Abstract
Purpose
To investigate the association between two Xeroderma pigmentosum group C polymorphism (XPC Lys939Gln and insertion/deletion PAT −/+ in intron 9) and bladder cancer (BC) susceptibility.
Materials and methods
Genotyping was performed in 208 BC patients and 245 controls by PCR–RFLP method.
Results
XPC PAT +/+ genotype was associated with elevated risk of BC (p = 0.021, OR = 2.49). XPC Lys939Gln AC + CC genotype was significantly associated with risk in invasive stage of BC (p = 0.041, OR = 2.52). Haplotype analysis revealed that variant genotypes C of XPC Lys939Gln and + of PAT, C+ were significantly associated with risk of BC (p = 0.004, OR = 1.70). The CC genotype of Lys939Gln was associated with high risk for recurrence in BCG-treated patients (HR = 3.21, p = 0.036) thus, showing reduced recurrence-free survival (AC + CC/AA = 36/60 months; log rank p = 0.045).
Conclusion
Polymorphisms and haplotypes in XPC appear to influence susceptibility to BC risk. The variant C allele at Lys939Gln may be responsible for early recurrence in BCG-treated patients.
Keywords: Bladder cancer, Bacillus Calmette–Guerin, DNA repair, Polymorphism, Recurrence, XPC
Introduction
Bladder cancer is one of the most common urological malignancies. In men, it is the fourth most common cancer after prostate, lung, and colorectal cancers, accounting for 6.2% of all cancer cases (Jemal et al. 2004). Median age at diagnosis is 69 years in male and 71 years in female (Lynch and Lohen 1995). According to epidemiological data, it is 3 times more common in men than in women and 90% of bladder tumors are transitional cell carcinoma (TCC) (Messing et al. 1995).
Superficial bladder cancer accounts for >85% of newly diagnosed cases. These cases comprise a heterogeneous group of tumors whose individual prognoses are difficult to predict (Chopin and Gattegno 2002). Nevertheless, following transurethral resection, tumor recurrence rate is about 70%, and 10% to 15% of such recurrences may be associated with progression to more invasive forms of cancer (Pashos et al. 2002). Earlier investigations revealed that administration of intravesical Bacille Calmette–Guerin (BCG) significantly reduced the risk of recurrence and progression. However, the response rate for BCG treatment is only 60% to 70%, and about one-third of responders develop recurrence and progression (Merz et al. 1995; Schenk-Braat and Bangma 2005). Consequently, numbers of pathologic and molecular markers have been studied as potential predictors of outcome in bladder cancer.
Among the various risk factors associated with bladder cancer, cigarette smoking is the major risk factor, accounting for about half of the cases in men and one-third of the cases in women. Cigarette smoking contains a variety of chemical carcinogens, including polycyclic aromatic hydrocarbons, aromatic amines, and N-nitrogen compounds that may cause DNA damage by forming DNA adducts. The formation of DNA adducts is associated with increased cancer risk confirmed by numerous basic and epidemiological studies. As a defense mechanism of cell, nucleotide excision repair (NER) is the key player in removing bulky DNA adducts and maintaining genome stability (Hoeijmakers 2001). There has been compelling evidence that deficient DNA repair capacity is a cancer predisposing factor (Berwick and Vineis 2000). The NER pathway has been extensively studied and the main component genes involved in human NER have been elucidated (Wood et al 2001).
The xeroderma pigmentosum complementation group C (XPC) protein plays a key role in nucleotide excision repair (NER) pathway. The functional DNA-binding domains of XPC interact with HR23B to form a complex that recognizes and binds to the sites of DNA damage (Sugasawa et al. 1998; van Hoffen et al 2003). Deficiency in XPC has been implicated in tumorigenesis. For example, XPC-defective mice were highly prone to skin cancer following exposure to UV radiation and also susceptible to common cancers, such as lung, esophageal, and bladder cancers when exposed to chemical carcinogens (Sands et al. 1995).
