glutathione s-transferase ml and its variants a and b as ...glutathione s-transferase ml and its...

ICANCERRESEARCH54,4103-4111,August1, 1994)

ABSTRACT

Glutathione S-transferase Ml (GSTM1) is a foreign compound-metabol@ enzyme with a heritable complete lack of activity In about 50% ofCaucasians. GSTM1 deficiency may predispose Individuals to urinarybladdercancer.Thus,a hospital-basedcase-controlstudywasperformedwith 296 patIents with bladder cancer and 400 controls, investigating thisGSTM1 deficiency in relation to environmental risk factors and types ofbladder cancer. Frequencies of the GSTM1 gene deletion (genotype,GSTM1O/O)andof the allelevariantsA (,s)andB (4i)of the GSTM1-active trait were determined using an intenial standard-controlled polymerase chain reaction technique. Moreover, in aH patients GSTM1 expres

slon was quantified In blood by an Immunoassay. Ofthe cases, 59.1% hadthe GSTMJO/O genotype, in contrast to 50.7% ofthe controls (odds ratio,1.40; 95% confidence limits, 1.02-1.92; P 0.017). The odds ratio afteradjustment for age and gender by logistic regression analysis was 1.54(95% confidence limits, 1.12-2.13). Occupational risk was defined asprevious employment in occupations with known increased bladder cancar risk,but the impactof GSTMJOIOwas not significantlydifferentinindividuals with rlskjobs versus these WithOut.The greater proportion ofthe GSTM1-deflclent individuals in the group with cancer was due to alowerfrequencyofcarrlersofGSTM1A.Theoddsratioforthesubgroupof individuals with the GSTM1B phenotype vei@cuscarriers of theGSTMIA phenotype In cases versus controls was 1.65 (95% confIdencelimits, 0.976-2.78; two-tailed Fisher's exact P 0.057). AnalysIs of functional GSTM1 activity In a subset of 370 blood samples with the modelsubstrate tsuns-stllbene oxide confirmed the genetic results and showedthat 9 of 10 individuals with p/is, heterodimers (genotype, GSTMJA/B)had activities above the median of all genetically GSTM1-active individuals (24 pmol/mln/1 x 10' lymphocytes; P < 0.01), indicating a gene doserelationship for GSTM1. GSTM1 expression in the urinary bladder endothelium detected by immunoassay and immunohistology correspondedto the genotype of the patients. It may be concluded from this study thatthe heritable GSTM1 deficiency is responsible for 17% (etiological fraclion; 95% confIdence limits, 2-30%) of bladder cancer cases.

INTRODUCTION

Bladder cancer was first related to occupational exposure to anilinederived dyes by the end of the 19th century (1) and could be experimentally induced with @3-naphthylaminein dogs in 1938 (2). According to epidemiological estimations, occupational exposure tochemicals may explain 16â€”24%of bladder cancers (3). Foreign compounds from tobacco smoking may be causative in up to 50% of thisdisease in Europe, but some drugs, alcohol, saccharin, coffee (4), andother factors have also been proposed as exogenous chemical riskfactors of bladder cancer (5). Many of these naturally occurring orsynthetic compounds require enzymatic activation to become ultimatecarcinogens that may fmally react with cellular macromolecules likeDNA. While oxidation by cytochrome P-450 enzymes is primarilyregarded as an activating process in carcinogenesis (6â€”8), mostphase-I! foreign compound-metabolizing enzymes are considered to

Received 8/11/93; accepted 5/23/94.Thecostsof publicationof thisarticleweredefrayedin partby thepaymentof page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Institut fÃ¼r Klinische Phar

makologie, UniversitAtsklinikumCharitÃ©,Humboldt-UniversitAt,Schumannstrasse 20/21,D-10098 Berlin, Germany.

be predominantly protective enzymes. In bladder cancer, especiallyhigh activity of arylamine N-acetyltransferase was thought to beprotecting (9). Similarly, GST2 may be expected to protect individualsfrom bladder cancer by deactivating electrophilic chemicals. Theglutathione S-transferase Ml belongs to the GST gene family formerly termed @1.or GST1 (10). GSTM1 appears in two allele variants,i.e., GSTMIÂ°A, coding for the phenotype GSTM1A (s), which biochemically means the presence of the homodimeric enzyme GSTM1a-la, and GSTM1 Â°B,coding for GSTM1B (iji), which refers to thehomodimeric enzyme GSTM1b-lb. In heterozygous carriers of oneGSTM1a and one GSTM1b subunit the heterodimeric enzymeGSTM1a-lb is also formed. GSTM1, which is absent in about half ofthe Caucasian population due to a gene deletion (11, 12), has highspecificity for certain carcinogens such as trans-stilbene oxide (13)and a highly carcinogenic metabolite of benzo(a)pyrene, namelyanti-benzo(a)pyrene.7,8-diol-9,10-epoxide. In vitro, it was shownthat lymphocytes containing this enzyme formed fewer DNA adductson exposure to aflatoxin B1 (14). GSTM1 isoenzymes are expressedin various tissues such as liver, gut, lymphocytes, and, as shown inthis presentation, urothelium. Indeed, according to one epidemiological study (15), GSTM1-deficient individuals seemed to be highlysusceptible to lung cancer, a finding which could not be confirmed byothers (16, 17), leading to the conclusion that the overall lung cancerrisk attributable to GSTM1 deficiency may be lower than initiallyestimated.

In the present study on bladder cancer, the phenotype and genotypeof GSTM1 and its allelic variants GSTM1*A (@) and GSTM1Â°B(ii)were studied in 296 bladder cancer cases and 400 controls in relationto smoking and occupational history, indicating possible exposure tocarcinogens. The procedures for molecular-epidemiological characterization of GSTM1 and its allelic variants (17) were further optimized, as described here. The hypothesis at initiation of the study wasthat individuals with two GSTM1 *() alleles are at higher risk ofbladder cancer, compared to those with one or two GSTM1 CA orGSTM1*B alleles. Furthermore, differences were expected in thefrequency of the GSTM1 deficiency depending on exposure to cigarette smoke or occupational exposure to carcinogens and pathologicalcharacteristics of bladder cancer.

