute extension at 72jC in a Perkin-Elmer 9700 z normal, estimated creatinine clearance 50 ml/min, serum Thermal Cycler (Perkin-Elmer, Boston, MA). Fluorescently bilirubin V1.25 Â upper limit normal, ALAT and ASAT V2.5 Â labeled products were electrophoretically separated on a 4% upper limit normal (<5 with liver metastasis or <10 with bone polyacrylamide gel on an ABI 377 DNA Sequencer. Fluorescent V metastasis), or alkaline phosphatase 2.5 Â upper normal limit bands were analyzed using GENESCAN 2.1 software to (<5 with liver metastasis or <10 with bone metastasis). Exclusion determine fragment length (Applied Biosystems). Genotypes criteria included known Gilbert’s disease, pregnancy, central were assigned as UGT1A1*1 (reference), UGT1A1*36, nervous system metastasis, active cardiac disease, or myocardial UGT1A7*28, and UGT1A7*37 for TA repeat numbers of 6, 5, infarction within the previous 12 months, active infections, 7, and 8, respectively. physical disorders of the gastrointestinal tract, or problems with UGT1A6. UGT1A6 genotype was determined with a malabsorption. Written informed consent was required, and the PCR-based assay that uses Pyrosequencing technology (Pyrose- study was approved by the institutional review boards at all of the quencing, Westborough, MA). UGT1A6 alleles are defined by participating sites in accord with an assurance filed with and permutations of three single-nucleotide polymorphisms (SNPs) approved by the U.S. Department of Health and Human Services. that alter the encoded amino acid sequence at nucleotide Drug Administration. Capecitabine (Xeloda, Roche positions 19T > G, 541A > G, and 552A > C (for amino acids Laboratories, Inc., Nutley, NJ) was given orally at a dosage of S7A, T181A, and R184S, respectively; ref. 15). For detection of 1,000 mg/m2 (cohort 1, 15 patients) or 900 mg/m2 (cohort 2, 52 the 19T > G SNP, a 238-bp fragment was amplified using primer patients) twice daily on days 2 through 15 of 3-week cycles. F-53 (5V-GATTTGGAGAGTGAAAACTCTTT-3V) and R184 Irinotecan (Camptosar, Pfizer, Inc., New York, NY) was given at (5V-biotin-CAGGCACCACCACTA-CAATCTC-3V). For the a dosage of 125 mg/m2 (cohort 1, 15 patients) or 100 mg/m2 remaining two SNPs, a 215-bp fragment was amplified using (cohort 2, 52 patients) as a 90-minute i.v. infusion on days 1 and primer F414 (5V-biotin-CTTTAAGGAGAGCAAGTTTGATG- 8 of each cycle. The doses of capecitabine and irinotecan were 3V) and R628 (5V-CCACTCGTTG-GGAAAAAGTC-3V). Ap- decreased as described for cohort 2 after the first 15 patients proximately 25 to 50 ng of genomic DNA was amplified in a (cohort 1) because of unacceptable toxicity. The maximum reaction mixture containing 20 mmol/L Tris-HCl (pH 8.4), 50 number of cycles given was 12. mmol/L KCl, 1.5 mmol/L MgCl , 0.2 mmol/L dNTPs, 0.2 Amol/ Evaluation of Response and Toxicities. Baseline tumor 2 L primers, and 2.5 units of Platinum Taq polymerase in a 50 AL measurements were obtained within 21 days before initial volume. Reactions were performed in a Perkin-Elmer 9700 treatment. Response assessments were obtained every two cycles Thermal Cycler with 45-second denaturation at 94jC, followed (6 weeks) with confirmation of response after 4 weeks according by 35 cycles of 94jC for 30 seconds, 56jC for 30 seconds, and to RECIST criteria (18). Toxicity was assessed weekly during the 72jC for 50 seconds. A final 3-minute extension at 72jC first two cycles of treatment according to the National Cancer completed the amplification.