The XPC gene, which encodes a 940-amino acid protein (Legerski and Peterson 1992; Li et al. 1996), spans 33 kb on chromosome 3p25, and contains 16 exons and 15 introns (Genbank accession no. AC090645). More than a 100 polymorphic variants in the XPC gene have been identified and the two most common polymorphisms are Lys939Gln (XPC A33512C, rs2228001) and a poly (AT) insertion/deletion polymorphism (XPC PAT −/+) in intron 9 which have been associated with risks of many human malignancies, including cancers of lung, bladder, breast, esophagus, skin, oral cavity, and head and neck (Zhang et al. 2008). The PAT+ polymorphism was associated with increased risks of skin (Blankenburg et al. 2005), head and neck (Shen et al. 2001), and esophageal cancer (Casson et al. 2005). The variant allele of Lys939Gln (A → C) was also associated with increased risks of skin (Blankenburg et al. 2005), breast (Zhang et al. 2005), and bladder cancers (Garcia-Closas et al. 2006). A potential rationale behind these gene-cancer risk associations is that these genetic variants may result in alterations in phenotypes like DNA repair capacity (DRC).
Polymorphisms in the coding and regulatory regions of DNA repair genes may modify gene expression, leading to altered DRC. Hence, in the present study we genotyped PAT and Lys939Gln polymorphism of the XPC gene in a hospital-based group matched case–control study to test the hypothesis that this polymorphism is associated with the risk of developing bladder cancer, assessing the main histologic types.
Materials and methods
Study subjects
The participants in this study were unrelated North Indian individuals of similar ethnicity from Lucknow and other adjoining cities of north India visiting Department of Urology at our center during May 2004 to June 2008. Histologically confirmed 208 transitional urothelial bladder cancer patients with mean age of 58.5 years and M/F ratio of 184:24 were recruited in the study. Subjects with previous cancer, metastasized to bladder from other origin, and those with previous radiotherapy were excluded. Healthy and unrelated individuals visiting the hospital for a routine checkup or health awareness camps and hospital employees were recruited as the controls (n = 245). All the controls with mean age of 56.8 years and M/F ratio of 212:33 were age-, sex-, and ethnicity-matched and had no evidence of malignancy or chronic disease. Informed consent from each subject was obtained when interviewing for demographic details and blood sample collection. The ethical committee of the institute approved the study.
Epidemiology data collection
Demographical details were obtained by interviewing each patient personally. The response rate was greater than 80% from the respondents taken as control. Smokers who had quit smoking for more than 1 year were defined as former smokers, and the other smokers categorized as current smokers. At the conclusion of the interview, a 5 ml of blood sample was drawn into coded EDTA vials.
Clinical data collection
The demographic and clinical characteristics of the patients are presented in Table 1. The clinical information about tumor size, number, stage and tumor grade, intravesical therapy and dates of recurrence, chemotherapy, radical cystectomy, pathological findings at cystectomy, and mortality were provided by the urologist in our Department. The tumor stages were classified as per American Joint Committee on Cancer’s 1997 TNM staging system (Sobin and Wittekind 2002).
Table 1.
Characteristics of bladder cancer patients and controls
| Variable | Cases n (%) | Controls n (%) |
|---|---|---|
| Sex | ||
| Male | 184 (88.5) | 212 (86.5) |
| Female | 24 (11.5) | 33 (13.5) |
| Age (years) | ||
| ≤60 | 132 (63.5) | 149 (60.8) |
| >60 | 76 (36.5) | 96 (39.2) |
| Smokinga | ||
| Never | 79 (40.1) | 132 (63.1) |
| Smoker | 83 (42.1) | 44 (21.2) |
| Chewer | 35 (17.8) | 33 (15.7) |
| Tumor numbera | ||
| Single | 110 (58.2) | |
| Multiple | 79 (41.7) | |
| Tumor size (cm)a | ||
| <1 | 43 (23.5) | |
| 1–3 | 94 (51.4) | |
| >3 | 46 (25.1) | |
| Stage | ||
| Ta | 61 (29.3) | |
| T1 | 84 (40.4) | |
| T2 | 63 (30.3) | |
| Grade | ||
| G1 | 78 (37.5) | |
| G2 | 37 (17.8) | |
| G3 | 93 (44.7) | |
aThe sum could not add up to the total due to some missing values
Out of the 208 patients enrolled in the study, 145 patients had superficial bladder cancer while the rest 63 had invasive bladder tumor. Patients with superficial BC were treated with intravesical Bacillus Calmette–Guerin (BCG) (n = 77). BCG immunotherapy treatment is administered to patients with low stage (Ta or T1) and high grade. Therefore patients with low stage and low grade were considered as non-BCG patients. Subsequently, the patients were examined by cystoscopy after every 3 months in first and second years and later at six-monthly intervals as long as there was no tumor recurrence. BCG treatment consisted of 6-weekly instillation (induction course [iBCG] n = 54); induction BCG + maintenance BCG. Since the number of patients receiving mBCG was too low, we did not categorize the patients according to BCG regime for statistical analysis. The end point of study included tumor recurrence, defined as a newly found bladder tumor following a previous negative follow-up cystoscopy, or end of study time (60 months). Patients with invasive BC (n = 63) were treated with radical cystectomy with or without adjuvant chemotherapy, which included cisplatin, gemcitabine followed by periodical cystoscopy. Blood sample was collected in EDTA from all subjects for genotyping at the time of enrollment and stored at −70°C.
