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Originally published as JCO Early Release 10.1200/JCO.2005.04.4206 on March 6 2006 © 2006 American Society of Clinical Oncology. Single Nucleotide Polymorphisms of RecQ1, RAD54L, and ATM Genes Are Associated With Reduced Survival of Pancreatic Cancer
From the Departments of Gastrointestinal Medical Oncology, Epidemiology, Surgical Oncology, Biostatistics, and Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX Address reprint requests to Donghui Li, PhD, Department of Gastrointestinal Medical Oncology—Unit 426, The University of Texas M.D. Anderson Cancer Center, PO Box 301402, Houston, TX 77230-1402; e-mail: dli{at}mdanderson.org
PURPOSE: Our goal was to determine whether single nucleotide polymorphisms (SNPs) in DNA repair genes influence the clinical outcome of pancreatic cancer. PATIENTS AND METHODS: We evaluated 13 SNPs of eight DNA damage response and repair genes in 92 patients with potentially resectable pancreatic adenocarcinoma. All patients were treated with neoadjuvant concurrent gemcitabine and radiotherapy with or without a component of induction gemcitabine/cisplatin at The University of Texas M.D. Anderson Cancer Center (Houston, TX) from February 1999 to August 2004 and observed through August 2005. Response to the pretreatment was assessed by evaluating time to tumor progression and overall survival. Kaplan-Meier plot, log-rank test, and Cox regression were used to compare survival of patients according to genotype. RESULTS: The RecQ1 A159C, RAD54L C157T, XRCC1 R194W, and ATM T77C genotypes had a significant effect on the overall survival with log-rank P values of .001, .004, .001, and .02, respectively. A strong combined effect of the four genotypes was observed. Patients with none of the adverse genotypes had a mean survival time of 62.1 months, and those with one, two, or three or more at-risk alleles had median survival times of 27.5, 14.4, and 9.9 months, respectively (log-rank P < .001). There is a significant interaction between the RecQ1 gene and other genotypes. All four genes except XRCC1 remained as independent predictors of survival in multivariate Cox regression models adjusted for other clinical predictors. CONCLUSION: These observations support the hypothesis that polymorphic variants of DNA repair genes affect clinical prognosis of patients with pancreatic cancer.
Pancreatic cancer is a rapidly fatal disease with a 5-year survival rate less than 5%.1 Surgical resection offers the only potentially curative treatment. The 5-year survival rate for patients after tumor resection is 10% for node-positive and 30% for node-negative resections. Other known prognostic factors for surgical patients include tumor size, tumor grade, and tumor stage.2 The poor prognosis of pancreatic cancer is a result of its metastasis-prone and therapy-resistant nature, which is partially explained by the frequent genetic and epigenetic alterations described in this tumor.3 On the other hand, host variations in DNA repair or drug metabolism may also influence clinical response to therapy and overall survival of the patients.4 Prognostic markers are needed to stratify patients on protocols and for use in clinical practice. Predictive markers identifying response to cytotoxic therapy has the potential to further provide a means of individualizing treatment and could lead to a better understanding of resistance mechanisms of both standard and novel treatment strategies. Gemcitabine is a standard of care in pancreatic cancer, offering a slightly better overall survival than fluorouracil for patients with locally advanced and metastatic tumors.5 Little is known about DNA repair pathways that may alter cytotoxicity or radiosensitivity of gemcitabine. Previous studies have shown that gemcitabine-induced radiosensitization and cytotoxic effect of mitomycin were absent in homologous recombination repair (HRR) –deficient cells, suggesting that HRR may play an important role in gemcitabine-mediated cell killing.6,7 HRR is also involved in the repair of DNA double-strand breaks (DSBs), which are the most common type of radiation lesions that lead to mammalian cell death.8,9 Previous clinical studies have shown that individual variation in DNA repair capacity conferred by single nucleotide polymorphisms (SNPs) affects the clinical response to platinum-based cancer therapy and overall survival of patients.10-13 Because gemcitabine- and radiation-induced DNA damage is most likely repaired through the base excision repair (BER) and HRR pathways, we hypothesized that genetic variations in these pathways may affect sensitivity to gemcitabine and radiotherapy, and thus overall prognosis. We tested this hypothesis in a relatively homogeneous population (ie, 92 patients with potentially resectable pancreatic adenocarcinoma who had undergone neoadjuvant gemcitabine-based chemoradiotherapy). We evaluated eight DNA repair genes: RecQ1, RAD54L, ATM, XRCC1, XRCC2, XRCC3, LIG3, and LIG4. We selected 13 SNPs of the eight genes on the basis of their known allele frequencies, functional significance, or previous reports of association with cancer risk or clinical outcome.