MATERIALS AND METhODS

Study Design and Inclusion Criteria. The hospital-based case-controlstudy was performed in patients from the Departments of Urology of theGeneral Hospital StÃ¤dtischesKrankenhaus NeukÃ¶llnand the UniversitStsklinikum Steglitz in Berlin. The study was approved by the Ethics Committee of the

Klinikum Steglitz and was started in January 1991. All inpatients with prey

alent bladder cancer who gave their written and informed consent wereincluded in this study, if they allowed enzymological and genetic analysis ofa blood sample. Only cases with histologically verified bladder cancer wereincluded, and patients with bladder cancer were excluded if they had aclinically apparent malignancy of any other origin. The control patients had avariety of diagnoses excluding cancer. Malignancies were excluded accordingto medical history and the routinely performed examinations. To avoid inter

2 The abbreviations used are: GST, glutathione S-transferase; ELISA, enzyme-linked

immunosorbent assay; GSTM1, glutathione S-transferase Ml (class @4;OR, odds ratio;PCR, polymerase chain reaction.

4103

Glutathione S-Transferase Ml and Its Variants A and B as Host Factors of

Bladder Cancer Susceptibility: A Case-Control Study

JÃ¼rgenBrockmÃ¶ller,' Reinhold Kerb, Nikolaos Drakoulis, Beate Staffeldt, and Ivar RootsInstitute of Clinical Pharmacology, UniversitÃ¤tsklinikumCharittt, HumbOldt University ofBerlin@D-10098 Berlin, Germany

on June 6, 2020. © 1994 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Table1 PrimersandPCR conditions usedfor analysis of the GSTMI@O, GSTM1 5A,and GSTM1BallelesAnnealingPrimerPrimer

sequenceComplementaryDNA positionaMgC12(@)Temperature(O()Time (@j@)Extension(@)CyclesGSTMI-specificp@b015'-CrGCCCrACflGA1TGATGGG-3'193-2131.56011.025G25'-CFGGATfl3TAGCAGATCATGC-3'370-350IFN@3c5'-GGCACAACAGGTAGTAGGCG-3'130-lWIFN-55'-GCCACAGGAGcTFCFGACAC-3'-40

toNested

procedure, specific for GSTMI A and GS7MJ5BOuterprimers065'-CTCAAAGC000AGATGAAGTC-3'582-5620.9600.72.0350185'-ACCATATGCAGCFGGGCATGA-3'335-355Inner

primers, GSTMI B(*)G75'-AGGCGTCCAAGCAG-3'547-5340.6541.51512G95'-TAAAGCfCrACFCAGAGT-3'419-436Inner

primers, GSTM1*,4(@)G8090.6 54 1.5 1.5 12

GLUTAThIONE S.TRANSFERASE MI IN BLADDER CANCER

ferences from the known ethnic differences in GSTM1 (18), only cases andcontrols of German origin were included. The controls were hospital patientswith similarity to the cases with respect to gender proportion and the range ofages (cases, 70.8 Â±11.6 years; controls, 64.0 Â±12.0 years; mean Â±SD). Mostpatients were permanent residents of the urban area of the formerly westernpart of Berlin. Data on lifetime occupational history, smoking history, andother anamnestic data were obtained by interviews of about 20-min duration.A subset of 104 controls for whom no data on smoking and occupation wereavailable were studied in the same time interval primarily in another context,but they fulfilled the other inclusion criteria for controls.

Tobacco Smoking. Data on smoking history, i.e., duration of smoking, ageat starting, time of stopping, and average number of cigarettes consumed perday, were recorded. As a cumulative measure, smoking is expressed aspack-years, with 1 pack-year corresponding to the smoking of one package (20cigarettes) daily for 1 year, i.e., smoking of 7300 cigarettes.

Occupational Risk Factors. Lifetime occupational history was recordedfor each cancerpatientand for 296 controls.The patientswere classifiedasâ€œexposedto occupational riskâ€•if they had been occupied for at least 2 years inone of the professions defined as a bladder cancer risk (given in Table 4). Theselection of presumptive high-bladder cancer risk employments was based onearlier epidemiological studies (3, 19, 20).

Classification of Bladder Cancer. The World Health Organization Inter

national Classification of Diseases in Oncology (21, 22), with differentiationaccording to histology, ThM classification, grading, and staging, was used. Asexpected, in most cases histology was of the papillary transitional cell carcinoma type, followed by nonpapillary transitional cell carcinoma, while variousother types were rare or absent (see Tables 5 and 6). Tumor staging wasdocumented as pTa, pTis, pTl, pT2, pT3, and pT4; the first three and the lattertwo categories were each evaluated together, as T@ 1 and T 3, respectively.Grading was classified as well differentiated (Gi), moderately well differentiated (02), or poorly differentiated (G3) bladder cancer (22). In the case ofmultiple tumors with different grades or stages, the more malignant was taken.Furthermore, because of differences in the natural course of bladder cancer(23) and as done by Kaisary et a!. (24), a binary classification of TNM stagingand tumor grading was used to distinguish two types of cancer, namely thosewith the combination ofT 1 and G 2 as â€œmoderatelyaggressive typeâ€•andthose with either T > 1 or G > 2 or with any metastasis as â€œaggressivetype.â€•

Phenotype Determinations of GSTM1. GSTM1 enzyme was quantitatively assayed in hemolyzed venous blood samples from all patients (25) usinga commercial ELISA reagent set (Mukit; Biotrin, Dublin, Ireland). For calibration, 0.l-@g portions of purified GS1'Ml supplied by Biotrin were dissolved in one batch of venous blood from a GSTM1-deflcient individual andwere diluted to concentrations of 0.05, 0.1, 0.5, 1, 10, and 50 mg/liter. Resultsare given as mg GSTM1/liter of whole blood. All of these measurements arebased on one batch ofthe GSTM1 standard, which was not further analyzed forpurity by us.

