European Journal of Cancer (2013) xxx, xxx– xxx

A v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m

journa l homepag e : www.e j cancer . com

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 Koreab Department of Urology, College of Medicine, Yeungnam University, Daegu, South Koreac Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan, South Koread Department of Food and Biotechnology, Chungju National University, Chungju, South Koreae 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

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KEYWORDS

Bladder cancerEpidemiologyGlutathione S-transferaseGSTT1

PolymorphismPrognosis

59-8049/$ - see front matter

tp://dx.doi.org/10.1016/j.ejca.

Corresponding author: Addesearch, 62 Kaeshin-dong, H

E-mail address: wjkim@chuf Co-first author.

lease cite this article in pressvasive bladder cancer patien

� 2013 E

2013.05.0

ress: Deeungduk-ngbuk.ac

as: Kangts, Eur J

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

lsevier Ltd. All rights reserved.

19

partment of Urology, Chungbuk National University, College of Medicine and Institute for Tumorgu, Cheongju 361-763, South Korea. Tel.: +82 043 269 6371; fax: +82 043 269 6144..kr (W.-J. Kim).

H.w. et al., Glutathione S-transferase M1 and T1 polymorphisms: Susceptibility and outcomes in muscleCancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.05.019

2 H.won Kang et al. / European Journal of Cancer xxx (2013) xxx–xxx

Please cite this article in press as: Kanginvasive bladder cancer patients, Eur J

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 ofinterest 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 assess

H.w. et al., Glutathione S-transferCancer (2013), http://dx.doi.org/10

the 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 theGSTT1 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-tions of GST polymorphism in MIBC have not been pub-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

ase M1 and T1 polymorphisms: Susceptibility and outcomes in muscle.1016/j.ejca.2013.05.019

H.won Kang et al. / European Journal of Cancer xxx (2013) xxx–xxx 3

consent was obtained from each subject. Collection andanalysis of all samples was approved by the InstitutionalReview Board of Chungbuk National University.

Tumours were staged according to the 2002 TMN clas-sification and the 1973 World Health Organization(WHO) grading system.17,18 All diagnoses were con-firmed by pathological analysis of frozen sections fromcystectomy and transurethral resection (TUR) specimens.Patients with clinically localised or locally advancedtumours and good Eastern Cooperative Oncology Group(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 (P2) 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,and those who were reluctant to undergo aggressive treat-ment, did not receive chemotherapy. Each patient was fol-lowed and managed according to standard practice. Inthis study, progression was defined as local regional recur-rence or new distant metastasis in the cystectomisedgroup, and P20% 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

A multiplex polymerase chain reaction (PCR) methodwas applied to detect the presence or absence of theGSTM1 and GSTT1 genes in genomic DNA samples, asdescribed previously.19 In brief, the primers used in thePCR were 50-GAACTCCCTGAAAAGCTAAAGC-30

(sense) and 50-GTTGGGCTCAAATATACGGTGG-30

(anti-sense) for GSTM1; 50-TTAGCTGACCTCG-TAGCCAT-30 (sense) and 50-GAAGTCCTTGGCCTT-CAGAA-30 (anti-sense) for GSTT1 and 50-GAAGAGCCAAGGACAGGTAC-30 (sense) and 50-CAA CTTCATCCACGTTCACC-30 (anti-sense) for beta-globin(b-globin). DNA (200 ng) was amplified in a total volumeof 20 lL, containing 10 pmol of each primer, 0.5 U Taqpolymerase, 2.5 mM dNTP and 10� PCR buffer. Follow-ing an initial denaturation step at 94 �C for 5 min, 40cycles of amplification were carried out at 94 �C for

Please cite this article in press as: Kang H.w. et al., Glutathione S-transferainvasive bladder cancer patients, Eur J Cancer (2013), http://dx.doi.org/10

60 s, 63 �C for 60 s and 72 �C for 60 s, with a final exten-sion step at 72 �C for 10 min. The amplified products wereelectrophoresed on a 2% agarose gel, and their sizes were219 bp for GSTM1, 372 bp for GSTT1 and 268 bp forb-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 ofGSTM1 and GSTT1 on survival and recurrence, adummy variable was created with three categories repre-senting the presence of alleles as follows: presence ofboth 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 inthe study. The mean age of MIBC patients was 67.8 years(range 34–87) and that of the controls was 67.9 years(range 34–89). The median follow-up period of the MIBCpatients was 16.1 months (range, 0.9–181.0). The frequen-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% in the MIBC group and 19.1% in the control group.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 ofbladder 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;

se M1 and T1 polymorphisms: Susceptibility and outcomes in muscle.1016/j.ejca.2013.05.019

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)

Smokinga

Never 121 (55.0) 39 (35.5)Prior or current 99 (45.0) 71 (64.5)

GST polymorphismGSTM1-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 theGSTM1 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

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 aGSTM1-null genotype had an approximately 1.3-foldhigher risk of MIBC (P = 0.043) (Table 2).

