A Molecular and Epidemiological Study on Bladder Cancer: P.53 Mutations, Tobacco Smoking, and Occupational Exposure to Asbestos1

Vol. 5. 33-39. Jwiuarv 1996 Cancer Epidemiology, Biomarkers & Prevention 33

A Molecular and Epidemiological Study on Bladder Cancer: p.53 Mutations, Tobacco Smoking, and Occupational Exposure to Asbestos1

Annamaria Kannio, Maaret Ridanp#{228}a,2 Heikki Koskinen, ical type of bladder cancer is ICC,3 accounting for >90% of all Timo Partanen, Sisko Anttila, Yrj#{246}Collan, bladder cancer. Tobacco smoking is the most important exter- Eino Hietanen, Harri Vainio, and nal cause of bladder cancer, with an estimated attributable Kirsti Husgafvel-Pursiainen3 fraction of 40% or more in males and 30% in women in Departments of Industrial Hygiene and Toxicology [A. K., M. R., H. V., developed countries (3). K. H-P.[. Occupational Medicine [H. K.. S. Al, and Epidemiology and A number of occupational exposures are known to be Biostatistics [T. P.1. Finnish Institute of Occupational Health, Topeliuksenkatu associated with an elevated risk of bladder cancer. Prominent 41 aA. FIN-(X)25() Helsinki, Finland, and Departments of Pathology IY. C.] excesses have been associated with exposure to aromatic and Clinical Physiology [E. H.]. Turku University Central Hospital. Kiinamyllynkatu 10, FIN-20520 Turku. Finland amines in several industrial processes (3). The role of asbestos exposure in urological cancers has been less well studied and remains unclear. Exposure to asbestos has been related to an Abstract increased risk of kidney cancer (4- 1 1 ). In occupationally exposed individuals, the lungs. lymph nodes, and pleura have the In this study, we found an unexpected association (crude highest concentrations of asbestos fibers, but high levels of odds ratio = 2.8; 95% confidence interval = 0.9-8.4) asbestos fibers are also found in the kidneys (12-14). Further- between definite work-related exposure to asbestos and more, asbestos fibers have been demonstrated in the urine of carcinoma of the urinary bladder in a small group of individuals exposed to asbestos (15, 16). A few studies have patients (n = 28) initially recruited as referents for an epidemiological feasibility study on the occupational causes been conducted on the possible association between bladder of lung cancer. We extended the study by using molecular cancer and occupational exposure to asbestos. mostly with negative results (17-19). methods to examine mutations in the p53 tumor suppressor A prospective case-referent feasibility study on occupa- gene in the same cases of bladder cancers. The same tional causes of lung cancer was initiated in Finland in 1988. number of archival samples of transitional cell carcinoma, mainly of grade 3, were added to the analysis. We failed to Consecutive bladder cancer patients attending the urological clinic were recruited as referents for the lung cancer cases. show any association between occupational exposure to Their past occupational exposure to asbestos was assessed from asbestos and p53 mutations among bladder cancer patients. job histories obtained in personal interviews (20). This frame- We observed an increasing occurrence ofp53 mutations in work, and the fact that the identification and collection of nonsmokers (5 of 17, 29%), former smokers (8 of 21, 38%), archival tissue samples from the epidemiological pilot study and current smokers (9 of 16, 56%) in that order; however, was possible, prompted us to conduct a molecular genetic study this was not statistically significant The most prevalent type of mutation was G:C to A:T transition. Tumor grade was using the same subjects. To date, mutations of the p53 gene appear to be among the most common genetic changes in not associated with the frequency of mutations, but the bladder cancer, with the reported frequency varying between 20 higher stage (T3-T4) tumors appeared to have mutations and 60% (21 , 22). As p53 has been suggested as a useful target more frequently than did the less invasive tumors (T1-T2). gene for studies focusing on the role of mutations in carcinogenesis (23, 24), we sought to use the p53 mutation pattern as Introduction a potential biomarker for environmental exposure (in this case The incidence of the cancer of the urinary bladder has been tobacco smoking and asbestos exposure) in bladder cancer. increasing in almost all countries in Europe, including Finland We report here the observations from the epidemiological ( I ). Bladder cancer is the third most common site of primary feasibility study. We examined archival tumor tissue samples cancer among men in Finland (2). The most common histolog- from 55 bladder cancer patients with mainly grade 2-3 TCC. Almost one-half of the patients had been exposed to asbestos at work. Mutations of the p53 gene were analyzed in relation to smoking habits and to interview-based asbestos exposure data. Received 5/23/95: revised 8/3 1/95: accepted 9/5/95. Mutation findings were also compared with the clinical features The costs of publication of this article were defrayed in part by the payment of of the tumors. page charges. This article must therefore be hereby marked advertisetnent in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I This study was supported in part by the Academy of Finland and the Finnish Subjects and Methods Cancer Foundation. 2 Present address: Department of Medical Genetics. University of Helsinki. P.O. Epidemiological Pilot Study Box 21. FIN-(XX)14 University of Helsinki, Finland. The cases were 28 bladder carcinomas out of 33 patients 3 The abbreviations used are: TCC. transitional cell carcinoma: OR, odds ratio; CI. confidence interval; DGGE. denaturing gradient gel electrophoresis; NSCLC. admitted consecutively for diagnostic or surveillance purposes non-small cell lung cancer; 4-ABP, 4-aminobiphenyl. to the Surgery Clinic, Turku University Central Hospital