Genotype analysis
Laboratory personnel were blinded to case and control status. Genomic DNA was extracted from peripheral blood samples as previously described (Miller et al. 1988). For quality control, genotyping was repeated randomly in at least 5% of the samples, and other lab personnel independently reviewed all results. To determine the XPC PAT polymorphism, intron 9 of the XPC gene was amplified by polymerase chain reaction (PCR) primers and conditions were previously described (López-Cima et al. 2007). The polymorphisms in XPC Lys939Gln (A33512C, rs2228001) were analyzed by PCR combined with restriction fragment length polymorphism (RFLP). PCR was performed in a total volume of 12 μl with 10 pmol of each primer, genomic DNA (100 ng), 0.25 mM each deoxynucleotide triphosphates, 1× PCR buffer containing 10 mM Tris–HCl, pH 8.6, 50 mM KCl, 1.5 mM MgCl2, and 0.5 units of Taq polymerase (Bangalore Genei, India). Amplified product was digested using restriction enzyme PvuII (MBI Fermentas Inc., USA). DNA fragments were resolved on 10% PAGE gels and stained with ethidium bromide.
Statistical analysis
Power of the study was calculated using Quanto software (Version 1.0) (http://hydra.usc.edu/gxe) with input of following variables: case–control study design, significance level (α) < 0.05 (2 sided), model of inheritance = log additive, allele freq = 0.16, genetic effect for odds ratio (OR) ≥ 1.65. Present study achieved 80% of the statistical power for the minor allele of XPC −/+, which exhibited lowest allele frequency among the two polymorphisms. The goodness-of-fit chi-square test was used to analyze any deviation from Hardy–Weinberg equilibrium in controls. Two sided chi-square test was used to look for statistical differences in genotype frequencies between patients and controls. Binary logistic regression model was used to estimate risk as odds ratio (OR) at 95% confidence intervals (CI). Haplotypes of each individual consisting of two SNPs in XPC Lys939Gln (A > C) and PAT (−/+) was constructed and maximum likelihood haplotype frequencies were estimated by expectation–maximization algorithm using Arlequin program version 2.000 (Excoffier and Slatkin 1995). Kaplan–Meier analysis was done to study recurrence-free survival and significance was calculated using log rank test. Cox proportional hazards model (using time to recurrence) was used to assess the effect of individual SNPs on the risk for recurrence. The reference hazard ratio (HR) of 1 was set for the wild-type genotype. A two-tailed p value of <0.05 was considered significant.
Results
Characteristics of subjects
A total of 245 controls and 208 cases were recruited for this study. There was no significant age difference between the cases (58.5 ± 12.5 years) and the controls (56. 8 ± 10.9 years) (p = 0.086). Though the cases had a significantly higher percentage of smokers (59.9%) than the controls (36.7%) (p = 0.01). The demographic details of the study subjects and clinical characteristics of the patients are presented in Table 1.