3.3. Relationship of GST genotype with

clinicopathological 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

Please cite this article in press as: Kang H.w. et al., Glutathione S-transferinvasive bladder cancer patients, Eur J Cancer (2013), http://dx.doi.org/10

(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). TheGSTM1 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 withGSTT1-positive/GSTM1-positive. Similar relationshipswere noted for the risk of CSD (Table 5).

After grouping by smoking status, smokers with theGSTT1-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 theGSTT1 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; 6T3N0M0 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 andP = 0.043, respectively) (Figures not shown).

ase M1 and T1 polymorphisms: Susceptibility and outcomes in muscle.1016/j.ejca.2013.05.019

Table 2GST genotypes and muscle invasive bladder cancer susceptibility according to smoking status.

Controls (%) Cases (%) OR (95% CI) a P-value

Total (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) 0.548

Never-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) 0.272

Prior 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-valuea Null (%) Positive (%) P-valuea

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, PN1, 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 5

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).

Please cite this article in press as: Kang H.w. et al., Glutathione S-transferainvasive bladder cancer patients, Eur J Cancer (2013), http://dx.doi.org/10

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

se M1 and T1 polymorphisms: Susceptibility and outcomes in muscle.1016/j.ejca.2013.05.019

Table 4Relationship between GSTT1 genotype and clinicopathologic parameters, progression, and cancer specific death in muscle invasive bladder canceraccording to smoking status.

Variables Never-smoker (n = 39) Prior or current smoker (n = 71)

Null (%) Positive (%) P-valuea Null (%) Positive (%) P-valuea

Grade 0.080 0.621G2 6 (42.9) 8 (57.1) 15 (60.0) 10 (40.0)G3 19 (76.0) 6 (24.0) 24 (52.2) 22 (47.8)

T stage 0.164 0.184T2 8 (80.0) 2 (20.0) 14 (53.8) 12 (46.2)T3 3 (37.5) 5 (62.5) 10 (76.9) 3 (23.1)T4, PN1 or M1 14 (66.7) 7 (33.3) 15 (46.9) 17 (51.5)

Progression 0.108 0.009*

Yes 16 (76.2) 5 (23.8) 22 (73.3) 8 (26.7)No 9 (50.0) 9 (50.0) 17 (41.5) 24 (58.5)

Overall survival 0.482 0.452Yes 7 (53.8) 6 (46.2) 11 (47.8) 12 (52.2)No 18 (69.2) 8 (30.8) 28 (58.3) 20 (41.7)

Cancer-specific death 0.320 0.014*

Yes 14 (73.7) 5 (26.3) 20 (74.1) 7 (25.9)No 11 (55.0) 9 (45.0) 19 (43.2) 25 (56.8)

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.

6 H.won Kang et al. / European Journal of Cancer xxx (2013) xxx–xxx

population and demonstrated that there was a statisti-cally significant increase in the frequency of the GSTM1

deletion genotype in MIBC patients compared to thecontrol group. Individuals with the GSTT1-null geno-type appeared to have an increased the predispositionto greater disease progression and cancer-specific death,and to have shorter survival.

Reports on the association between genetic variabil-ity in specific GST genes (GSTM1, GSTT1 and GSTP1)

Table 5Combination effect of GST genotypes on disease progression and cancer sstatus.