Patient Recruitment and Data Collection The study involved 92 patients who, at the time of diagnosis, had potentially resectable adenocarcinoma of the head of the pancreas and had not received any treatment for pancreatic cancer. All patients were enrolled onto one of two phase II clinical trials (ID98-020 and ID01-341) of preoperative (neoadjuvant) chemoradiotherapy at The University of Texas M.D. Anderson Cancer Center (Houston, TX) conducted sequentially from February 1999 to August 2004 and were observed through August 31, 2005. These 92 patients represent the subset of patients enrolled in these clinical trials who consented to blood donation. Patients in the ID98-020 trial (n = 43) had received gemcitabine-based chemoradiotherapy consisting of weekly gemcitabine (400 mg/m2) for 4 weeks and radiation (30 Gy in 10 fractions) for 2 weeks. Patients in the ID01-341 trial (n = 49) had received induction therapy of gemcitabine (750 mg/m2/d) and cisplatin (30 mg/m2/d) every 2 weeks for 4 weeks, followed by chemoradiotherapy with weekly gemcitabine (400 mg/m2) for 4 weeks and radiation (30 Gy in 10 fractions) for 2 weeks. Clinical information was collected from the medical records with the patients’ consent. Clinical stage was defined on the basis of the initial computed tomography imaging and defined as stated in the American Joint Committee on Cancer (AJCC) Cancer Staging Manual using the TNM staging system.14 Pathologic stage was determined after surgery in those patients who underwent a successful resection of their primary tumor, and also used the TNM staging system.14 Tumor progression was defined as local tumor progression, tumor recurrence, metastasis and death as a result of the disease. Patients without tumor progression at the end of the follow-up were censored. The preoperative treatment effect was evaluated histologically in resected tumors according to previously published criteria15 (ie, tumors with more than 90% viable carcinoma cells were defined as treatment effect grade 1, 51% to 90% as grade 2A, 10% to 50% as grade 2B, and less than 10% as grade 3). Postsurgical treatment or treatment received after tumor recurrence were not considered in this study.
DNA Extraction and Genotyping
Survival Measurements Dates of death were obtained and cross checked using at least one of the following sources: the Social Security Death Index (www.deathindexes.com/ssdi.html), inpatient medical records, or the M.D. Anderson Cancer Center tumor registry. Time to progression and overall survival time were calculated from the date the patient had enrolled in the trial to the date tumor progression was first recorded, or the date of death or last follow-up, respectively. When patients with tumor resection were analyzed as a separate group, overall survival time was calculated from the date of tumor resection to date of death or last follow-up.
Statistical Methods
Patient Characteristics and Survival Analysis The median age of the 92 patients was 65 years (range, 38 to 83 years). The patient characteristics and clinical features of the tumor are summarized in Table 2. The median time interval from the initial pathologic diagnosis to enrollment onto the trial was 16 days (range, 1 to 91 days). Of the 92 patients, 62 had their primary tumor surgically resected after preoperative treatment; the remaining 30 patients did not undergo resection as a result of disease progression found at restaging after preoperative therapy (23 patients) or at the time of operation (seven patients). All 62 patients who underwent pancreatic resection had a grossly complete resection; however, pathologic evaluation of the surgical specimens demonstrated a microscopically positive margin (R1 resection) in seven (11.3%) of the 62 patients.
There were 57 deaths (62.0%) among the 92 cases. The median follow-up time was 33 months for the living patients. The MST of the 92 patients was 20.3 months (95% CI, 14.7 to 25.9). Tumor size larger than 1 cm, poor differentiation, serum level of CA19-9 at diagnosis more than 1,000 units/mL, tumor not resected, and node-positive resection (stage IIB) were significantly associated with reduced overall survival (Table 2).