Table 1 Asbestos cx posure of bl adder cancer ca ses and refer ents. Data fr om the feasibil ity study (group I).

Cases Referents Asbestos exposure OR 95% Cl Ft (‘:4) t (%)

Definite 16 (57) 9 (32) 2.75 0.90-8.37 Possible I (4) 2 (7) 0.77 0.06-.9.6() Definite plus possible 17 )6l) 11 (39) 2.39 0.82-6.98 None II (39) 17 (61) 1.00

All 28 (100) 28 (100)

(Turku, Finland) between October 1988 and December 1988. Table 2 Occupations of bladder cancer patients and referents who had been There were 23 (82%) men and 5 (1 8%) women; the median age exposed to asbestos. Data from the feasibility study (group I). was 70 years (range, 41-79 years). Nine (32%) of the patients were current smokers, 12 (43%) former smokers, and 7 (25%) Cases Referents Occupation - had never smoked. Twenty-eight population referents (23 men ‘I (‘A’) it ((4.) and 5 women) were selected from 58 persons who were invited Construction worker I I (69) 6 (67) for routine radiographical screening for tuberculosis in three Shipyard worker 2 (13) 1 (II) towns in the health district of Turku University Central Hos- Brake repairer I (6) 2 (22) pital. The median age of the referents was 64 years (range, Foundry worker 1 (6) 52-83 years). Eight (29%) of the referents were current smok- Steam engine maintenance worker I (6) ers, eight (29%) were exsmokers, and 12 (42%) had never All 16 (100) 9 ) I(S)) smoked. Lifelong job histories, comprising job titles, task de- scriptions and duration. and smoking data were obtained from personal interviews with the subjects. completed by a trained interviewer. Patients who had quit smoking 1 year before the was performed once with xylene, followed by two 95% and two interview were classified as exsmokers. The cases were inter- 70% ethanol rinses, and samples were left to dry. Tissues were viewed at the bedside, the referents in connection with the pelleted between each extraction. The samples were incubated in screening procedure. Therefore, the interviewer was not 1 mg/mI protease (Sigma Chemical Co.), 10 mrsi Tris-HC1 (pH blinded to the bladder cancer referent status of the subjects. 7.5), 1 mM EDTA, 0. 1 M NaCl, and I % SDS for 3 days at 50#{176}C, followed by phenol-chloroform extraction and ethanol precipita- p53 Mutation Analysis tion. DNA was diluted into Tris-EDTA buffer, pH 7.4. The quality Tumor Samples. For diagnostic purposes, urinary bladder of DNA was determined on 1% agarose gel. Two .d of genomic cancer samples were obtained during surgery to remove tumors DNA were used in a 50 pi PCR reaction using 2.5 units Taq DNA or by biopsy at the Turku University Central Hospital between Polymerase (Promega) with 1-1.5 mrsi MgCl.,, 25 p concentra- 1975 and 1993. Formalin-fixed paraffin-embedded archival tis- tion of each dNTP (Pharmacia), 50 mrvi KC1, 10 mM Tris-HC1 (pH sue samples, which contained tumor tissue, were obtained for 9.0), 0.1% Triton X-l00, and 0.5-1.0 p.M of each primer (Institute this study from 27 bladder cancer patients recruited to the of Biotechnology, University of Helsinki, Helsinki, Finland). One feasibility study. Representative samples of nontumor tissue of each primer pair contained a 40-bp GC-rich sequence (GC- were not available. In this group (group 1 ), there were nine clamp), which incorporated into the final PCR product. The three- current smokers, ten former smokers, and eight who had never step amplification cycle (1 mm at 96#{176}Cfordenaturation. 1 mm at smoked (23 men and 4 women; mean age, 70 years; range 54-60#{176}C for annealing, and I mm at 74#{176}C)for synthesis was 41-77 years); 46 tissue samples were received. Twenty-three of repeated 30-40 times. The primer sequences for exons 6-8 are the cases were classified as grade I -3 (TCC) of the bladder, one given in Ridanp#{228}#{228}et a!. (27) and for exon 5 in Beck et a!. (28). TCC in situ, one papilloma, one urethra polyp, and one TCC of DGGE Gel Runs and Sequencing. The gels (1-mm thick) the ureter. Group 2 consisted of random cases of TCC, mainly contained 7.5% acrylamide in I X Tris-acetate-EDTA buffer grade 3. Of these 28 patients, 7 were current smokers, 1 1 were (pH 8.0), N,N’-diallyltartaramide as the cross-linker and vary- former smokers, and 9 were nonsmokers, four of whom had ing denaturant concentrations ( I 00% denaturant corresponds to never smoked; for 1 case no data on smoking were available (23 7 M urea and 40% deionized formamide). N,N,N’,N’-tetrameth- men and 5 women; mean, 70 years; range, 43-90 years). Smok- ylethylenediamide and ammonium persulfate were used to ac- ing histories for group 2 were obtained from hospital records tivate the gels polymerization (29). The gels were run in 1 X and based on a patient’s smoking status when admitted to the Tris acetate-EDTA buffer at 60#{176}Casdescribed by Ridanp#{228}#{228}et hospital. Thirty-one tissue samples were received for analysis. a!. (27). The running time for exon 5 was 5 h at 10 W or 150 Tumors were graded histopathologically (25) and staged using V for the parallel gel (40-70% gradient of denaturants). Each the tumor-node-metastasis classification (26). Occupational p53 mutation found in parallel gel runs was also confirmed in histories of the patients in group 2 were not sought. perpendicular gels. The gels were stained with ethidium bro- DNA Extraction and PCR Amplification from the Paraffin- mide and photographed by using a UV transilluminator. For embedded Archival Tissues. Two 25-gm-thick sections were sequencing, PCR amplification was carried out with a biotiny- sliced from each paraffin block and placed in an Eppendorf tube. lated primer. Direct sequencing (Sequenase version 2.0 proto- The microtome blade was carefully cleaned with ethanol between col; United States Biochemical) of the PCR product was per- samples to avoid cross-contamination. Two parallel samples were formed by using magnetic beads (Dynabeads M280 cut from each block. For mutation analysis, DNA was isolated and Streptavidin; Dynal) as solid support. Sequencing gels con- extracted two to three times from each block. Deparaffinization sisted of 4.5% polyacrylamide and 7 M urea (Bio-Rad). The

