1408 Vol. 11, 1408–1415, February 15, 2005 Clinical Cancer Research Nucleotide Excision Repair Gene Polymorphisms and Recurrence after Treatment for Superficial Bladder Cancer Jian Gu,1 Hua Zhao,1 Colin P. Dinney,2 Yong Zhu,3 fewer putative high-risk alleles as the reference group, Dan Leibovici,2 Carlos E. Bermejo,2 individuals with six to seven risk alleles and individuals with eight or more risk alleles had higher recurrence risks, with H. Barton Grossman,2 and Xifeng Wu1 hazard ratios of 0.92 (0.54-1.57) and 2.53 (1.48-4.30), 1 2 Departments of Epidemiology and Urology, The University of Texas respectively (P for trend < 0.001). There was also a M.D. Anderson Cancer Center, Houston, Texas, and 3Department of Epidemiology and Public Health, Yale University, New Haven, significant trend for shorter recurrence-free survival time Connecticut with increasing number of variant alleles (log rank test, P = 0.0007). When we stratified the patients according to intravesical Bacillus Calmette-Guerin treatment, we found a ABSTRACT significant trend for shorter recurrence-free survival time in Purpose: Interindividual differences in DNA repair patients with variant alleles of XPA or ERCC6 polymor- capacity not only modify individual susceptibility to carci- phisms who received Bacillus Calmette-Guerin treatment nogenesis, but also affect individual response to cancer (log rank test, P = 0.078 and 0.022, respectively). There were treatment. Nucleotide excision repair (NER) is one of the no significant individual or joint associations between these major DNA repair pathways in mammalian cells involved in polymorphisms and progression. the removal of a wide variety of DNA lesions. Polymorphisms Conclusions: These data suggest that interindividual in NER genes may influence DNA repair capacity and affect differences in DNA repair capacity may have an important clinical outcome of bladder cancer treatment. impact on superficial bladder cancer recurrence. A pathway- Experimental Design: To test the influence of NER gene based approach is preferred to study the effects of individual polymorphisms on superficial bladder cancer outcome polymorphism on clinical outcomes. (recurrence and progression), we conducted a follow-up study of 288 patients with superficial bladder cancer. INTRODUCTION Median follow-up among patients who were recurrence- Bladder cancer is the fifth most commonly diagnosed free at the end of observation was 21.7 months from malignancy in the United States, with more than 55,300 new diagnosis. The specific polymorphic loci examined include cases diagnosed in 2004 (1). Superficial bladder cancer accounts XPA [A/G at 5V untranslated region (UTR)], XPC (poly AT, for >85% of newly diagnosed cases. These cases comprise a Ala499Val, Lys939Gln), XPD (Asp312Asn, Lys751Gln), XPG heterogeneous group of tumors whose individual prognoses are (His1104Asp), ERCC 1 (G/T at 3V UTR), and ERCC6 difficult to predict (2). Nevertheless, following transurethral (Met1097Val, Arg1230Pro). resection, tumor recurrence rate is about 70%, and 10% to 15% of Results: The ERCC6 (Met1097Val) polymorphism had a such recurrences may be associated with progression to more significant impact on recurrence: carriers of at least one invasive forms of cancer (3, 4). Clinical and pathologic variant allele (Val) had a significantly higher recurrence risk prediction variables of disease recurrence have been extensively than carriers of the wild-type allele (Met/Met; hazard ratio, studied. In a patient cohort of 1,529 patients, tumor size, number 1.54; 95% confidence interval, 1.02-2.33). There were no of tumors, and presence of carcinoma in situ have been correlated overall statistically significant differences in the distributions with disease recurrence (5). Administration of intravesical Bacille of the other polymorphisms between patients with and Calmette-Guerin (BCG) has been found to significantly reduce without recurrence. However, when we combined these the risk of recurrence and progression. However, the response variant genotypes, there was a significant trend for an rate for BCG treatment is only 60% to 70%, and about one-third increased recurrence risk with an increasing number of of responders develop recurrence and progression (6, 7). A putative high-risk alleles. Using individuals with five or number of pathologic and molecular markers have been studied as potential predictors of outcome in bladder cancer. Mutant cell cycle and proliferative markers, including Ki-67, cytokeratin 20 Received 6/4/04; revised 1/7/05; accepted 1/13/05. and 34h, and growth factors, such as epidermal growth factor, Grant support: National Cancer Institute grants CA 74880, CA 85576 