GST genotype Disease progression

GSTM1 GSTT1 Yes No OR (95% CI)a

Total (n = 110)

Present Present 2 (12.5) 14 (87.5) 1.00�

Null Present 11 (36.7) 19 (63.3) 4.05 (0.77–21.26)Present Null 17 (58.6) 12 (41.4) 9.92 (1.89–51.93)Null Null 21 (60.0) 14 (40.0) 10.50 (2.06–53.52)

Never-smoker (n = 39)

Present Present 1 (33.3) 2 (66.7) 1.00�

Null Present 4 (36.4) 7 (63.6) 1.14 (0.77–16.95)Present Null 5 (62.5) 3 (37.5) 3.33 (0.20–54.53)Null Null 11 (64.7) 6 (41.2) 3.67 (0.27–49.29)

Prior or current smoker (n = 71)

Present Present 1 (7.7) 12 (92.3) 1.00�

Null Present 7 (36.8) 12 (63.2) 7.00 (0.74–65.94)Present Null 12 (57.1) 9 (42.9) 16.00 (1.75–146.66)Null Null 10 (55.6) 8 (44.4) 15.00 (1.59–141.16)

* P < 0.05.� Reference; null = alleles that have a deletion of the GSTM1 or GSTT1 ga Logistic regression models were used to estimate OR with the correspon

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and susceptibility to bladder cancer have been conflict-ing, and the results are inconsistent among different eth-nic groups.20 Salinas-Sanchez et al. investigated thevalue of GSTT1 and GSTM1 genotypes in bladder can-cer, and influence of smoking and occupational fac-tors.21 They suggested that GSTM1 null genotype andGSTM1/GSTT1 null/null genotype had 1.7 and 3.5 oddsratio of bladder cancer, respectively. When stratified bysmoking habit, smokers with both null types an odds

pecific death in muscle invasive bladder cancer according to smoking

Cancer-specific death

P-value Yes No OR (95% CI)a P-value

2 (12.5) 14 (87.5) 1.00�

0.098 10 (33.3) 20 (66.7) 3.50 (0.66–18.49) 0.1400.007* 16 (55.2) 13 (44.8) 8.62 (1.65–44.97) 0.011*

0.005* 18 (51.4) 17 (48.6) 7.41 (1.46–37.57) 0.016*

1 (33.3) 2 (66.7) 1.00�

0.923 4 (36.4) 7 (63.6) 1.14 (0.77–16.95) 0.9230.398 5 (62.5) 3 (37.5) 3.33 (0.20–54.53) 0.3980.327 9 (52.9) 8 (47.1) 2.25 (0.17–29.77) 0.538

1 (7.7) 12 (92.3) 1.00�

0.089 6 (31.6) 13 (68.4) 5.54 (0.58–52.96) 0.1370.014* 11 (52.4) 10 (47.6) 13.20 (1.45–120.62) 0.022*

0.018* 9 (50.0) 9 (50.0) 12.00 (1.28–112.66) 0.030*

enes; OR = odds ratio; CI = confidence interval.ding 95% CI.

ase M1 and T1 polymorphisms: Susceptibility and outcomes in muscle.1016/j.ejca.2013.05.019

H.won Kang et al. / European Journal of Cancer xxx (2013) xxx–xxx 7

ratio of 4.7. This result showed the similarity to the cur-rent study, in which GSTM1-null individuals exhibitedincreased risk for MIBC and an association with ciga-rette smoking. However, the current study differs fromprevious studies in several aspects. First, the case popu-lation included only MIBC patients, whereas previousstudies targeted all cases of bladder cancer regardlessof invasiveness. Bladder cancer has heterogeneous bio-logical and functional characteristics; therefore, it mustbe classified according to muscle invasiveness. Second,the present study was designed to assess the prognosticutility of GST polymorphisms with a focus on cancerprogression, cancer-specific death in MIBC. To ourknowledge, this is the first study of the prognostic utilityof GST polymorphisms in MIBC. We found that thehomozygous GSTT1 deletion was closely associatedwith disease progression and cancer-specific death inMIBC.

Fig. 1. The probability of progression free survival and cancer specific scancer. (a) Progression free survival according to GSTT1 genotype (log-ragenotype (log-rank test; P = 0.893); (c) Cancer specific survival accordingsurvival according to GSTM1 genotype (log-rank test; P = 0.547).