Genotype Frequency and Effects on Overall Survival
Combined Genotype Effects A strong gene-dosage effect was observed when the RecQ1 159 AC/CC, RAD54L 157 CT/TT, XRCC1 199W, and ATM 77TC genotypes were analyzed in combination. Patients with none of the adverse genotypes had a mean survival time of 62.1 months, whereas those with one, two, or more at-risk alleles had MSTs of 27.5, 14.4, and 9.9 months, respectively (P(LR) < .001; Table 3; Fig 2). Further analysis revealed a significant interaction between the RecQ1 A159C genotype and the remaining three SNPs. A strong protective effect of the RecQ1 159AA genotype was observed. In the presence of the AA genotype, the overall survival time was 62.1 (mean), 40.3 (mean), and 10.3 (median) months for those with zero, one, or two adverse genotypes of the RAD54L, ATM, and XRCC1 SNPs, respectively (PLR < .001). Among patients with the RecQ1 159 AC/CC genotypes, however, the MSTs were 20.3, 14.6, and 9.9 months, respectively, if they also had zero, one, or two adverse genotypes of the other three genes (PLR = .17). The likelihood ratio P value for the statistical interaction between RecQ1 and the number of other adverse genotypes was .003.
Genotype Effects on 2-Year Survival and Time to Tumor Progression Two-year survival was evaluated among 72 patients, and the survival rate was 39%. RecQ1 159 AC/CC and ATM 77TC genotypes was significantly associated with a reduced 2-year survival rate, 21% and 15%, respectively (Table 4). The odds ratio of death was 7.5 (95% CI, 2.5 to 22.9) and 6.4 (95% CI, 1.9 to 22.1) for the two adverse genotypes, respectively. Patients with two or more adverse genotypes of the RecQ1, RAD54L, ATM and XRCC1 genes had a 2-year survival rate of only 8% (odds ratio, 20.7; 95% CI, 4.1 to 105; P < .001). The RecQ1 A159C and ATM T77C variant genotypes were also significantly associated with a shorter time to disease progression (Table 4).
Cox Regression Models Finally, we conducted a multivariate analysis of the effects of genotype on survival using Cox proportional hazards models adjusted for other clinical factors. As shown in Table 5, RecQ1 A159C, RAD54L C157T, and ATM T77C genotypes remained significant independent predictors of survival among all patients regardless of their surgical status. The hazard ratios for the RecQ1 159 AC/CC, RAD54L 157 CT/TT, and ATM 77TC genotypes were 3.0 (95% CI, 1.4 to 6.3), 2.9 (95% CI, 1.4 to 6.0), and 1.9 (95% CI, 1.0 to 3.8), respectively, after adjusting for tumor size and CA19-9 level. Tumor grade was not included in this model or in the model for patients with nonresected tumors because information on tumor grade was missing from 28% of the 92 patients. Among patients who had undergone tumor resection, the RecQ1 and RAD54L genotypes remained as significant independent predictors for survival after adjusting for other clinical factors (Table 5).
In this study, we evaluated the effect of 13 SNPs of eight DNA repair genes on the overall survival of patients with resectable pancreatic adenocarcinoma who had received preoperative gemcitabine-based chemoradiotherapy. We demonstrated that the RecQ1 A159C variant genotype, both alone and in combination with other genes, was associated with significantly decreased overall survival in this patient population. The RecQ1 159 AA genotype displayed a strong protective effect and a significant interaction with other genotypes. Three SNPs of the RAD54L, ATM, and XRCC1 genes were also found to significantly affect overall survival. The RecQ1 gene belongs to the DNA helicase family, which includes four additional members: Werner (WRN), Bloom (BLM), RecQ4, and RecQ5.16 RecQ helicases are important tumor suppressors that are not only involved in DNA HRR but also in S-phase checkpoint and telomere maintenance.17,18 Of the five human RecQ helicases identified, three are associated with genetic disorders characterized by an elevated incidence of cancer or premature aging: Werner, Bloom, and Rothmund-Thomson syndrome.19 Although the biologic significance of the WRN and BLM helicases has been investigated extensively, less information is available concerning the functions of the other human RecQ helicases. RecQ1 is known to be able to unwind a diverse set of DNA substrates,20,21 to catalyze efficient strand annealing between complementary single-stranded DNA molecules, and to interact with several important factors required for DNA mismatch repair.22 It is possible that a deficient RecQ1 gene may confer an aggressive tumor phenotype through rapid accumulation of genetic alterations and genomic instability. On the other hand, a deficient RecQ1 function may confer a higher cellular sensitivity to genotoxic stress, thus a better clinical