XPC Lys939Gln (A > C) and PAT (− > +) polymorphisms in bladder cancer
The genotypic distributions of XPC Lys939Gln and PAT polymorphisms in controls were in Hardy–Weinberg equilibrium. The genotype and allele frequencies of XPC Lys939Gln and PAT polymorphism in healthy controls and patients with bladder cancer are shown in Table 2. The variant allele frequency (C) of XPC Lys939Gln and (+) of PAT was higher in cases as compared to controls (32.7 vs. 30.2 and 49.3 vs. 24.5, respectively). Individuals with XPC PAT (+/+) genotype were at higher risk of bladder cancer (p = 0.025, OR = 2.43). XPC Lys939Gln was not significantly associated with the risk of bladder cancer. No association was observed in the case of combination of heterozygotes and homozygous variant genotypes. Also in the case of alleles, no association was seen with susceptibility to bladder cancer risk.
Table 2.
XPC Lys939Gln (A > C) and PAT (− > +) polymorphisms and susceptibility to bladder cancer
| Genotype | Cases n (%) (n = 208) | Controls n (%) (n = 245) | p value | Crude OR (95% CI) | p value | Age–gender–smokinga
adjusted OR (95% CI) |
|---|---|---|---|---|---|---|
|
XPC Lys939Gln (A > C) | ||||||
| AA | 97 (46.6) | 113 (46.2) | Reference | Reference | ||
| AC | 86 (48.2) | 116 (47.3) | 0.326 | 0.82 (0.55–1.21) | 0.278 | 0.80 (0.53–1.19) |
| CC | 25 (12.2) | 16 (6.5) | 0.201 | 1.57 (0.78–3.17) | 0.156 | 1.66 (0.82–3.37) |
| AC + CC | 111 (53.4) | 132 (53.8) | 0.683 | 0.92 (0.63–1.34) | 0.75 | 0.94 (0.64–1.37) |
| A allele | 280 (67.3) | 342 (69.8) | Reference | |||
| C allele | 136 (32.7) | 148 (30.2) | 0.502 | 1.10 (0.83–1.45) | ||
| XPC PAT (−/+) | ||||||
| −/− | 105 (50.5) | 134 (54.7) | Reference | Reference | ||
| −/+ | 80 (38.5) | 100 (40.8) | 0.751 | 1.06 (0.71–1.58) | 0.667 | 1.09 (0.73–1.62) |
| +/+ | 23 (11.0) | 11 (4.5) | 0.021 | 2.49 (1.15–5.41) | 0.025 | 2.43 (1.11–5.32) |
| (−/+) + (+/+) | 103 (49.5) | 111 (45.3) | 0.34 | 1.19 (0.82–1.74) | 0.36 | 1.19 (0.81–1.73) |
| − allele | 290 (69.7) | 368 (78.5) | Reference | |||
| + allele | 206 (49.3) | 122 (24.5) | 0.07 | 1.31 (0.97–1.75) | ||
aSmoking (ever/never smokers) adjusted
Association of an early age onset of bladder cancer with XPC
The median age of the cases was 60. The cases were further sub-grouped into two categories on the basis of median age (i) <60, (ii) >60. Each gene was analyzed in both the groups. The risk was evident in the age group of <60, where XPC PAT +/+ genotypes were associated with elevated risk of bladder cancer (p = 0.04, OR = 3.58). No association was seen in the age group of >60 (Table 3).
Table 3.