Please cite this article in press as: Kang H.w. et al., Glutathione S-transferainvasive bladder cancer patients, Eur J Cancer (2013), http://dx.doi.org/10

In our study, subgroup analysis was performed tocreate more homogeneous patient groups. Patients werestratified according to smoking status, treatment modal-ity and the prognostic value of GST polymorphisms oncancer survival in MIBC were examined in each group.Above all, whether the relationship is modified by smok-ing history should be assessed for the reason that theseenzymes play a crucial role in the detoxification of majorclasses of tobacco carcinogens in urine. The predictivevalue of the GSTT1 genotype was restricted to the smo-ker group. Smokers with the GSTT1-null genotypeshowed higher rates of progression and cancer-specificdeath, as well as shorter PFS and CSS. This finding sup-ports the fact that these enzymes play a crucial role inthe detoxification of major classes of tobacco carcino-gens. In addition, because radical cystectomy and che-motherapy are the most influential prognostic factorsfor MIBC, survival analysis was re-performed according

urvival according to GST polymorphisms in muscle invasive bladdernk test; P = 0.009); (b) Progression free survival according to GSTM1

to GSTT1 genotype (log-rank test; P = 0.030); and (d) Cancer specific

se M1 and T1 polymorphisms: Susceptibility and outcomes in muscle.1016/j.ejca.2013.05.019

Fig. 2. The effect of GST polymorphisms on progression free survival and cancer specific survival in subgroup analysis. (a) Progression freesurvival and cancer specific survival according to GSTT1 genotype in smoker group (log-rank test, P = 0.021 and 0.030, respectively); (b)Progression free survival and cancer specific survival according to GSTT1 genotype in the subgroup of patients who received chemotheraphy (log-rank test, P = 0.036 and 0.054, respectively); and (c) Progression free survival and cancer specific survival according to GSTT1 genotype in thesubgroup of patients who did not undergo radical cystectomy (log-rank test, P = 0.027 and 0.056, respectively).

8 H.won Kang et al. / European Journal of Cancer xxx (2013) xxx–xxx

to treatment history. When the stratification was appliedto patients who did not undergo radical cystectomy, theGSTT1 genotype was identified as a reliable prognosticmarker for predicting PFS. Interestingly, amongpatient groups that received chemotherapy, the fre-quency of progression was significantly higher for thosewith the GSTT1-null genotype than those with theGSTT1-positive genotype. It has been suggested thatgenetic polymorphisms in certain GST genes reducethe effectiveness of detoxifying cytotoxins generated bychemotherapeutic agents.22,23 Yang et al. reported thatamong patients with invasive breast carcinoma whowere treated with chemotherapy, women who werehomozygous for the variant GSTP1 105Val allele (associ-ated with lower substrate-specific catalytic activity) hada 60% reduction in mortality risk compared with womenwho were homozygous for the Ile allele.24 Ambrosoneet al. suggested that GST enzyme activity influences dis-ease recurrence and overall survival among women whoreceived chemotherapy or radiation therapy for breastcancer. Women who were null for both GSTM1 andGSTT1 had a lower risk of recurrence and death. Inaddition, there has been a lot of research on geneticpolymorphisms of DNA repair genes (Nucleotide exci-sion repair; NER) and survival in advanced non-smallcell lung cancer patients treated with chemotherapy.25–28

Biologically, chemotherapy mostly effects cytotoxicityvia DNA damage, and therefore elevated DNA repair

Please cite this article in press as: Kang H.w. et al., Glutathione S-transferinvasive bladder cancer patients, Eur J Cancer (2013), http://dx.doi.org/10

capacity would reduce drug efficacy. The disparate roleof DNA repair in cancer susceptibility and drug resis-tance, a double-edged sword, should be considered.29

On the other hand, GST expression plays an importantrole in determining the cytotoxicity of chemotherapeuticdrugs. Davies et al. showed that children who lackedGSTT1 exhibited greater drug toxicity and reduced sur-vival after chemotherapy for acute myeloid leukaemia(AML) compared with children with at least 1 GSTT1

allele.13 As a result, GST polymorphisms may be linkedto the efficacy and toxicity of cancer drugs. The resultsof the present study are in agreement with the findingsof Davies’ study group. Although the exact reason isunknown, one possibility is that cisplatin-based chemo-therapy in MIBC is usually not curative and the overall5-year survival is only 5–15%30,31; consistent with this,chemotherapy status is not associated with disease pro-gression or CSS in our cases. Therefore, the GSTT1-positive genotype has a survival benefit through thereduction of adverse side-effects rather than throughreduced effectiveness.

In order to avoid the complementary effect of twoGST genes, we performed combined analyses; subjectswith the GSTT1-null genotype were seen to have poorcancer outcomes regardless of their GSTM1 genotype.The null genotype in both genes did not show a signifi-cant synergistic effect on disease progression and cancer-specific death. Therefore, the GSTM1 genotype is not a

ase M1 and T1 polymorphisms: Susceptibility and outcomes in muscle.1016/j.ejca.2013.05.019

Table 6Multivariate Cox regression analysis for prediction of disease progression and cancer specific death in muscle invasive bladder cancer.