Table 3 Tobacco smoking and cI inical status of the bladder can cer cases who had p53 mutations

Sample no. Sex/age (yr) Smoking” Asbestos exposure’ Graded Stage Exon with mutation

Group I UB 1 I M/56 CS + I ND 6,7 UB26 M/63 CS + 2 T, 7 UBI3 M/70 CS + 3 T4 5 UB24 M/57 ES + I T, 6 UBI M/41 ES + 2 T3 6 UB9 M177 ES + 2 T3 6

UBI9 F/52 CS - I T1 6

UB2O F173 CS - 3 T 6.8

UBI6’ M/64 CS - 3 ND 6

UB7 M/51 ES - 2 T, 6

UB25 F/60 NS - 3 T, 6

UBI5 M178 NS - 2 ND 5

Group 2 UB35 M167 ES ND 2 T3 8 UB32 M/65 CS ND 3 T 7 UBMY’ M/43 CS ND 3 T2 S UB54 M/60 CS ND 3 ND 6 UB4I M/7l ES ND 3 T 5 UB36 F/67 ES ND 3 T, 6 UB52 M/76 ES ND 3 T2 8 UB42 M15l NS ND 3 T3 8 UB56 M170 NS ND 3 T 8 UB47 M/78 NS ND 3 ND 8

“ M. male: F. female. ‘, CS. current smoker: ES. exsmoker; NS, nonsmoker. , +, exposed: -. not exposed: ND, not determined.

‘I Histological grading according to WHO recommendations (25). ,. Pathological staging according to the tumor-node-metastasis classification (26): ND, not determined. , TCC originating from ureter.