This enzyme content determination was functionally checked in 370 patients by an activity measurement employing a GSTM1-specific conjugationassay with the substrate trans-stilbene oxide (50 @Msubstrate concentration),as described previously (13, 17, 25).

GSTM1 Tissue Concentrations and Immunohistochemistry. In 74 ofthe cancer patients, GSTM1 concentration was additionally determined inbladder cancer tissue samples by the aforementioned ELISA method. Thetissue was obtained from endoscopic resections, frozen (â€”20Â°C)after removal,and homogenized at a concentration of about 10 mg/mi wet tissue weight instandard phosphate-buffered saline. In addition to these measurements onhomogenates, an immunohistochemical evaluation of GSTM1 content wasperformed with paraffm-embedded material by the alkaline phosphatase-antialkaline phosphatase method, using the human GSTM1-specific rabbit antiserum from Biotrin.

PCR Analyses of GSTMJ Allelic Conformation. Two different reactionsets were applied; the first was used to distinguish between GSTM1-active andGSTM1-deficient individuals and the second set of reactions differentiated,among the GSTM1-active individuals, the GSTM1A phenotype (isoenzyme

GSTM1a-la, formerly @;genotype, GSTMJA/GSTM1A or GSTMJ*A/GSTM1@O), the GSTM1B phenotype (isoenzyme GSTM1b-lb, formerly s@i

genotype, GSTM1*B/GSTMJ*B or GSTM15B/GSTM1@O),and theGSTM1AIB phenotype (isoenzyme pltfr, genotype, GSTMJ*A/GSTMJ*B).

PCR primerswere designed accordingto the known GSTM1 complementary DNA sequences (11, 12), taking into account the homologous complementary DNA sequences of human muscle and brain-specific GSTM2 andGSTM3 isoenzymes (26, 27) (Table 1). Genomic DNA was prepared fromleukocytes by proteinase K-phenol-chloroform extraction. DNA (100 ng) wasamplified in a final volume of 25 @slcontaining 10 mM TrisHQ, pH 8.3 atroom temperature, 50 mM KC1, 1.5 mr@iMgCl2 (or modified; see Table 1),0.01% (w/v) gelatin, 63 @Mlevels of each deoxynucleoside triphosphate, 0.25@LMlevels of each primer, and 1.0 unit of Taq polymerase (Ampli.Taq;

Perkin-Elmer, Uberlingen, Germany), overlaid with one drop of mineral oil.The PCR primers (Table 1) were from Molbiol (Berlin, Germany) and wereused without further purification. PCRs were carried out in a programmablewater bath (Autogene II; Grant Instruments, Cambridge, UK); after 5-mmpretreatment at 94Â°C,the amplifications were done as given in Table 1,followed by a post-treatment of 30-s annealing and 7-mm extension. Twenty,.d of the amplification mixture were subjected to electrophoresis on a 4%agarose gel (NuSieve GTG; FMC Bioproducts, Rockland, ME) at 140 V for60 mm.

The GSTM1-speciflc PCR with primers Gi and 02 (amplified product size,271 base pairs) was performed as described previously (17, 25, 28), but theassay design was modified for an internal standard-controlled PCR (29).Coamplification of a segment of the a-interferon gene primers (interferon

primers 3 and 5) resulted in a constant 170-base pair band in all samples, andGSTM1deficiencyis concludedfromtheabsenceof thespecific 271-basepair

5'-AGGCGTCCAAGCAC-3' 547-5345'-TAAAOCFCFACrCAGAGT-3' 419-436

a According to Scidegard et aL (11); new nomenclature is according to the system of Mannervik el aL (10).

b Genes of brain- and muscle-specific GST isoenzymes (GSTM3 and GSTM4) are not amplified by these PCRs.C @3-Interferon amplification served as an internal standard; interferon (IFN) primers were as described by Neubauer et aL (29).

4104



.1252bp-@

GLUTATHIONE S-TRANSFERA5E MI IN BLADDER CANCER

fragment (see Fig. 1). Independently from this reaction, a nested PCR revealedthe allelic variants A (@)and B (ti,) in exon 7. These variants of GSTM1 aredue to an exchange from the basic lysine (in p@)tothe nearly neutral asparagine(in ii,) at amino acid 172. By this analysis, GSTM1-active individuals could be

differentiated into carriers of one or two GSTM1 *A alleles (on the proteinlevel, GSTM1a-la homodimers),carriersof one or two GSTMJB alleles(GSTM1b-lb homodimers), or carriers ofone A and one B allele (GSTM1a-lbheterodimers; pji(i). This reaction included first a 35-cycle amplification with

primers 06 and 018. The yield of the resulting 1252-base pair amplifiedproduct confirmed the GSTM1-active genotype. The following 12-cycle amplification with each 1-id amplification mixture taken from the first PCR assayresulted in a 214-base pair fragment with primers G7 and G9 (iii) and, in asecond tube, an amplified product ofthe same size with primers 08 and 09 (@)if the respective alleles were present. Electrophoretic analyses of the first andsecond amplification mixtures are illustrated in Fig. 2. Because point mutationsmay be introduced artificially during PCR, the @.tIi@i-differentiatingPCRs wereperformed in duplicate with most samples. Results of these second reactionsets, however, never differed from those for the first reaction.

Statistics. Minimal sample size was determined using a type I error of 5%and a type II error of 10%. With a presumed frequency of the GSTM1-defective trait ofSO% in the reference group and a hypothesized (15) frequencyof 65% in the group with bladder cancer, a minimal number of 226 each forcases and controls was calculated before the study was started, using equationsderived by Schiesselman (30, 31). The final sample size was larger, becausethe patients were also studied with respect to another polymorphic enzyme(cytochrome P-450 2D6) in a study which requireda larger sample size.Significance of the disproportions was determined by the exact Fisher test (onesided if not explicifly specified), with 95% confidence intervals calculated asgiven by Thomas (32). Approximated 95% confidence intervals of the oddsratios adjusted according to the method of Mantel and Haenszel (33) werecalulated with the BMDP4F program (34). The test for homogeneity (35) wasalso calculated with the BMDP4F program.