response to cytotoxic therapy. It is tempting to speculate that RecQ1 may play a role in resolving gemcitabine-induced DNA replication arrest. Because the functional significance of the RecQ1 SNPs investigated in this study is currently unknown, the biologic interpretation of the data is difficult. The RecQ1 A159C SNP is located on the 3' untranslated region of the gene and exerted its effect in a dominant-inheritance mode (ie, one variant allele is required to alter the chance of survival). It is possible that this SNP may affect translation efficiency and mRNA stability, or it may be in linkage disequilibrium with other SNPs of the same gene or other important genes located in the same chromosome region. Further investigations to confirm our observations in other patient populations as well as on the biologic functions of this SNP and this gene are warranted. RAD54L belongs to the Asp-Glu-Ala-Asp (DEAD)–like helicase superfamily,19 and it plays a role in the HRR of DNA DSBs. RAD54L has been proposed as a candidate tumor suppressor gene in tumors with a nonrandom deletion of chromosome 1p32.23-25 The RAD54L C157T SNP has previously been associated with increased risk of meningioma.25 The variant allele of this SNP was associated with a significantly reduced overall survival in this study, which could be related to a better repair of DNA DSBs and thus poorer clinical response to therapy. However, functionally, the C157T SNP is a silent polymorphism (Ala730Ala) that does not induce any amino-acid change. We can only speculate that the observed effect on survival was related to linkage disequilibrium of this SNP with other SNPs of the same gene or with other genes on the same chromosome. A haplotype analysis will help to explain this observation. The ATM gene encodes a protein kinase that plays a key role in the detection and repair of DNA DSBs and in maintaining genome integrity.26,27 A previous study28 has shown that polymorphic variants of the ATM gene are associated with increased in vitro chromosomal radiosensitivity. Another study showed that the ATM 77TC heterozygote was associated with a reduced radiosensitivity among patients with breast cancer compared with the TT homozygote.29 Interestingly, we observed significantly reduced survival time among TC heterozygote carriers relative to that among TT and CC homozygote carriers, which is consistent with a poor response to radiotherapy for the TC heterozygote. Because the T-77C SNP is located in an intron region, it is conceivable that its functional difference may be mediated by affecting RNA splicing. However, to date, no functional studies have been reported linking altered protein function or cellular phenotype with the presence of this SNP. Further analysis of the ATM haplotypes is underway. The XRCC1 protein plays an important role in BER.30,31 The XRCC1 194W allele has been previously associated with a reduced risk of cancer but has not been evaluated as a predictor of clinical outcome.32 In this study, we observed an association between the 194W allele and a shorter overall survival time, even though this effect disappeared after other clinical and genetic factors had been adjusted for (Table 5). The Q399R genotype has been shown to affect clinical response to platinum-based cancer therapy,13 but we did not observe any significant effect of this SNP on clinical outcome in this study. The weak effect or lack of effect of the XRCC1 gene SNPs observed in this study are consistent with those of a previous report that the BER pathway does not significantly affect the repair of gemcitabine-induced DNA damage and cell killing.6 XRCC2 and XRCC3 are RAD51 paralogs that play mediating roles in homologous recombination. Ligase IV and ligase III are components of the nonhomologous end joining machinery required for DNA replication and repair.33,34 None of the SNPs of these genes had any significant effect on patient survival or other clinical parameters in this study, suggesting a minor role of these genes in clinical response to gemcitabine-based chemoradiotherapy in patients with resectable pancreatic cancer. This study is limited by its small size, and some of the observations may be made by chance alone. However, the remarkable effect these genotypes have on the overall survival of this relatively homogeneous patient population and the gene dosage effects observed in this study warrant further confirmation in a larger study. If confirmed, such information may provide opportunities for discovery of novel therapeutic targets and genetic profiles that can direct the choice of therapy and predict the treatment tolerance, response, and overall outcome.
The authors indicated no potential conflicts of interest.