S Exon 6 (codon 213) polymorphism also detected.

sequencing primers used were taken from Ridanpaa et a!. (27). with definite exposure to asbestos ranged from 6 to 44 years At least three separate PCR products from each sample were (median, 20 years) among the cases and from 10 to 47 years sequenced to confirm the result. (median, 21 years) among the referents. Of the 28 primary bladder cancer cases in group 1 , 17 (61%) had been exposed to asbestos at work. Of these, 16 Statistical Analysis (94%) were considered to have been definitely exposed. A In bivariate binary analyses, simple logit routines were used to crude OR of 2.8 (95% Cl = 0.9-8.4) was calculated for estimate the OR and their 95% CI for the outcome parameters. definite exposure to asbestos against no asbestos exposure and A small number of multivariate maximum likelihood logistic 2.4 (95% CI = 0.8-7.0) for the pooled category of definite to models were fitted to the mutation data. ORs and 95% CIs for possible exposure against no asbestos exposure (Table 1). the dependent parameter, the presence/absence of p53 muta- p53 Mutations in TCC Patients. Altogether, 77 paraffin- tion, were estimated conditionally on the determinants (previ- embedded tissue samples from 55 patients with urothelial can- ous occupational exposure to asbestos, tobacco smoking. tumor cer were analyzed for mutations in exons 5-8 of the p53 gene differentiation, and stage) and the potential confounders (gen- by using DGGE. In the group of patients recruited for the der and age). The following codings were applied: gender (male feasibility study, a paraffin-embedded tissue sample containing versus female); age at the time of tumor sampling (67-90 years tumor tissue was obtained from 27 cases (Table 3; group 1). A versus younger); tobacco smoking (current or former versus p53 mutation in the tumor tissue was found by DGGE analysis no); asbestos exposure (yes versus no or unknown); missing in 12 (44%) cases. The TCC cases analyzed for mutation in data for asbestos exposure (missing versus known); tumor group I included 15 patients who were classified as having differentiation (grade 3 versus grades 1-2); and tumor stage (T1 been definitely exposed to asbestos; 6 (40%) of them had a versus T,-T4). The missing asbestos exposure variate was mutation, and they were either current or former smokers (Ta- added because data on exposure to asbestos were missing for a ble 3). In addition, we analyzed mutations in 28 random TCC considerable number of subjects. samples, mainly of grade 3 (Table 3; group 2); in this group, 10 (36%) mutations were found. Results From the 22 archival samples, which repeatedly tested Asbestos Exposure in Bladder Cancer Patients. Table 1 positive for mutation using DOGE, we were able to sequence shows the distribution of asbestos exposure among the bladder 14 (Table 4). A representative DGGE gel run of DNA frag- cancer cases and their referents (group 1 ). Table 2 shows the ments amplified from exon 6 showing alterations from the wild latest occupations for those bladder cancer cases and referents type is presented in Fig. 1A. In Fig. 1, a polymorphism in exon who were classified as having been definitely exposed to as- 6 (codon 213) can be distinguished from a mutation in the same bestos at work. The duration of employment in occupations exon by the pattern of migrating DNA fragments. Examples of

8 Table 4 Types of j,53 mutations sequenced from the transitional cell carcinoma sainples

. , Amino acid Sample no. Exon ( odon Base change change 7-. UB4I 5 155 ACC-CCC Thr- Pro UBI3 5 173 GTG-GGG Val-Gly UBI5 5 173 GTG-’GGG Val-’Gly UB36 6 196 CGA-CCA Arg-’ Pro 6 UB2O” 6 213 CGA-*CAA Arg-’Gln UB26 7 234 TAC-TCC Tyr-. 5cr FBI I’ 7 236 TAC-*GAC Tyr-’Asp UB32 7 248 CGG-’TGG Arg-.Trp n=5 5 UB42 S 271 GAG-#{176}AAG Glu-#{176}Lys UB47 S 273 CGT-#{176}TGT Arg-’Cys ri UB35 S 28)) AGA-’ACA Arg-Thr 0 UB52 8 280 AGA-AAA Arg-Lys 4 [ UBSO S 283 CGC-*TGC Arg-Cys UB2O” S 29() CGC-CAC Arg- His E “ Boldface and underlining indicate base change.