Logistic regression analyses were calculated by the BMDP (34) subroutineLR (BMDP program, release 7.0). Variables included were age, gender,smoking (as pack-years), previous employment in bladder cancer-risk occupations, GSTM1 activity, and GSTMJ allelic variants A and B. The improvement of the logistic regression model produced by including interactionsbetween, for example, gender and smoking, GSTM1 and smoking, GSTM1and risk occupations, and GSTM1 and age was evaluated (36, 37). Theetiological fraction, i.e., the proportion of all cases that may be attributable to

GSTM1 deficiency, was calculated (31, 38) from the OR under the assumptionthat GSTM1 deficiency can be approximately treated like exposure to a riskfactor (Pr). Then, the etiological fraction (EF) is defined as EF @E(OR â€”1)/(1'@ (OR â€”1) + 1), assumingthatP5 (i.e., GSTM1deficiency) inthe control group is similar to that in the target population.

RESULTS

Deficiency of GSTM1. The PCR methods described above allowed an internal standard-controlled classification of either GSTM1-active (homozygous or heterozygous) or GSTM1-deficient(GSTM1 *0/0) individuals. The genotype was the basis of the epidemiological interpretation but, to exclude misclassifications, genotyp

Fig. 1. Electrophoretic analysis ofthe internal standard-controlled PCR procedure usedfor detection of GSTM1 deficiency. Accurate performance of PCR with samples A to Jwas ensured by amplification of the internal standard (lower 170-base pair band) in allsamples; samples A and I represent DNA samples from GSTM1-deficient individuals. St,size standard of 100-base pair homopolymers, with the lowest band corresponding to 100base pairs.

GSTM1*8

St ABCDEFG St

Second

214 bp

214 bp@

Fig. 2. Illustration of the procedure for differentiation of the GSTMJ A and GSTMJ Balleles.Upper,1252-basepairfragmentfromtheGSTMJ-specificfirstamplification.Theabsence ofany amplified product(lane C)indicates the presence oftwo GSTMIO alleles.Middle, GSTMJA-specific second reaction. Lower, GSTMI @ff.sp@ific second reaction.Thus, lanes A to G refer to persons with the GSTM1A, GSTM1A/B, GSTM1-null,GSTM1A, GSTM1B, GSTM1A/B, and GSTM1A phenotypes, in this order. Si, DNA sizestandard.

271 bp -l7Obp

4105

Firstamplification:

GSTM1*A

ing was biochemically controlled by two other methods, as describedbelow.

Of the 296 patients suffering from bladder cancer, 59.1% wereclassified as heritably GSTM1 deficient, a proportion which wassignificantly higher than the 50.7% for the controls (n = 400), with acrude odds ratio of 1.40 (95% confidence limits, 1.02â€”1.92;one-tailedFisher's exact P = 0.017). This constellation leads to an etiologicalfraction of 0.17 (95% confidence interval, 0.02â€”0.30) of GSTM1deficiency in bladder cancer causality.

A stratification for variables potentially confounding the impact ofGSTM1 in bladder cancer is given in Table 2. Although the odds ratiofor GSTM1 differed slightly in men (OR = 1.50) and women(OR = 1.26), these two strata fulfilled the criterion of homogeneityand the gender-adjusted odds ratio (Mantel-Haenszel OR = 1.41) wasthe same as the crude odds ratio. Stratification for age revealed a morecomplex situation. GSTM1 deficiency was a highly significant bladder cancer risk factor in the group of age 55â€”65years (P = 0.01), itwas not significant in the group of age 65â€”75years, and it was againsignificant (P = 0.04) in patients older than 75 years. Since thedifferences between these strata were not significant by testing forhomogeneity, the age-adjusted Mantel-Haenszel OR was determinedas 1.67.

Adjustment for both gender and age was done by logistic regressionanalysis and revealed an odds ratio for GSTM1 deficiency of 1.54(95% confidence limits, 1.12â€”2.13). The logistic regression analysis

including age, gender, and GSTM1 status did not show correlationsbetween these three variables. Among other criteria for adequacy ofthe logistic regression model, the Hosmer-Lemeshow goodness of fitx2value(37)was0.96.Theseresults,includingananalysisforthepreviously (24) defined subgroups of aggressive and moderate bladdercancer, are summarized in Table 3.

Impact of GSTM1 in Relation to Smoking and OccupationalRisk Factors. Of the cancer patients studied here, 70% had eversmoked more than 1 pack-year of cigarettes. Among the 93 nonsmokers with bladder cancer there were 61.3% GSTM1-deficient individ



Table 2 Modulation ofbladder cancer risk by GSTMJdeficiencyTotal

numberGSTM1 @O/O(%)ORfor95%confidenceControlsCasesControls

CasesGSTM1O/Oâ€•limitsPStratification

bygenderMale25020248.858.81.50Female1509454.0

59.61.26ORMH1.411.04â€”1.930.031Age55

years1033559.269.21.5755â€”65years1156149.670.52.4365â€”75years1057654.356.61.10>75

years7712336.452.01.90ORMH1.671.19â€”2.310.002Cigarette

smoking'@Nonsmokers479344.761.31.961â€”20

PYâ€•996458.665.61.3520â€”5OPY1009751358.9135>50PY494453.1

45.50.74ORMH1330.94â€”1.870.11Possible

occupationalexposurescNotin risk jobs24019255.063.0139In

riskjobs5610442.951.91.44ORMH1.411.00â€”1.980.05

Odds ratio95%confidence

limitsPâ€•Crude

odds ratio, whole group 1.401.02â€”1.920.017â€•

Table 4 Impact of GSTM1 deficiency on bladder cancer risk: stratification forriskoccupationsbased on all 296 bladder cancer patients and 296controlsNumberCases