Association of an early age of bladder onset with XPC Lys939Gln (A > C) and PAT (− > +) gene polymorphisms
| Age < 60 | Cases (132) n (%) | Controls (149) n (%) | p value | Age–gender–smokinga adjusted OR (95% CI) |
|---|---|---|---|---|
| XPC Lys939Gln (A > C) and PAT (− > +) gene genotypes according to age at diagnosis | ||||
| Lys939Gln | ||||
| AA | 64 (48.5) | 65 (43.6) | ||
| AC | 50 (37.9) | 73 (49.0) | 0.091 | 0.64 (0.38–1.07) |
| CC | 18 (13.6) | 11 (7.4) | 0.357 | 1.48 (0.64–3.44) |
| AC + CC | 68 (51.5) | 84 (56.4) | 0.342 | 0.794 (0.49–1.27) |
| PAT | ||||
| −/− | 64 (48.5) | 80 (53.7) | Reference | |
| −/+ | 57 (43.2) | 65 (43.6) | 0.514 | 1.17 (0.71–1.93) |
| +/+ | 11 (8.3) | 4 (2.7) | 0.04 | 3.58 (1.06–12.1) |
| (+/−) + (+/+) | 68 (51.5) | 69 (46.3) | 0.319 | 1.27 (0.79–2.04) |
| Age > 60 | Cases (76) | Controls (96) | ||
|---|---|---|---|---|
| Lys939Gln | ||||
| AA | 33 (43.4) | 48 (50) | ||
| AC | 36 (47.4) | 43 (44.8) | 0.583 | 1.19 (0.62–2.27) |
| CC | 7 (9.2) | 5 (5.2) | 0.516 | 1.53 (0.42–5.51) |
| AC + CC | 43 (56.6) | 48 (50) | 0.58 | 1.18 (0.64–2.18) |
| PAT | ||||
| −/− | 41 (54.0) | 54 (56.2) | ||
| +/− | 23 (30.2) | 35 (36.5) | 0.642 | 0.85 (0.43–1.67) |
| +/+ | 12 (15.8) | 7 (7.3) | 0.153 | 2.14 (0.75–6.10) |
| (+/−) + (+/+) | 35 (46) | 42 (43.8) | 0.818 | 1.07 (0.58–1.97) |
aSmoking (ever/never smokers) adjusted
Association of genotypes with tumor stage/grade
As the frequency of variant homozygote for DNA repair genes was low with further stratification on the basis of clinical characteristics, we included them into one group with heterozygote. The patients with similar stage but with different grades respond to treatment differently (Larsson et al. 2003). Hence, we stratified the patients into three categories according to stage/grade (TaG1 [low risk], TaG2,3 + T1G1–3 [moderate risk], and T2+ [high risk]). TaG1 was taken as a reference. XPC Lys939Gly AC + CC was observed to be associated with elevated risk with invasive stage (T2+) of bladder cancer (p = 0.041, OR = 2.52). The risk was also observed with TaG2,3 + T1G1–3 stage of bladder but it was not statistically significant (p = 0.065, OR = 2.12). Overall, statistically no significant association was observed in case of XPC PAT −/+ with any of the stages of bladder cancer (Table 4).
Table 4.
Association of XPC Lys939Gln (A > C) and PAT (− > +) polymorphisms with tumor grade/stage categories of BC patients
| XPC | n (%) | n (%) | p value | Crude OR (95% CI) | p value | Adjusted OR (95% CI) |
|---|---|---|---|---|---|---|
| Lys939Gln | AA | AC + CC | ||||
| TaG1 | 22 (22.7) | 13 (11.7) | 1.00 | – | – | |
| TaG2-3, T1G1-3 | 48 (49.5) | 62 (56.3) | 0.069 | 2.08 (0.94–4.59) | 0.065 | 2.12 (0.95–4.75) |
| T2+ | 27 (27.8) | 36 (57.0) | 0.055 | 2.30 (0.98–5.40) | 0.041 | 2.52 (1.03–6.14) |
| PAT | −/− | (−/+) + (+/+) | ||||
| TaG1 | 19 (18.1) | 16 (15.5) | 1.00 | – | – | |
| TaG2-3, T1G1-3 | 47 (44.8) | 63 (61.2) | 0.352 | 1.44 (0.66–3.15) | 0.337 | 1.46 (0.67–3.21) |
| T2+ | 39 (37.1) | 24 (23.3) | 0.407 | 0.69 (0.29–1.63) | 0.325 | 0.64 (0.26–1.55) |
Haplotype analysis of XPC Lys939Gln and PAT with risk of bladder cancer
Haplotype frequency of XPC Lys939Gln and PAT polymorphisms are presented in Table 5. Haplotype A− (A of Lys939Gln and − of PAT) was taken as reference. The haplotype C+ (C of Lys939Gln and + of PAT) showed 1.74-folds increase in risk for bladder cancer (OR = 1.74, p value = 0.004).
Table 5.