ATM (ataxia telangiectasia mutated): The protein, encoded by ATM gene, is a kinase that coordinates DNA repair by activating other proteins. HRR (homologous recombination repair): A DNA repair pathway that repairs the broken ends of DNA by using information on the intact sister chromatid or homologous chromosome. Masscode: The Masscode (BioServe, Laurel, MD) system is a high-throughput genotyping process, which uses a polymerase chain reaction–based, allele-specific discrimination assay measured by means of cleavable mass spectrometry tags. RAD54L: The protein encoded by this gene has been shown to play a role in homologous recombination related repair of DNA double-strand breaks. RecQ1: The protein encoded by this gene is a member of the RecQ DNA helicase family, which comprises enzymes involved in various types of DNA repair. SNP (single nucleotide polymorphism): Genetic polymorphisms are natural variations in the genomic DNA sequence present in greater than 1% of the population, with SNP representing DNA variations in a single nucleotide. SNPs are being widely used to better understand disease processes, thereby paving the way for genetic-based diagnostics and therapeutics. XRCC1 (X-ray repair complementing defective repair in Chinese hamster cells 1): The protein encoded by this gene is involved in the efficient repair of DNA single-strand breaks formed by exposure to ionizing radiation and alkylating agents.
Supported by National Institutes of Health (NIH) RO1 Grant No. CA098380 (D.L.), SPORE P20 Grant No. CA101936 (J.L.A.), NIH Cancer Center Core Grant No. CA16672, and a research grant from the Lockton Research Funds (D.L.). Presented in part at the 13th SPORE Investigators Workshop, Washington, DC, July 9-12, 2005. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Greenlee RT, Murray T, Bolden S, et al: Cancer statistics 2000. CA Cancer J Clin 50:7-33, 2000[Abstract] 2. Cleary SP, Gryfe R, Guindi M, et al: Prognostic factors in resected pancreatic adenocarcinoma: Analysis of actual 5-year survivors. J Am Col Surg 198:722-731, 2004[CrossRef] 3. Bardeesy N, DePinho RA: Pancreatic cancer biology and genetics. Nat Rev Cancer 2:897-909, 2002[CrossRef][Medline] 4. Phillips KA, Van Bebber SL: Measuring the value of pharmacogenomics. Nat Rev Drug Discov 4:500-509, 2005[CrossRef][Medline] 5. Burris HA III, Moore MJ, Andersen J, et al: Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: A randomized trial. J Clin Oncol 15:2403-2413, 1997 6. Crul M, van Waardenburg RC, Bocxe S, et al: DNA repair mechanisms involved in gemcitabine cytotoxicity and in the interaction between gemcitabine and cisplatin. Biochem Pharmacol 65:275-282, 2003[CrossRef][Medline] 7. Wachters FM, van Putten JW, Maring JG, et al: Selective targeting of homologous DNA recombination repair by gemcitabine. Int J Radiat Oncol Biol Phys 57:553-562, 2003[CrossRef][Medline] 8. Shenouda G: Strand-break repair and radiation resistance, in Panasci LC, Alaoui-Jamali MA (eds): DNA Repair in Cancer Therapy. Totowa, NJ, Humana Press, 2004, pp 257-272 9. Peterson CL, Cote J: Cellular machineries for chromosomal DNA repair. Genes Dev 18:602-616, 2004 10. Bosken CH, Wei Q, Amos CI, et al: An analysis of DNA repair as a determinant of survival in patients with non-small-cell lung cancer. J Natl Cancer Inst 94:1091-1099, 2002 11. Camps C, Sarries C, Roig B, et al: Assessment of nucleotide excision repair XPD polymorphisms in the peripheral blood of gemcitabine/cisplatin-treated advanced non-small-cell lung cancer patients. Clin Lung Cancer 4:237-241, 2003[Medline] 12. Park DJ, Stoehlmacher J, Zhang W, et al: Xeroderma pigmentosum group D gene polymorphism predicts clinical outcome to platinum-based chemotherapy in patients with advanced colorectal cancer. Cancer Res 61:8654-8658, 2001 13. Gurubhagavatula S, Liu G, Park S, et al: XPD and XRCC1 genetic polymorphisms are prognostic factors in advanced non-small-cell lung cancer patients treated with platinum chemotherapy. J Clin Oncol 22:2594-2601, 2004 14. Greene FL, Page DL, Fleming ID, et al: Exocrine pancreas, in AJCC Cancer Staging Manual. New York, Springer, 2002, pp 157-164 15. Evans DB, Rich TA, Byrd DR, et al: Preoperative chemoradiation and pancreaticoduodenectomy for adenocarcinoma of the