ControlsGSTM1O/O

GSTM1 active GSTM1@O/OGSTM1activeAuto

mechanic 5 9 42Bus,taxi,ortruckdriver7 13 56Chemical

industry worker 22Chemicaldry cleaning 12workerElectronic

industry 5 1 31worker,electricianFurniture

manufacturing 4 2 23workerGasoline

station attendant 21Gasworker 11Hairdresser

3 21Leather& shoe 11manufacturing

workerMetalindustry worker 14 10 56Painter

7 3 35PlasticsmanufacturingworkerPrinting

worker 1 23Rubberindustry worker 21Sweep,fire-fighter 1 32Total

54 50 24 32

GLUTAThIONE S-TRANSFERASE Ml IN BLADDER CANCER

a From the respective Mantel-Haenszel x@ values (33), using the BMDP4F program (34).

b@ ratios of GSTM1-deficient over GSTM1-active individuals in cases versus controls in the respective stratum and odds ratios adjusted according to the method of Mantel andHaenszel (ORMH) (33).

C These data were available for all cases and for 296 control patients.

d PY, pack-years.

chronic bronchitis (n = 83; 53.0%), pneumonia (n 20; 25.0%),myocardial infarction (n 45; 46.7%), nephrolithiasis (n 22;50%), prostatic hyperplasia (n = 23; 39.1%), intoxications (ii = 11;27.3%), diabetes mellitus (n = 14; 71.4%), heart failure (n = 12;50%), hypertension (n 24; 54.2%), and cardiac arrhythmias(n 16; 37.5%), followed by several less frequent diagnoses. The

1.54 1.12-2.13 0.009 differences in the proportion of carriers of GSTM1 *0/0 among these

1.34 0.89-2.00 Not significant diagnoses for the controls were not significant by x@ analysis.

1.75 1.17-2.61 0.006 GSTM1 in Different Subgroups of Cancer. The different types

of bladder cancer are presented in Table 5. Over-representation of theGSTM1-deflcient patients was pronounced among the group withmore advanced types of cancer; 20 of 29 patients (69%) with a tumor

uals, in contrast to 44.7% among the nonsmoking controls (Table 2).GSTM1 deficiency tended to be even less frequent among the heavysmokers (>50 pack years).

Analysis of lifetime occupational history showed that 35.1% of caseshad ever worked for at least 2 years in a profession identified to beassociated with increased bladder cancer risk. With a respective proportion of 18.9% of control persons occupied in these professions, the oddsratio for occupational risk was 2.73 (95% confidence limits, 1.78â€”4.17).The proportion of GSTMJâ€•O/Oindividuals in the specified risk occupations is given in Table 4, to allow exploratory analysis and comparisonwith other epidemiological data. GSTM1 deficiency was not strikinglyfrequent in motor exhaust-related occupations (auto mechanics and drivers), while the proportion of GSTM1 deficiency was pronounced inprofessions working with dyes. However, altogether there was not evena trend of higher prevalence of GSTM1-deficient bladder cancer patients,compared to the bladder cancer group without presumptive occupationalrisk (compare Tables 2 and 4).

Control Patients. The most frequent diagnoses of the referencepatients were bronchial asthma (n = 27; 44.4% with GSTM1 Â°O/O;thispercentage is given also for the other diseases), acute courses of

Table 3 GSTMI 0/Oand bladder cancer risk: summary of logisticregression results

Adjusted for age and genderWhole groupAggressive tumorsCModerately aggressive tumors

Adjusted for age, gender, cigarette smoking, and possible occupational exposureWhole group 1.60 1.1 1â€”2.29 0.01Aggressive tumors 1.38 0.89â€”2.13 Not significantModerately aggressive tumors 1.77 1.13â€”2.76 0.01

a Improvement of x@ by including GSTMJ genotype in the logistic model of bladder

cancer risk prediction.b One-tailed Fisher's exact P for the crude odds ratio.C See â€œMaterials and Methodsâ€• for definition and Table 5 for numbers.

4106



Table5 Impaciof GSTMl in various subsets of the cancergroupGSTM1@O/OTotal95%

confidencenumberNumber%bOdds ratioalimitsWhole

cancer group296175 59.1See Tables2 and3TypeAggressive14785

57.81.330.98â€”1.99Moderate1499060.41.480.99â€”2.21Grading019252

56.51.260.78â€”2.050270456431.751.00â€”3.09031347858.21350.89â€”2.05SizeTa,

Tis, Ti185113 61.11.521.05â€”2.21T2824251.21.010.62â€”1.69T3292069.02.150.91â€”5.50Transitional

cell, all279164 58.81381.00â€”1.91Papillartype224132 58.91390.99â€”1.97Flat

type5532 58.21350.73â€”2.51Other

types17ii 64.71.780.59â€”5.97Squamouscell4375.0â€”â€˜@carcinomaAdenocarcinoma41

25.0â€”Undifferentiated71143â€”carcinomaUnclassified21

50.0â€”

Table 6 Frequencies of the GSTMIisoenzymes A (a) and B (4') in differentstrata and subsets of bladder cancer andcontrolsGSTM1-active

individualsGSTM1AGSTM1BGSTMIA/BTotalnumberNumber%@Number

%Number%ControlsTotal40013433.553

13.3102.5Male2498835.33514.152.0Female1514731.118

11.953.3CancerTotal2966923.345

15.272.4Male2034120.23617.763.0Female932830.19

9.711.1Cancer

grading andstagingTypeAggressive1473725.222

15.032.0Moderate1493221.52315.442.7Grading01921920.718

19.633.302701521.4811.422.9031343526.119

14.221.5SizeTa,

Tis, Ti1854021.62714.652.7T282242931417.122.4T329517.24

13.80Histological

typesTransitionalcell, all2796523.34415.862.2Papillartype2245223.234

15.262.7Flattype551323.61018.20Rare

histological forms19421.015315.3a

@ of total in the respective rows.