Frequency distribution of haplotype XPC Lys939Gln (A > C) and PAT (− > +) in cases and controls
| Haplotype | Patients | Controls | p value | OR (95% CI) | ||
|---|---|---|---|---|---|---|
| n | % | n | % | |||
| A− | 202 | 48.56 | 280 | 57.03 | Reference | – |
| C− | 88 | 21.15 | 87 | 18.08 | 0.056 | 1.40 (0.99–1.98) |
| C+ | 78 | 18.75 | 62 | 12.54 | 0.004 | 1.74 (1.19–2.54) |
| A+ | 48 | 11.54 | 61 | 12.36 | 0.685 | 1.09 (0.71–1.65) |
Modulation of genotype variants and outcome after BCG immunotherapy
To analyze the association of XPC (Lys939Gln A > C, PAT) polymorphisms and risk of recurrence in superficial BC patients, further analysis was restricted to 145 superficial BC patients with a median follow-up of 14 months (3–60 months). BCG immunotherapy is conventionally used to reduce the rate of recurrence in superficial BC (Schenk-Braat and Bangma 2005; Table 6). Our data also supported the finding as BCG-treated patients showed reduced risk of recurrence (HR = 0.589, p = 0.046). Subsequently, we analyzed the association of genotypes and risk of recurrence after BCG immunotherapy. We grouped patients into BCG-treated (n = 77) and non-treated (n = 56). In case of XPC PAT +/+ genotype, significant elevated risk was evident in patients receiving no treatment (p = 0.009, OR = 6.80). The results demonstrated that the XPC Lys939Gln CC genotype exhibited risk for recurrence in BCG-treated group (HR = 3.21, p = 0.03). As recurrence risk was observed in BCG-treated patients, further recurrence-free survival was calculated by Kaplan–Meier analysis. The results showed a lower median recurrence-free survival of 36 months for CC genotype as compared to 60 months for AA genotype carrying patients (log rank p = 0.045; Fig. 1; Table 7).
Table 6.
Cox regression analysis for risk of recurrence/progression and treatment variables
| Variables | No recurrence n (%) | Recurrence n (%) | HR (95% CI), p value |
|---|---|---|---|
| Sex | |||
| Male | 70 (86.4) | 56 (87.5) | Reference |
| Female | 11 (13.6) | 8 (12.5) | 0.74 (0.33–1.54), 0.394 |
| Age (years) | |||
| ≤60 | 55 (67.9) | 40 (62.5) | Reference |
| >60 | 26 (32.1) | 24 (37.5) | 1.27 (0.75–2.13), 0.364 |
| Grade | |||
| G1 | 36 (44.4) | 35 (54.7) | Reference |
| G2 | 15 (18.5) | 14 (21.9) | 0.842 (0.48–1.45), 0.538 |
| G3 | 30 (37.1) | 15 (23.4) | |
| Tumor number | |||
| Single | 45 (60) | 34 (54.8) | Reference |
| Multiple | 30 (40) | 28 (45.2) | 2.64 (0.93–2.76), 0.05 |
| Tumor size (cm) | |||
| <1 | 13 (17.6) | 24 (40.7) | Reference |
| 1–3 | 39 (52.7) | 27 (45.7) | 3.19 (1.11–9.17), 0.268 |
| >3 | 22 (29.7) | 8 (13.6) | 2.28 (0.68–7.61), 0.033 |
| Type of treatment | |||
| Non-treated | 25 (30.9) | 31 (48.4) | Reference |
| BCG | 49 (60.5) | 28 (43.8) | 0.589 (0.35–0.99), 0.046 |
| Mitomycin C | 7 (8.6) | 5 (7.8) | 0.838 (0.32–2.16), 0.715 |
Fig. 1.
Kaplan–Meier curve of influence of XPC Exon 15 A > C polymorphism and risk of recurrence in BCG-treated patients; median recurrence-free survival for AA = 60 months and AC + CC = 36 months, log rank p value = 0.0447
Table 7.