Variables Disease progression Cancer-specific death

HR (95% CI)a P-value HR (95% CI)a P-value

Age 0.992 (0.960–1.025) 0.629 0.987 (0.954–1.022) 0.472Gender (male versus female) 1.500 (0.725–3.104) 0.275 1.384 (0.637–3.007) 0.412Smoking (No versus Yes) 0.975 (0.492–1.932) 0.943 0.811 (0.387–1.696) 0.577GSTT1 (positive versus null) 3.110 (1.551–6.235) 0.001* 3.029 (1.445–6.348) 0.003*

GSTM1 (positive versus null) 1.060 (0.576–1.953) 0.850 0.855 (0.439–1.667) 0.646Grade (G2 versus G3) 1.240 (0.647–2.377) 0.518 1.533 (0.755–3.114) 0.237

T stageT2 1� - 1� –T3 2.104 (0.899–4.926) 0.087 2.816 (1.135–6.990) 0.026T4 or PN1 or M1 2.941 (1.398–6.189) 0.004 3.984 (1.728–9.185) 0.001*

Chemotherapy (No versus Yes) 0.703 (0.351–1.407) 0.320 0.784 (0.372–1.654) 0.523Radical cystectomy (No versus Yes) 0.365 (0.187–0.712) 0.003* 0.209 (0.103–0.426) <0.001*

* P < 0.05.� Reference; null = alleles that have a deletion of the GSTM1 or GSTT1 genes; HR = hazard ratio; CI = confidence interval.a Multivariate Cox regression analysis was used to estimate HR with the corresponding 95% CI.

H.won Kang et al. / European Journal of Cancer xxx (2013) xxx–xxx 9

critical prognostic factor in MIBC. Smoking and theGSTT1-null genotype showed a joint effect, with ele-vated risks of disease progression (from 9.92 to 16.0)and cancer-specific death (from 8.62 to 13.2).

The prevalence of the GSTM1- and GSTT1-nullgenotypes varies among ethnic groups. The frequenciesof 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. The frequencies of nullstatus for both GSTM1 and GSTT1 were 31.8% in theMIBC group and 19.1% in the control group, respec-tively. These observations are consistent with previouspopulation-based studies.9,32,33

In a previous study, we reported that GSTT1 was aprognosticator for recurrence and progression inpatients with NMIBC. In patients with NMIBC, theGSTT1-positive genotype was an independent predictorfor recurrence and progression.16 The results of the cur-rent study were not consistent with this previous evi-dence. These results imply that different molecularmechanisms are involved in disease progression andprognosis in NMIBC and MIBC.

A limitation of the present study is that quantitativecigarette consumption (i.e. measured in pack-years)was not assessed. The exact influence of cigarette smok-ing on disease progression, metastasis, and survival-related prognosis in MIBC should be elucidated basedon the number of cigarettes or the duration of the smok-ing habit. Another concern is that MIBC patients’cohort is inhomogeneous because various treatmentmodalities are selected according to their performancestatus and life expectancy. To minimise this problem,we excluded any patients received radiation therapy orneoadjuvant chemotherapy. Also, subgroup analysiswas performed according to treatment modalities tominimise the different effects of therapeutic modalities.However, analysis was not sufficient in part of sub-

Please cite this article in press as: Kang H.w. et al., Glutathione S-transferainvasive bladder cancer patients, Eur J Cancer (2013), http://dx.doi.org/10

groups because of limited number of patients. To over-come these concerns, we plan to collect more MIBCtissue samples in order to ensure the prognostic valueof GST polymorphisms.

5. Conclusions

The present study suggests that the GSTT1 genotypeis a useful prognostic marker for MIBC. This findingmay be an important tool with regard to the individua-lised prediction of prognosis for patients with MIBC.The underlying molecular biological mechanism of theGSTT1 genotype and its prognostic value should be fur-ther investigated.

Conflict of interest statement

None declared.

Acknowledgements and funding

This research was supported by the Basic Science Re-search Program of the National Research Foundationof Korea (NRF) that is funded by the Ministry of Edu-cation, Science, and Technology of Korea (2012-0000476 and 2012R1A1A4A01008753).

This work was supported by a Grant from the Next-Generation BioGreen 21 Program (No. PJ009621),Rural Development Administration, Republic of Korea.

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