vascular endothelial growth factor, and fibroblast growth factor (X. Wu), CA 91846 (C. Dinney and X. Wu), and CA 86390 (M. Spitz). receptor-3, have been found to influence recurrence. p53, p21, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked RB, cadherins cathepsins, metalloproteinases, BCL-2, cyto- advertisement in accordance with 18 U.S.C. Section 1734 solely to keratins, and fibroblast growth factor receptor-3 have been found indicate this fact. to be predictors of tumor progression (8). Requests for reprints: Xifeng Wu, Department of Epidemiology, Unit A possible explanation for the unpredictability of the clinical 1340, The University of Texas MD Anderson Cancer Center, 1155 Pressler Boulevard, Houston, TX 77030. Phone: 713-745-2485; Fax: course of superficial bladder cancer may be related to the fact that 713-792-4657; E-mail: [email protected]. host genetic differences may render some individuals at higher D2005 American Association for Cancer Research. risk than others. Functional polymorphisms may account for Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Clinical Cancer Research 1409 phenotypic differences that lead to enhanced cancer risk. The Genotyping. Genomic DNA was first isolated from associations between genetic polymorphisms and cancer risk have peripheral blood lymphocytes by proteinase K digestion been extensively reported; however, few studies have focused on followed by isopropanol extraction and ethanol precipitation; polymorphisms as predictors of specific clinical events. the sample was then coded and then subjected to genotyping. One of the pathways for carcinogenesis is related to DNA The specific polymorphic loci examined include XPA (A/G at damage and repair. DNA repair systems are essential for 5V UTR, rs1800975), XPC (poly AT, Ala499Val, Lys939Gln), maintaining genomic integrity. The ability to monitor and repair XPD (Asp312Asn, Lys751Gln), XPG (His1104Asp), ERCC1 (G/T carcinogen-induced DNA damage is an important determinant of at 3V UTR, rs3212986), and ERCC6 (Met1097Val, Arg1230Pro). susceptibility to carcinogenesis. There is considerable evidence Most of the polymorphisms, except for XPA, XPC (poly AT in suggesting that reduced DNA repair capacity may play a role in intron 11), and XPD (Asp312Asn), were determined using cigarette smoking–related cancer risk (9, 10). Interestingly, in TaqMan real-time PCR. The primer and probe sequences, terms of cancer recurrence and survival, efficient DNA repair which are available upon request, were either obtained from capacity is not always a favorable prediction factor. Catto et al. National Cancer Institute’s SNP500 database or designed using (11) reported that reduced expression of two mismatch repair Primer Express Software. The probes were labeled fluorescently genes, hMLH1 and hMSH2, was associated with fewer with either FAM or VIC on the 5V end and a nonfluorescent recurrences of superficial bladder cancer. In addition, Bosken minor groove binder quencher on the 3V end (Applied et al. (12) found that effective host DNA repair capacity was Biosystems, Foster City, CA). Typical amplification mixes (5 associated with poorer survival in patients with non–small cell AL) contained sample DNA (5 ng), 1Â TaqMan buffer A, 200 lung cancer who were treated with chemotherapy. Am deoxynucleotide triphosphates, 5 mmol MgCl2, 0.65 units Nucleotide excision repair (NER), one of the major DNA of AmpliTaq Gold, 900 nmol/L each primer, and 200 nmol/L repair pathways in mammalian cells, is involved in the removal of each probe. The thermal cycling conditions consisted of 1 cycle a wide variety of DNA lesions, including bulky carcinogen for 10 minutes at 95jC, 40 cycles for 15 seconds at 95jC, adducts resulting from tobacco exposure. All key NER factors and 1 minute at 60jC. SDS version 2.1 software (Applied have been cloned, and the core of the ‘‘cut-and-paste’’ reaction Biosystems) was used to analyze end-point fluorescence. Water has been reconstituted in vitro (13). Most NER genes are control, internal controls, and previously genotyped samples polymorphic, and some of the polymorphisms have been were included in each plate to ensure accuracy of genotyping. extensively studied in terms of their associations with cancer risk Genotyping for XPA, XPC (poly AT in intron 11), and XPD as well as clinical outcomes in specific cancer types (14–17). (Asp312Asn) were done as described previously using PCR- However, most studies have used a candidate gene approach, RFLP (14–16). investigating one single nucleotide polymorphism (SNP) in a Statistical Analyses. v2 analyses and Fisher’s exact test single gene. In the current study, we applied a pathway-based were used to assess