Association of XPC Lys939Gln (A > C) and PAT (− > +) genotypes on the outcome of BCG immunotherapy
| Genotype | No recurrence | Recurrence | HR | p value (95% CI) | ||
|---|---|---|---|---|---|---|
| n | % | n | % | |||
| XPC Lys939Gln (A > C) | ||||||
| All patients | ||||||
| AA | 35 | 43.3 | 21 | 32.8 | 1.00a | |
| AC | 36 | 44.4 | 33 | 51.6 | 1.37 | 0.295 (0.75–2.49) |
| CC | 10 | 12.3 | 10 | 15.6 | 1.69 | 0.181 (0.78–3.68) |
| AC + CC | 46 | 56.7 | 43 | 67.2 | 1.78 | 0.052 (1.02–3.07) |
| Non-BCG | ||||||
| AA | 13 | 40.6 | 14 | 39.0 | 1.00a | |
| AC | 14 | 43.8 | 19 | 52.7 | 1.19 | 0.682 (0.51–2.75) |
| CC | 5 | 15.6 | 3 | 8.3 | 1.56 | 0.515 (0.40–5.96) |
| AC + CC | 19 | 59.4 | 22 | 61.0 | 1.07 | 0.772 (0.46–2.46) |
| BCG | ||||||
| AA | 22 | 44.9 | 7 | 25.0 | 1.00a | |
| AC | 22 | 44.9 | 14 | 50.0 | 1.77 | 0.260 (0.65–4.81) |
| CC | 5 | 10.2 | 7 | 25.0 | 3.21 | 0.036 (1.07–9.61) |
| AC + CC | 27 | 55.1 | 21 | 75.0 | 3.98 | 0.031 (1.02–10.7) |
| XPC PAT (−/+) | ||||||
| All patients | ||||||
| −/− | 35 | 43.2 | 30 | 46.9 | 1.00a | |
| −/+ | 38 | 46.9 | 30 | 46.9 | 0.819 | 0.482 (0.47–1.42) |
| +/+ | 8 | 9.9 | 4 | 6.3 | 1.70 | 0.332 (0.58–4.97) |
| (−/+) + (+/+) | 46 | 56.8 | 34 | 53.2 | 0.78 | 0.395 (0.47–1.34) |
| Non-BCG | ||||||
| −/− | 19 | 59.4 | 11 | 30.5 | 1.00a | |
| −/+ | 11 | 34.4 | 21 | 58.3 | 1.07 | 0.868 (0.47–2.43) |
| +/+ | 2 | 6.2 | 4 | 11.2 | 6.80 | 0.009 (1.61–28.8) |
| (−/+) + (+/+) | 13 | 40.6 | 25 | 69.5 | 4.05 | 0.024 (0.48–20.2) |
| BCG | ||||||
| −/− | 16 | 32.7 | 19 | 67.9 | 1.00a | |
| −/+ | 27 | 55.1 | 9 | 32.1 | 0.534 | 0.171 (0.21–1.31) |
| +/+ | 6 | 12.2 | 0 | 0 | NC | 0.983 (0) |
| (−/+) + (+/+) | 33 | 67.3 | 9 | 32.1 | NC | 0.983 (0) |
HR hazards ratio, NC not calculated
aUsed as reference category
Discussion
Carcinogenesis is a multistage process, which involves various molecular events related to the fundamental alterations in cell physiology including self-support in growth signals, insensitivity to growth-inhibitory signals, escape from apoptosis, infinitive replication, sustained angiogenesis, and tissue invasion and metastasis (Hanahan and Weinberg 2000).
Xeroderma pigmentosum complementation group C (XPC) is an important DNA nuclear excision repair (NER) gene that recognizes the damage caused by a variety of bulky DNA adducts. The present study elucidated that individuals with XPC PAT +/+ genotypes were at significant increased risk of bladder cancer (p = 0.025, OR = 2.43, 95% CI = 1.11–5.32). The PAT+ polymorphism has been shown to be associated with increased risks of skin (Blankenburg et al. 2005), head and neck (Shen et al 2001), and esophageal cancer (Casson et al 2005). Qiao et al. 2002 studied XPC genotype-related DRC using a host-cell reactivation assay and found that healthy subjects with the homozygous variant genotype of the PAT polymorphism (+/+) exhibited lower DRC as compared to wild-type carriers (−/−).
No risk was observed in XPC Lys939Gln CC gene polymorphism (p = 0.156, OR = 1.66). A recent meta-analysis of 34 case–control studies by Zhang et al. 2008 showed XPC Lys939Gln allele C and XPC PAT+ allele to be associated with lung, breast, bladder, colorectal, esophageal, and other cancer risks. The lack of associations found in XPC Lys939Gln CC gene polymorphism (p = 0.156, OR = 1.66) in our study is a finding consistent with the results of a previous study of XPC polymorphisms A939C in bladder cancer (Sak et al. 2005), but our results contradict the findings of another study, in which a significantly elevated risk of bladder cancer was observed with a variant allele of Lys939Gln (Fontana et al. 2008). Therefore, it can be speculated that functional variation of XPC may influence the individual susceptibility of bladder cancer.
