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Glutathione S-Transferase M1 and T1 Polymorphisms in Brazilian African Descendants

The glutathione S-transferase gene family has an important role in the biotransformation and detoxification of different xenobiotics and endogenous compounds. Two polymorphic genes of this family, GSTM1 and GSTT1, present null alleles that
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  Glutathione S-transferase M1 and T1 polymorphisms:Susceptibility and outcomes in muscle invasive bladder cancerpatients Ho won Kang a,f  , Phil Hyun Song b,f  , Yun-Sok Ha a,e , Won Tae Kim a ,Yong-June Kim a , Seok-Joong Yun a , Sang-Cheol Lee a , Yung Hyun Choi c ,Sung-Kwon Moon d , Wun-Jae Kim a, ⇑ a Department of Urology, College of Medicine, Chungbuk National University, Cheongju, South Korea b Department of Urology, College of Medicine, Yeungnam University, Daegu, South Korea c Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan, South Korea d Department of Food and Biotechnology, Chungju National University, Chungju, South Korea e The Section of Urologic Oncology and Dean and Betty Gallo Prostate Cancer Center, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ, USA KEYWORDS Bladder cancerEpidemiologyGlutathione S-transferase GSTT1 PolymorphismPrognosis Abstract  Background:  We investigated whether genetic polymorphisms in the  glutathione S transferase mu  ( GSTM1 ) and  theta  ( GSTT1 ) genes modulated risk, disease progression andsurvival in primary muscle invasive bladder cancer (MIBC). Methods:  GSTM1  and  GSTT1  polymorphisms were analysed by multiplex polymerase chainreaction (PCR) using blood genomic DNA in 110 MIBC patients and 220 gender- and age-matched healthy controls. The influence of the genetic polymorphisms on patient survivalwas evaluated by Kaplan–Meier survival curves and Cox Proportional Hazard models. Wealso evaluated whether cigarette smoking and treatment modality modified the associationbetween genotype and prognosis. Results:  GSTM1 -null individuals exhibited increased risk for MIBC and an association withcigarette smoking.  GSTT1 -null subjects showed significant disease progression and cancer-specific death. In the combined analysis,  GSTT1- null genotype was an independent risk factorfor disease progression and cancer specific death regardless of   GSTM1  genotype. Significantdifferences in progression-free survival (PFS) and cancer-specific survival (CSS) were seenbased on  GSTT1  genotype. The survival impact of the  GSTT1  genotype was only valid forsmokers. The  GSTT1- null genotype was an independent prognostic factor for shorter PFS 0959-8049/$ - see front matter    2013 Elsevier Ltd. All rights reserved. ⇑ Corresponding author:  Address: Department of Urology, Chungbuk National University, College of Medicine and Institute for TumorResearch, 62 Kaeshin-dong, Heungduk-gu, Cheongju 361-763, South Korea. Tel.: +82 043 269 6371; fax: +82 043 269 6144. E-mail address: (W.-J. Kim). f  Co-first author.European Journal of Cancer  (2013)  xxx , xxx  –  xxx Available at journal homepage: www.ejcancer.comPlease cite this article in press as: Kang H.w. et al., Glutathione S-transferase M1 and T1 polymorphisms: Susceptibility and outcomes in muscleinvasive bladder cancer patients,  Eur J Cancer  (2013),  in patients who received chemotherapy and those who did not undergo radical cystectomy. Bymultivariate Cox regression analysis,  GSTT1 -null genotype was a predictive factor for diseaseprogression and cancer specific survival regardless of treatment modality. Conclusions:  The  GSTM1 -null genotype plays an important role in genetic susceptibility toMIBC and the  GSTT1 -null genotype is associated with disease progression and shorter sur-vival in MIBC.   2013 Elsevier Ltd. All rights reserved. 1. Introduction Although only 20% of bladder cancer patients arediagnosed as having muscle invasive bladder cancer(MIBC), the main cause of death in MIBC patients ismetastasis. 1,2 Bladder cancer has diverse biological andfunctional characteristics. Therefore, it is very difficultfor urologists to estimate the success rate of treatmentsand to counsel patients about prognosis. Prognosticinformation obtained from conventional histopatholo-gical parameters such as tumour stage or grade andlymph node status is insufficient to predict outcomes. 