GLUTAT1HONES-TRANSFERASE Ml IN BLADDER CANCER

type. There were, however, no differences among the latter groupswith respect to the GSTM1 traits.

GSTMJ alleles A and B. All GSTM1-active individuals werecharacterized with respect to their GSTM1 allelic status by use of thenested PCR procedure, and the results are given in Table 6 for varioussubgroups. These analyses resulted in the unexpected finding that thesmaller proportion of GSTM1-active individuals among the patientswith bladder cancer was entirely due to a smaller proportion ofcarriers of the GSTMI Â°Aallele. Direct comparison within the bladdercancer group of carriers of one or two GSTMI *B alleles (homodimeric GSTM1b-lb enzyme) versus carriers of one or twoGSTM1 *A alleles (homodimeric GSTM1a-la enzyme) revealed anodds ratio of 1.65 (95% confidence limits, 0.976â€”2.78; two-tailedFisher's exact P = 0.057).

Phenotypical Confirmations. In all 696 patients included in thisstudy, the genetic result (active or deficient) was controlled by analysis of the phenotypical expression of GSTM1, using immunologicalquantification of the enzyme in whole blood (Fig. 3). The histogramof the GSTM1 concentrations in blood illustrates that >90% of allindividuals could be classified as either active (GSTM1 8 mg/liter)or deficient (GSTM1 < 1 mg/liter) only by application of this immunoassay. Classifications based on molecular genetic results allowed,however, an unambiguous binary distinction of all individuals, evenof the few individuals with intermediate immunoreactivity.

Correspondence between the phenotypical assignments and thegenetic results was further ensured by the GSTM1-specific conjugation assay with the substrate trans-stilbene oxide, using venous bloodlymphocytes as the enzyme source. For practical reasons, this wasonly done in a selection of 123 bladder cancer cases and 247 controls.Fig. 4 shows the correlation of genotyping with the conjugationactivity in these 370 patients. A gene dose effect could be demonstrated by the fact that the special group of carriers of two activeGSTM1 alleles, namely the GSTM1Â°A/Bcarriers, were mostly amongthose with high activities. Nine of 10 had activities above the median

a Crude odds ratios compared with the whole control group (n 400).

b@ of total in the respective rows.C _, not calculated because of small sample size.

stage of T3 were GSTM1 deficient. This was, however, not significant, compared with the total control group, and no trends in theGSTM1 Â°O/Ofrequency with increasing tumor grade or stage wereseen. The predominant histological type of bladder cancer was transitional cell carcinoma, which was found in 94% of the cases andwhich could be further differentiated into a papillar type and a flat

4107



@,.

GLiffAThIONE S-TRANSFERASE Ml IN BLADDER CANCER

was no quantitative correlation of the GSTM1 *A or GSTM1 Â°Bgenotypes with the GSTM1 content in the urothelium. The mean GSTM1concentration in the samples of T 1 was 176 mg/kg (n = 16), andin those of T > 1 it was 161 mg/kg (n = 14); the mean GSTM1concentration in the samples of G@ 2 was 207 mg/kg (n = 22),compared to 64 mg/kg in those of G > 2 (n = 8). The latter data mayshow a tumor-associated down-regulation of GSTM1 expression butthe differences in GSTM1 concentration according to both criteria,grade and stage, were not significant (t-test), due to the relativelysmall sample size. The homogenate approach, however, had thedisadvantage of variable content of connective tissue, and theoretically the source of the GSTM1 concentrations measured in homogenates may even have been lymphocytes that had invaded the bladderwall. However, immunohistochemical analysis revealed that GSTM1was expressed in the transitional epithelium, and only a few positivelystained single cells, which may have been lymphocytes, were seen(Fig. 5).

DISCUSSION

Over-representation of the Defective GSTM1 Trait in BladderCancer. There was a statistically significant over-representation ofGSTM1-defective patients in the group with bladder cancer, as shownby genotyping and confirmed by two methods of phenotypical analysis. The detected proportion of 59.1% GSTM1-deficient bladdercancer patients exceeded the proportion of 53% found previously in

Bladder cancer patientsn = 296

Control patientsn = 400

n

60

20@

20-

4@:

60@

0.01@@ @oWhole blood GSTM1-content, mg/I

Fig. 3. Distribution of the GSTM1 blood concentrations immunologically testedamong bladder cancer and control patients, in relation to the genotypical classifications.U, GSTM1-deflcientindividuals; @,personswith one or two GSTMJA alleles;0,persons with one or two GSTMJ @Balleles; 0, persons with a heterozygous GSTMI 5A/Bgenotype. The blood concentration of GSTM1 serves as a surrogate parameter for theGSTM1 concentration in tissues such as the bladder urothelium (see Fig. 5).

n

60 a GSTM1'O/O n=214m GSTM1A/AorGSTMIA10 n-99

@ GSTM1B/B orGSTM1B/0 nâ€”47

@ GSTM1'A/B n=10

40

20

I.@

_l@@ @-@ @-@-

10 20 30 40 50 60pmole/min/1 million lymphocytes

Fig. 4. Distribution of GSTM1 activity in lymphocytes, measured as the activity forconjugation of reduced glutathione with the GSTM1-specific substrate trans-stilbeneoxide. A substrate concentration ofSO gistand about i0@lymphocytes were used. Negativevalues indicate the scatter of the assay in the case of deficient enzyme activity (25).

#

v*@@

* .

i@.

â€˜, t, @_ *@(

@,@ @.@ .@@

@;; â€˜@

S

-@

.@

* @â€˜(â€˜@ 4@@@

@.. .@@@@

% ** %@

.@@ a,

4

value of all genetically GSTM1-active individuals (i.e., above 24pmol/min/10â€• lymphocytes), which was significant with P < 0.01(Fisher test). The formerly found antimode of 10 pmol/min/106 lymphocytes (25) could be confirmed.