On the basis of age stratification, the patients carrying the XPC +/+ genotype with age <60 (p = 0.04, OR = 3.58) were observed to be at 3-folds elevated risk of bladder cancer. This suggested that bladder cancer is associated with early onset of age in XPC PAT +/+. No association was observed with XPC Lys939Gln on the basis of age stratification. Polymorphisms in the coding and regulatory regions of XPC gene may alter the gene expression and thereby modulate the DNA repair function. The variant allele of PAT (+) is associated with lower/reduced DNA repair capacity, the probable mechanism could be that DNA damage due to exogenous and endogenous agents might not be able the trigger the repair pathways effectively resulting in reduced ability to repair DNA lesions and an increased risk of developing cancer.
Association of the genotypes with disease stages was carried out to explore the influence of variant genotypes on disease phenotype. Patients with invasive stage (T2+ category) of bladder cancer and XPC (Lys939Gln) CC genotypes were at very high risk with statistically significant association (OR = 2.52, p = 0.041) in comparison to non-invasive group (TaG1). This perhaps suggests that XPC (Lys939Gln) C allele carriers have low enzyme activity and thus, are unable to annul the effect of DNA damage. Similar results were seen in a study of French cohort, where the Gln allele of the XPC 939 polymorphism was found to be associated with an increase in bladder cancer risk (Fontana et al. 2008).
XPC PAT +/+ showed no significant association with any of the stage/grade categories.
We have also analyzed interaction of XPC genotypes with tobacco exposure to investigate the modulation of risk. In the present study there was no association with smoking for bladder cancer risk (Data not shown).
In a retrospective study, taking into account the follow-up data of each patient, comparison was done on the basis of recurrence/no recurrence of bladder tumor in superficial bladder cancer patients. In case of XPC PAT, statistically significant risk was observed with the variant allele carrier (+/−) + (+/+) genotype in non-treated patients (p = 0.024, OR = 4.05), that is, chances of tumor recurrence is higher in patients carrying the PAT +/+ allele in non-treated category.
We further observed that CC genotype at Lys939Gln showed 3.12-folds increase in recurrence risk (HR = 3.12, p = 0.036) in BCG-treated patients. Since recurrence risk for BC patients on BCG immunotherapy was observed, Kaplan–Meier recurrence-free survival analysis was conducted. This demonstrated AA genotype recurrence-free survival of 60 months in comparison to median recurrence-free survival of 36 months for CC genotype (p = 0.045). These results clearly suggest that mutant C allele at Lys939Gln (A > C) might be responsible for early recurrence of bladder tumor in patients on BCG immunotherapy.
Haplotype analysis has an edge over single-locus analysis in terms of disease susceptibility by assessment of interaction among two or more genes that are closely located to each other. Haplotype C+ was found to be associated with increased risk of bladder cancer in our study (p = 0.004, OR = 1.74). In view of frequency distribution of XPC genotypes, the variant allele of XPC is thus at higher risk.
In summary, this study provided further evidence that the XPC genotypes and haplotypes may contribute to susceptibility to bladder cancer. We found that the variant alleles of the +/+ XPC Lys939Gln and PAT polymorphism was associated with lower DRC. These results provide biological plausibility from previous epidemiologic studies linking XPC polymorphisms with altered cancer risk. We agree that relevant data is sparse about the effects of deficient DNA repair capacity on treatment outcome. Further, larger studies are needed to confirm our findings and some mechanistic studies are warranted to investigate the functions of XPC SNPs and mechanisms underlying its associations with bladder cancer risk.
Acknowledgments
The study was supported by the grant of Department of Science and Technology, New Delhi Govt. of India. RG is thankful to Council of Scientific and Industrial Research, New Delhi for JRF.
Conflict of interest statement
None.
Abbreviations
- XPC
Xeroderma pigmentosum group C
- BCG
Bacillus Calmette–Guerin
- BC
Bladder cancer
- MMC
Mitomycin-C
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