3,4 The limited value of established prognostic markersrequires the analysis of new molecular parameters of interest for predicting the prognosis of bladder cancerpatients in the clinical setting. 5,6 Although the precisereason why specific individuals develop bladder cancerand progress to invasive disease with poor prognosisremains unknown, important determinants of popula-tion risk to bladder cancer may include genetic andepigenetic alterations in proto-oncogenes and tumoursuppressor genes, loss of heterozygosity for specificalleles and genetic polymorphisms. 6 The influence of genetic polymorphisms in  glutathi-one S transferase mu  ( GSTM1 ) and  theta  ( GSTT1 ) onsusceptibility to numerous cancers has received particu-lar interest because they play a role in the detoxificationof carcinogenic species. 7 Bladder cancer is oftendescribed as polyclonal field cancerisation associatedwith highly concentrated urine that contains endo- orexogenous cytotoxic and potentially carcinogenic chem-icals. 8,9 Because the metabolism of tobacco-related car-cinogens may be influenced by the activity of GSTs,polymorphisms in  GSTM1  and  GSTT1  may modifythe risk of bladder cancer associated with these carcino-gens. The  GSTM1 -null genotype has been consistentlyassociated with an increased risk of bladder cancer inpooled and meta-analyses. On the other hand, studiesinvestigating the importance of   GSTT1  in bladder carci-nogenesis have been more limited and inconsistent. 10 Specific enzymes that are known to be important incarcinogenesis can also play a critical role in diseaserecurrence and progression after initial treatment.  GST  polymorphisms may influence survival via GST-medi-ated detoxification of chemotherapeutic agents andsomatic change in tumour tissue. 11 Based on thishypothesis, trials have recently been conducted to assessthe relationship between  GST   polymorphisms and clini-copathological characteristics and prognoses in patientswith solid tumours (ovarian cancer, lung cancer, breastcancer, oesophageal cancer) and haematologic malig-nancies. 11–15 We have previously reported that the GSTT1  genotype could be a useful prognostic markerfor recurrence and progression in non-muscle invasivebladder cancer (NMIBC). 16 However, the prognosticvalue of   GST   polymorphisms in patients with bladdercancer has not yet been sufficiently elucidated.To the best of our knowledge, the prognostic implica-tionsof  GST  polymorphisminMIBChavenotbeenpub-lished. The aim of this study is to investigate thecontribution of   GSTM1  and  T1  genotypes to MIBC sus-ceptibility, as well as the usefulness of   GST   polymor-phisms as prognostic markers in MIBC and theirinteraction with smoking. 2. Materials and methods  2.1. Study population A case–control study was conducted including 110cases with primary MIBC and 220 controls. The con-trols were selected from among patients with non-malig-nant diseases and two-to-one matched with similar ages.Cases were recruited from the patients with MIBC atour institution who were histologically verified as havingurothelial carcinoma. To reduce confounding factorsaffecting the analyses and to delineate a more homoge-nous study population, any patients diagnosed with aconcomitant carcinoma  in situ  (CIS), received radiationtherapy or neoadjuvant chemotherapy, as well aspatients with short-term follow-up periods (less than6 months) were excluded. A questionnaire structuredto obtain a detailed smoking history was provided toeach subject by well-trained interviewers. Patients weredivided into three categories based on smoking statusas follows: smokers (current smokers and patients whoquit smoking within 10 years), ex-smokers (those whoquit smoking at least 10 years previously), and non-smokers (those who had never smoked). However,because the number of ex-smokers was relatively small( N   = 13; 11.8%) to be analysed as an independentgroup, ex-smokers were included in the smokers group.The Ethics Committee of Chungbuk National Univer-sity approved this protocol and written informed 2  H.won Kang et al./European Journal of Cancer xxx (2013) xxx–xxx Please cite this article in press as: Kang H.w. et al., Glutathione S-transferase M1 and T1 polymorphisms: Susceptibility and outcomes in muscleinvasive bladder cancer patients,  Eur J Cancer  (2013),  consent was obtained from each subject. Collection andanalysis of all samples was approved by the InstitutionalReview Board of Chungbuk National University.Tumourswerestagedaccordingtothe2002TMNclas-sification and the 1973 World Health Organization(WHO) grading system. 