pancreas. Arch Surg 127:1335-1339, 1992 16. Kaneko H, Fukao T, Kondo N: The function of RecQ helicase gene family (especially BLM) in DNA recombination and joining. Adv Biophys 38:45-64, 2004[CrossRef][Medline] 17. Khakhar RR, Cobb JA, Bjergbaek L, et al: RecQ helicases: Multiple roles in genome maintenance. Trends Cell Biol 13(9):493-501, 2003 18. Hickson ID: RecQ helicases: Caretakers of the genome. Nat Rev Cancer 3:169-178, 2003[CrossRef][Medline] 19. Mohaghegh P, Hickson ID: DNA helicase deficiencies associated with cancer predisposition and premature ageing disorders. Hum Mol Genet 10:741-746, 2001 20. Cui S, Arosio D, Doherty KM, et al: Analysis of the unwinding activity of the dimeric RECQ1 helicase in the presence of human replication protein A. Nucleic Acids Res 32:2158-2170, 2004 21. Sharma S, Sommers JA, Choudhary S, et al: Biochemical analysis of the DNA unwinding and strand annealing activities catalyzed by human RECQ1. J Biol Chem 280:28072-28084, 2005 22. Doherty KM, Sharma S, Uzdilla LA, et al: RECQ1 helicase interacts with human mismatch repair factors that regulate genetic recombination. J Biol Chem 280:28085-28094, 2005 23. Rasio D, Murakumo Y, Robbins D, et al: Characterization of the human homologue of RAD54: A gene located on chromosome 1p32 at a region of high loss of heterozygosity in breast tumors. Cancer Res 57:2378-2383, 1997 24. Matsuda M, Miyagawa K, Takahashi M, et al: Mutations in the RAD54 recombination gene in primary cancers. Oncogene 18:3427-3430, 1999[CrossRef][Medline] 25. Leone PE, Mendiola M, Alonso J, et al: Implications of a RAD54L polymorphism (2290C/T) in human meningiomas as a risk factor and/or a genetic marker. BMC Cancer 3:6, 2003[CrossRef][Medline] 26. Golding SE, Rosenberg E, Khalil A, et al: Double strand break repair by homologous recombination is regulated by cell cycle-independent signaling via ATM in human glioma cells. J Biol Chem 279:15402-15410, 2004 27. Shiloh Y: ATM and related protein kinases: Safeguarding genome integrity. Nat Rev Cancer 3:155-168, 2003[CrossRef][Medline] 28. Gutierrez-Enriquez S, Fernet M, Dork T, et al: Functional consequences of ATM sequence variants for chromosomal radiosensitivity. Genes Chromosomes Cancer 40:109-119, 2004[CrossRef][Medline] 29. Angele S, Romestaing P, Moullan N, et al: ATM haplotypes and cellular response to DNA damage: Association with breast cancer risk and clinical radiosensitivity. Cancer Res 63:8717-8725, 2003 30. Caldecott KW, McKeown CK, Tucker JD, et al: An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. Mol Cell Biol 14:68-76, 1994 31. Cappelli E, Taylor R, Cevasco M, et al: Involvement of XRCC1 and DNA ligase III gene products in DNA base excision repair. J Biol Chem 272:23970-23975, 1997 32. Goode EL, Ulrich CM, Potter JD: Polymorphisms in DNA repair genes and associations with cancer risk. Cancer Epidemiol Biomarkers Prev 11:1513-1530, 2003 33. Audebert M, Salles B, Calsou P: Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining. J Biol Chem 279:55117-55126, 2004 34. Wang H, Rosidi B, Perrault R, et al: DNA ligase III as a candidate component of backup pathways of nonhomologous end joining. Cancer Res 65:4020-4030, 2005 Submitted September 29, 2005; accepted December 13, 2005.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test. The association between overall survival time and time to disease progression with genotypes was estimated using the Kaplan-Meier method and assessed using the log-rank test. Median follow-up time was computed with censored observations only. Median survival time (MST) was calculated, and mean survival time was presented when MST could not be calculated. Hazard ratios and 95% CIs were estimated using univariate or multivariate Cox proportional hazards models. Known or potential clinical prognostic factors such as tumor size, tumor grade, tumor stage, the effects of preoperative treatment on tumors, and serum CA19-9 values at diagnosis were included in the multivariate model when appropriate. Gene-gene interactions were determined by generating a cross-product term and tested by using the likelihood ratio test. All statistical testing was conducted with SPSS software, version 12.0 (SPSS Inc, Chicago, IL), and statistical significance was defined as P 
.05. 