GSTM1 Expression in Bladder Cancer Tissue. For 74 of thepatients, samples of cancer tissue were available and were analyzed astissue homogenates for GSTM1 content, by immunological quantification (ELISA). Of these, only 30 samples were GSTM1 positive,with a mean tissue GSTM1 concentration of 169 mg/kg (this meandoes not include concentrations for the GSTM1 *@/@samples). There

T@@@ :@@@@

# *#*, â€¢@@ @. @.

Fig. 5. Immunohistochemical analysis ofGSTM1 in bladder tumor samples. A, samplefrom a patient who was genetically GSTM1 active. The red- or orange-stained cells denotethe presence of immunoreactive GSTM1. In none of the genetically GSTM1-defectiveindividuals could GSTM1-specific staining be seen. B, tissue of a homozygous GSTM1-defective individual, processed in the same batch and with the same procedure as in A.

4108

4.,

4@1@ r4@@@@ :@@ .@@



Table 7Epidemiologicalstudies on GSTMI deficiency asa bladder cancer riskfactorBladder

cancercasesControlsTotal

Deficientnumber (%)Total

Deficientnumber (%)Odds

ratioâ€•(95% confidence

limits)Pâ€•MethodAdditional dataInvestigators, year,country213c

61199c 481.7 (1.1â€”2.5)0.007GenotypingBell et aL (39), 1993,USALafuente

et aL (40), 1993, SpainDaly et aL (41), 1993, EnglandThong ci al. (42), 1993, EnglandThis study, 1994, Germany

GLIffAThIONE S-TRANSFERASE Ml IN BLADDER CANCER

Community controls; datafor African Americans

75 66.7 75 45.4 2.41 (1.18-4.93) 0.007 Phenotyping GSTM1 in larynx cancer53 84.9 52 59.6 3.81 (1.53â€”9.34) 0.0002 Genotyping Healthy controls97 40.2 225 41.8 0.84(050â€”1.40) Not significant Genotyping Other types of cancer

@ _@_L@ @@:?_ 1.40 (1.02â€”1.92) 0.017 Geno and phenotyping giIi@s isoenzyme differentiation

a Frequency of GSTM1-active and GSTM1-deficient individuals among cancer patients and controls; crude odds ratios.

b Calculated by the one-tailed exact Fisher test.C Only data for Caucasians and hospital controls are given here, for reasons of comparability.

lung cancer patients in Berlin (17) using a similar analytical methodology. A large proportion of GSTM1-defective individuals amongbladder cancer patients has recently also been reported by othergroups, as summarized in Table 7 (39â€”41). Only one report showedan opposite trend (42) of under-representation of the GSTM1 *0/0genotype in bladder cancer. With a sample size of 97 cases that studyhad a relatively low statistical power but, including it, the five studiesin Table 7 failed testing for homogeneity; thus, a common odds ratiofor GSTM1 as bladder cancer risk factor could not be calculated.

The crude odds ratio of 1.4 (Tables 2 and 3) was less than initiallyassumed for the sample size calculations (see â€œMaterialsand Methodsâ€•).Nevertheless, due to the high prevalence of the risk factorGSTM1 deficiency (about 50% of all individuals), this odds ratio is ofimportance in bladder cancer epidemiology. The impact of GSTM1 isexpressed by the etiological fraction of 0.17, which is based only onthe odds ratio found and on the proportion of GSTM1 *0/0 carriers(50.7%) in the control population. We have no hints that our controlsdo not reflect the target population with respect to GSTM1 frequency.The estimate of 17% of the bladder cancer cases being due to GSTM1deficiency is, however, relatively wide, with 95% confidence limits of2â€”30%.

Despite this obvious impact of GSTM1 deficiency, any predictivescreening for GSTM1 as a bladder cancer risk factor seems useless,because bladder cancer has an annual incidence rate of around 15/100,000 in Germany. With an etiological fraction of 0.17, only3/100,000 might eventually profit from the genetic information abouttheir increased risk and the remaining 49,997/100,000 with GSTM1deficiency would be falsely stated to be at higher risk for bladdercancer. Screening might be considered if certain occupationally defined subgroups were found to be at an especially high risk. Our studycould not, however, identify such subgroups, with respect to eithersmoking or occupation.

Genotype versus Phenotype Analysis. The correspondence between the genotype and phenotype of GSTM1 was established earlier(11, 17,25, 43) andwasconfirmedin thisstudyby functionalassaysand immunological quantifications. This methodical effort was necessary since most molecular genetic PCR assays can in principle notprove an active genotype. Thus, a phenotypical confirmation was still

required to exclude rare mutations. Among the almost 700 patients inFig. 3, only a few cases of ambiguous classification were found,mostly placed near the antimode. If we had not shown this phenotype

genotype correspondence for GSTM1 in bladder cancer, the high

proportion of 66.7% GSTM1-deflcient individuals found in anotherstudy (40) or the very high proportion of GSTM1-deficient individuals among patients with lung cancer found earlier (15) might befalsely explained by GSTM1 mutations which remained undetectedby our PCR assay. For future studies in our ethnic group we wouldrely completely on genotyping, because undetected mutations resulting in phenotypic changes are obviously rare or may even not exist.

In general, characterization by genotype should be the best methodof studying the impact of a polymorphic enzyme on cancer susceptibility in a case-control study. The genotypic configuration is a timeinvariant parameter. In contrast, enzyme activities may theoreticallybe affected by tumor-associated metabolic and hormonal changes andby many other factors. Thus, genotyping completely eliminates theproblem of reversed causality bias (44), i.e., differential misclassification between cases and controls. Furthermore, phenotyping provides values on a continuous scale. Although such continuous valuesmay be used for a ranked risk prediction in epidemiology, they areusually dichotomized (6). Figs. 3 and 4 show that the antimodebetween GSTM1 deficiency and activity is not absolutely defined,because of biological and analytical variability, while the answer fromPCR is binary.