17,18 All diagnoses were con-firmed by pathological analysis of frozen sections fromcystectomyandtransurethralresection(TUR)specimens.Patients with clinically localised or locally advancedtumoursandgoodEasternCooperativeOncologyGroup(ECOG) performance status (0 or 1) underwent radicalcystectomy and complete pelvic lymph node dissection.Patients who were not eligible for radical cystectomydue to metastatic disease, poor life expectancy, or poorECOG performance status ( P 2) underwent TUR orbiopsy for histopathological diagnosis. Patients withpT3, pT4, or node-positive disease based on the analysisof radical cystectomy specimens, or with metastatic dis-ease but good performance status, received at least 4cycles of cisplatin-based chemotherapy. Alternatively,patients with poor general health and advanced age,andthosewhowerereluctanttoundergoaggressivetreat-ment,didnotreceivechemotherapy.Eachpatientwasfol-lowed and managed according to standard practice. Inthisstudy,progressionwasdefinedaslocalregionalrecur-rence or new distant metastasis in the cystectomisedgroup, and  P 20% increment of mass or new distantmetastasis in the non-cystectomised group.  2.2. Samples and DNA extraction Before the operation, a blood sample of 5 ml was col-lected from each patient into 0.1 ml EDTA, frozen inliquid nitrogen, and stored at  80   C until use. GenomicDNA was extracted from human whole blood for geno-typing using a genomic DNA purification kit (Promega,Madison, WI) in accordance with the manufacturer’sinstructions.  2.3. Genotype assays of polymorphism Amultiplex polymerase chain reaction (PCR)methodwas applied to detect the presence or absence of the GSTM1  and  GSTT1  genes in genomic DNA samples, asdescribed previously. 19 In brief, the primers used in thePCR were 5 0 -GAACTCCCTGAAAAGCTAAAGC-3 0 (sense) and 5 0 -GTTGGGCTCAAATATACGGTGG-3 0 (anti-sense) for  GSTM1 ; 5 0 -TTAGCTGACCTCG-TAGCCAT-3 0 (sense) and 5 0 -GAAGTCCTTGGCCTT-CAGAA-3 0 (anti-sense) for  GSTT1  and 5 0 -GAAGAGCCAAGGACAGGTAC-3 0 (sense) and 5 0 -CAA CTTCATCCACGTTCACC-3 0 (anti-sense) for beta-globin( b -globin).DNA(200 ng)wasamplifiedinatotalvolumeof 20  l L, containing 10 pmol of each primer, 0.5 U Taqpolymerase,2.5 mMdNTPand10  PCRbuffer.Follow-ing an initial denaturation step at 94   C for 5 min, 40cycles of amplification were carried out at 94   C for60 s, 63   C for 60 s and 72   C for 60 s, with a final exten-sionstepat72   Cfor10 min.Theamplifiedproductswereelectrophoresed on a 2% agarose gel, and their sizes were219 bp for  GSTM1 , 372 bp for  GSTT1  and 268 bp for b -globin. Both positive and negative samples wereanalysed in each experiment, and  GSTM1  and  GSTT1 genotypes were not scored unless the internal referencegene ( b -globin) product was evident.  2.4. Statistical analysis The Chi-square method was used to test the frequen-cies of   GSTM1  and  GSTT1  genotypes between case andcontrol groups. To examine the combined effects of  GSTM1  and  GSTT1  on survival and recurrence, adummy variable was created with three categories repre-senting the presence of alleles as follows: presence of both  GSTM1  and  GSTT1 , presence of one of the  GST  genes and null status for both  GSTM1  and  GSTT1 .Logistic regression models were used to estimate theodds ratio (OR) with the corresponding 95% confidenceinterval (CI). The Kaplan–Meier method was used toestimate cancer-specific survival (CSS), and differenceswere assessed using log-rank statistics. The prognosticvalue of   GSTM1  and  GSTT1  genotypes was analysedusing univariate and multivariate Cox ProportionalHazard regression models. Statistical analyses were per-formed using SPSS 12.0 software (SPSS Inc., Chicago,IL), and a  P  -value of <0.05 was considered significant. 