GSTM1 Activity in the Urothelium Mother glutathione S-transferase, GST type A, is overexpressed in tumors such as hepatocarci.nomas, and GST type A may therefore be considered an early mdicator of malignant cell transformation (45). In contrast, concentrationdeterminations in 74 bladder cancer tissue samples failed to indicateoverexpression of GSTM1 in the urothelium with dependence eitheron different grades or on stages of the tumors. GSTM1 may exert aprotective function locally, i.e., within the bladder urothelium, andmoreover may act systemically, i.e., by detoxifying foreign compounds in the liver or other remote tissues. Also, in another tissuewhich is a frequent target of chemical carcinogens, namely the lung,the presence of GSTM1 was recently illustrated by Nazar-Stewartet aL (43). Those authors confirmed a gene dose relationship forGSTM1, showing that persons with very high catalytic activities ofGSTM1 were especially frequently carriers of two active alleles, asdetermined by quantitative evaluation of restriction fragment patterns.

Differentiation of the GSTM1Â°A and GSTMJÂ°B Alleles. PCRrevealed that the carriers of the GSTMJ *A allele were under-represented in the group with bladder cancer (Table 6). Assuming that theGSTM1a-la isoenzyme (@) is indeed more protecting than theGSTM1b-lb isoenzyme (*)â€one might calculate that 220 (74.3%) ofour 296 cancer patients were deficient of any GSTMJ Â°Aallele, cornpared to 256 (64.0%) of the 400 controls (odds ratio, 1.63; 95%confidence limits, 1.15â€”2.30),which was significant at P = 0.004even when the two-tailed test was used. The under-representation ofGSTM1 *A versus GSTMJ*B in bladder cancer should, however, beinterpreted with caution, since this constellation was not hypothesizedat the initiation of this study and there is currently no mechanisticexplanation for greater protective capacity of GSTM1a-la versusGSTM1b-lb, at least not with the specific model substrate transstilbene oxide (see Fig. 4). A study with isolated GSTM1a-la orGSTM1b-lb from a yeast expression system provided, similarly, nodifferential activity towards the model substances tested (46). However, the difference in the isoelectric points of the two isoenzymescorresponds to 1 order of magnitude in the H@ concentration (iso

4109



OLUTATHIONE S-TRANSFERASE Ml IN BLADDER CANCER

electric point of 6.6 for GSTMIa versus 5.5 for GSTM1b), which mayinfluence dirnerization of GSTM1 subunits, ligand binding properties,and binding affinities for other, not yet characterized, substrates.

Impact of Risk Occupations and Cigarette Smoking. Earlierepidemiological studies showed that only 5â€”10%and no more than30% of bladder cancers are attributable to occupation-related chemicals (3, 19, 47). In our study, 35.1% of the cases and 18.9% of thecontrols had ever worked for more than 2 years in occupations thathave been associated with increased bladder cancer risk (Table 4). Ofthe cancer patients studied in Berlin, 69% had ever smoked, butstratification according to extent or duration of smoking gave nofurther information (Table 2). Proportions of GSTM1-deficient mdividuals were not statistically different between nonsmokers (i.e.,never-smokers), moderate smokers, and heavy smokers, and there waseven a trend to a decreased impact of GSTM1 on bladder cancer riskwith increasing total cigarette consumption. The causalities behindthese observations remain unknown, but it is evident that carcinogensother than those from cigarette smoking or occupation have an important tumor-inducing or -promoting role in bladder cancer and adifferential role of GSTM1 with these substances might be postulated.The involvement of GSTM1 isoenzymes in the synthesis of leukotriene C4 from leukotriene A4 may be further studied to explain the roleof GSTM1 in bladder cancer (48). Furthermore, glutathione S-transferases play an ambiguous role; most toxins and carcinogens aredetoxified by these enzymes, but there are some substrates (especiallyhalogenated compounds) which are known to be toxified by glutathione S-transferases. The data of Daly et a!. (41) agree with our findingthat the protective role of GSTM1 is not especially evident in smokers, whereas Bell et a!. (39) found that the odds ratio for bladdercancer in relation to GSTM1 deficiency was roughly doubled only insmokers.

Are There Differences between Types of Bladder Cancer?Transitional cell carcinoma with different grades of differentiationand stages of tumor invasiveness may represent entities with differentetiology and prognosis and differences in the tumor molecular genetics. Thus, in the case of the p53 tumor suppressor gene, certainmutations are seen mostly in tumors of G 3 or T 3 (49, 50). Thus,types of mutations and initiating events in carcinogenesis (includingthe impact of detoxification by GSTMI) may differ in the aggressiveand moderate types of bladder cancer. In the present study, of 147cases with the more aggressive types 57.8% were deficient inGSTM1, a proportion equal to that for the whole group of cancerpatients (Table 5). Remarkably but statistically not significantly, inthe small group with tumors classified as T 3 (n = 29), 69% of theindividuals were GSTM1 deficient, and it may be an interestingsubject of further studies to determine whether the defined types ofmutations within the tumors, e.g., p53 mutations, differ in relation tothe GSTM1 status.

ACKNOWLEDGMENTS

This study would not have been possible without the cooperation of the

Departments of Urology at the Krankenhaus NeukOlln and the Klinikum

Steglitz, and we thank especially Prof. Dr. P. Schabert and Dr. K-P. Dieck

mann for their cooperation. The scientific contributions of C. Sawatzki, K.Jayanata, G. Lenting, J. LÃ¼bker,and S. Wieczorek are gratefully acknowledged. Furthermore, the highly skilled technical assistance of B. Schwanebergis appreciated. The authors thank Dr. S. Patt for his help with the histologicalprocedures and Dr. A. Neubauer for his valuable suggestions regarding thedifferential PCR technique. Last but not least, we thank Dipl.-Math. M. Nitz

for all his help with data processing.

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1994;54:4103-4111. Cancer Res Jürgen Brockmöller, Reinhold Kerb, Nikolaos Drakoulis, et al. Factors of Bladder Cancer Susceptibility: A Case-Control Study

-Transferase M1 and Its Variants A and B as HostSGlutathione

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