3. Results 3.1. Baseline characteristics Table 1 lists the baseline characteristics of the 110MIBC patients and 220 population controls included inthestudy.ThemeanageofMIBCpatientswas67.8 years(range 34–87) and that of the controls was 67.9 years(range34–89).Themedianfollow-upperiodoftheMIBCpatientswas16.1 months(range,0.9–181.0).Thefrequen-cies of the  GSTM1 -null and  GSTT1 -null genotypes were59.1% and 58.2% in the MIBC group and 46.8% and58.2% in the control population, respectively. The fre-quency of both  GSTM1-  and  GSTT1 -null status was31.8%intheMIBCgroupand19.1%inthecontrolgroup.Sixty-four percent (71/110) of the MIBC patients weresmokers and 35.5% (39/110) were non-smokers. Of the110 MIBC patients, 61 (55.5%) cases underwent radicalcystectomy and 48 (43.6%) patients received adjuvantchemotherapy. During follow-up, 51 of the 110 MIBCpatients (46.4%) had progression and 46 (41.8%) died of bladder tumours. 3.2. Interactions of GST genotypes with smoking and MIBC susceptibility Smoking history was significantly associated withsusceptibility of MIBC (OR = 2.22; 95% CI, 1.39–3.57; H.won Kang et al./European Journal of Cancer xxx (2013) xxx–xxx  3Please cite this article in press as: Kang H.w. et al., Glutathione S-transferase M1 and T1 polymorphisms: Susceptibility and outcomes in muscleinvasive bladder cancer patients,  Eur J Cancer  (2013),  P   = 0.001) (Table 1). The  GSTM1 -null genotype wasmore common in the MIBC group than in the controlgroup (OR = 1.30, 95% CI = 1.01–1.68,  P   = 0.037).After grouping by smoking status, smokers with a GSTM1 -null genotype had an approximately 1.3-foldhigher risk of MIBC ( P   = 0.043) (Table 2). 3.3. Relationship of GST genotype withclinicopathological parameters, disease progression and cancer-specific death Associations between the  GST   genotype and histopa-thological parameters such as tumour stage and gradewere not seen.  GSTT1 -null patients exhibited greaterfrequencies of disease progression and CSD( P   = 0.002,  P   = 0.006, respectively) (Table 3). However,the  GSTM1  genotype was related to neither disease pro-gression nor CSD.After stratification by smoking status, the associationbetween the  GSTT1  genotype and prognosis was statis-tically significant only in smokers (Table 4). The GSTM1  genotype was not correlated with MIBC prog-nosis regardless of smoking history ( P   > 0.05 in eachcase; data not shown). 3.4. Combination effects of GST genotypes on disease progression and CSD in MIBC  The results of logistic regression analysis indicatedthat the risk of disease progression was higher in sub- jects with  GSTT1- null/ GSTM1- positive and  GSTT1- null/ GSTM1- null genotypes than in those with GSTT1- positive/ GSTM1 -positive. Similar relationshipswere noted for the risk of CSD (Table 5).After grouping by smoking status, smokers with the GSTT1- null/ GSTM1- positive genotype were at approx-imately 16-fold and 13-fold higher risk for disease pro-gression and CSD, respectively ( P   = 0.014,  P   = 0.022,respectively). 3.5. Genotypes of GSTM1 and GSTT1, and MIBC survival  Kaplan–Meier estimates revealed significant differ-ences in time to progression and CSS according to the GSTT1  genotype. Patients with the  GSTT1 -null geno-type had significantly poorer PFS and CSS than thosewith the  GSTT1 -positive genotype (log rank test, P   = 0.009 and  P   = 0.030, respectively) (Fig. 1a and c).However, no individual effect of the  GSTM1  genotypewas found in relation to time to disease progressionand CSD (Fig. 1b and d).In the subgroup analysis according to smoking his-tory, smokers with a  GSTT1- null genotype had signifi-cantly reduced PFS and CSS compared to those withthe  GSTT1 -positive genotype (log rank test,  P   = 0.021and  P   = 0.030) (Fig. 2a). Moreover, the  GSTT1- nullgenotype was found to be an independent prognosticfactor for shorter PFS in patients who received chemo-therapy or did not undergo radical cystectomy (log-ranktest,  P   = 0.036,  P   = 0.027, respectively) (Fig. 2b and c).In addition, subgroup analysis was performed accordingto TNM stage (localised BC;  6 T3N0M0 versusadvanced BC; T4 or any N1 or M1), grade (grade 2 ver-sus 3) and treatment combination (cystectomy only ver-sus cystectomy + chemotherapy versus TURB onlyversus TURB + chemotherapy). The prognostic valueof the  GSTT1  genotype in terms of PFS appeared onlyin advanced/metastatic BC, grade 3 disease, and TURBalone group (log-rank test,  P   = 0.022,  P   = 0.012 and P   = 0.043, respectively) (Figures not shown). Table 1Baseline characteristics.Parameters Controls( n  = 220)Cases( n  = 110)Mean ± SD age (years, range) 67.9 ± 10.5(34–89)67.8 ± 9.7 (34– 87)Median follow-up period (months,range) – 16.1 (0.9– 181.0)GenderMale 175 (79.5) 88 (80.0)Female 45 (20.5) 22 (20.0)Smoking a Never 121 (55.0) 39 (35.5)Prior or current 99 (45.0) 71 (64.5) GST   polymorphism GSTM1- null 103 (46.8) 65 (59.1) GSTT1 -null 128 (58.2) 64 (58.2)GradeG2 – 39 (35.5)G3 – 71 (64.5)TNM stageT2N0M0 – 36 (32.7)T3N0M0 – 21 (19.1)T4 or higher than N0M0 – 53 (48.2)Radical cystectomyNo – 49 (44.5)Yes – 61 (55.5)ChemotherapyNo – 62 (56.4)Yes – 48 (43.6)ProgressionNo – 59 (53.6)Yes – 51 (46.4)SurvivalAlive – 36 (32.7)Deceased 74 (67.3)Cancer-related – 46 (41.8)Non-cancer-related – 28 (25.5)SD = standard deviation; null = alleles that have a deletion of the GSTM1  or  GSTT1  genes. a Odds ratio (OR) = 2.22; 95% confidence interval (CI), 1.39–3.57; P   = 0.001 on the chi-square test.4  H.won Kang et al./European Journal of Cancer xxx (2013) xxx–xxx Please cite this article in press as: Kang H.w. et al., Glutathione S-transferase M1 and T1 polymorphisms: Susceptibility and outcomes in muscleinvasive bladder cancer patients,  Eur J Cancer  (2013),  On multivariate analysis,  GSTT1 -null genotype wasindependent predictors for disease progression [hazardratio (HR), 3.110; 95% CI, 1.551–6.235;  P   = 0.001] andCSS [(HR, 3.029; 95% CI, 1.445–6.348;  P   = 0.003)](Table 6). 4. Discussion The current study investigated whether  GST   geno-types influence on genetic susceptibility and the clinicalcourse of disease in MIBC patients in the Korean Table 2 GST   genotypes and muscle invasive bladder cancer susceptibility according to smoking status.Controls (%) Cases (%) OR (95% CI)  a P  -valueTotal ( n  = 330) GSTM1 Present 117 (53.2) 45 (40.9) 1.00  Null 103 (46.8) 65 (59.1) 1.30 (1.01–1.68) 0.037 * GSTT1 Present 92 (41.8) 46 (41.8) 1.00  Null 128 (58.2) 64 (58.2) 1.00 (0.76–1.31) 0.548Never-smoker ( n  = 160) GSTM1 Present 54 (44.6) 11 (28.2) 1.00  Null 67 (55.4) 28 (71.8) 1.58 (0.92–2.71) 0.091 GSTT1 Present 56 (46.3) 14 (35.9) 1.00  Null 65 (53.7) 25 (64.1) 1.29 (0.81–2.05) 0.272Prior or current smoker ( n  = 170) GSTM1 Present 63 (63.6) 34 (47.9) 1.00  Null 36 (36.4) 37 (52.1) 1.33 (1.00–1.77) 0.043 * GSTT1 Present 36 (36.4) 32 (45.1) 1.00  Null 63 (63.6) 39 (54.9) 0.81 (0.56–1.16) 0.270 * P   < 0.05.  Reference; null = alleles that have a deletion of the  GSTM1  or  GSTT1  genes; OR = odds ratio; CI = confidence interval. a Logistic regression models were used to estimate OR with the corresponding 95% CI.Table 3Relationship between  GST   genotypes and clinicopathological parameters, progression and cancer specific death in muscle invasive bladder cancer.Variables  GSTM1  (%)  GSTT1  (%)Null (%) Positive (%)  P  -value a Null (%) Positive (%)  P  -value a Grade 0.428 0.314G2 24 (51.5) 15 (38.5) 21 (53.8) 18 (46.2)G3 41 (57.7) 30 (42.3) 43 (60.6) 28 (39.4)T stage 0.520 0.776T2 24 (66.7) 12 (33.3) – 22 (61.1) 14 (38.9) – T3 12 (57.1) 9 (42.9) 13 (61.9) 8 (38.1)T4, P N1, or M1 29 (54.7) 24 (45.3) 29 (54.7) 24 (45.3)ProgressionYes 32 (62.7) 19 (37.3) 0.298 38 (74.5) 13 (25.5) 0.002 * No 33 (55.9) 26 (44.1) 26 (44.1) 33 (55.9)Overall survivalYes 23 (63.9) 13 (36.1) 0.539 18 (50.0) 18 (50.0) 0.303No 42 (56.8) 32 (43.2) 46 (62.2) 28 (37.8)Cancer-specific deathYes 28 (60.9) 18 (39.1) 0.845 34 (73.9) 12 (26.1) 0.006 * No 37 (57.8) 27 (42.2) 30 (46.9) 34 (53.1)null = alleles that have a deletion of the  GSTM1  or  GSTT1  genes. * P   < 0.05. a P  -values were based on the chi-square test. H.won Kang et al./European Journal of Cancer xxx (2013) xxx–xxx  5Please cite this article in press as: Kang H.w. et al., Glutathione S-transferase M1 and T1 polymorphisms: Susceptibility and outcomes in muscleinvasive bladder cancer patients,  Eur J Cancer  (2013),
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