,, Patient with two sequenced tiutattons. If) ‘ Patient with one sequenced and one DGGE.detected mutation. c_ C

n=2 A 2-

. . #{149}‘r 1I_ wt wt -:‘- ‘- . -

0 - Wild type UB47 UBS2 UB35 G:C>A:T A:T>C:G G:C>C:G

ACCT A(T A Fig. 2. Distribution of base substitutions in the 14 p53 mutations sequenced from a total of 22 samples showing a mutation in the DGGE analysis.

showed that mutations most often occurred in exons 6 and 8; we detected 1 1 mutations in exon 6 and 7 mutations in exon 8 using DGGE (Table 3; Fig. 1). In addition, we observed two cases exhibiting a codon 213 polymorphism. Of these two, patient UB5O also had a somatic mutation in exon 8 in the tumor (Table 3). In total, the mutational analysis revealed that 22 of the 55 cases studied had a mutation, with an overall mutation fre- L quency of 40%. When the patients were divided into nonsmok- 273: 280: 280: ens, former smokers, and current smokers, the mutation fre-

CGT -+ TGT AGA -+ AAA AGA-IACA quency was found to vary with smoking history. We found that 29% (5 of 17) of the nonsmokers, 38% (8 of 2 1 ) of the former

Fig. 1. Examples ofji53 mutations detected in archival bladder tumor samples. smokers, and 56% (9 of 16) of the current smokers had a p.53 A. DGGE analysis (if exon 6. Lwie.s 3-6 and 9. tumor DNA with a mutation: mutation in the tumor (Fig. 3). Two patients (UB I 1 and UB2O) Lanes 1 and 8, wild-type DNA: Lane 2, a codon 2 I 3 polymorphism: Lane 7, lung were found to have two separate p53 mutations (Tables 3 and tumor DNA used as a positive control for exon 6 mutation. The wild-type (nt) 4). The multivariate logistic modeling of the p53 mutation hands are indicated. B, sequence analysis of mutations in exon 8. The codons and the base substitutions are indicated. The case numbers refer to Tables 3 and 4. outcome also indicated that tobacco smoking was associated with p53 mutation, although not with statistical significance (Table 5). When pathological grade and tumor stage were analyzed, sequence analysis are shown in Fig. lB. Most of the sequenced the TCC cases studied were distributed as follows: 5 grade 1 mutations had occurred at G:C base pairs. where nine. i.e., tumors, 18 grade 2 tumors, and 29 grade 3 tumors; 25 were >60%, of the mutations were found. Seven G:C to A:T tran- stage T2-T4 (invasive type) tumors, and 20 were stage T1 sitions were seen; five of them occurred at cytidine phosphate tumors. Crude and adjusted logistic analyses both showed tu- guanosine dinucleotides (CpG: Fig. 2). All the sequence van- mon stage to be the strongest determinant for the presence of ations identified were missense mutations resulting in amino mutation, although not to a statistically significant degree acid substitutions (Table 4). (Table 5). Of the stage T3-T4 tumors, 56% (9 of 16) exhibited The distribution of mutations within the p53 gene a mutation, compared to 28% (8 of29) ofthe T-T, tumors. Six

1 00 transformation of bladder tissue (34-36). Similar mechanisms have been suggested for asbestos-related pulmonary carcinogenesis (37). It has been reported recently that lung tumors from 90 patients exposed to asbestos more frequently demonstrated p53 accumulation in immunohistochemistry than did tumors from non-

80 exposed patients (38). Our data suggest that the frequency ofp53 mutations in the bladder tumors is dependent on the history of tobacco smoking. 70. The lowest mutation frequency (29%) was found in tumors from nonsmokers. and the frequency was 38% in exsmokers ci, and 56% in current smokers. In earlier studies, associations 0 60. 9/16 between p53 mutation and tobacco smoking have been observed in cancers of the lung, esophagus, and bladder, as well

as in cancers of the head and neck (21 . 39-41 ). Nuclear E 50. overexpression or mutations have been found in 40-45% of bladder tumors from smokers as compared to a lower level

40 8/21 among exsmokers and nonsmokers (30-35%: Refs. 2 1 . 42). p53 Mutation Pattern. A clustering of mutations in the fifth conserved domain (codons 270-288) of the p53 gene has been 30 5/17 found, and the sequence around codon 280 in exon 8 has been suggested as a hotspot for mutations in bladder tumors from

20 smokers (21 ). In line with this, we found four mutations between codons 270-288. Two patients, a current and a former smoker, had a mutation in the second base of codon 280 (G to C and G to 10’ A substitutions, respectively). and one current smoker had a mutation in codon 283 (C to T). In addition, we found two tumors from nonsmokers with mutations in this area: a codon 27 1 muta- S tion (G to A) and a codon 273 mutation (C to T) mutation. Bladder Cs ES NS tumor mutations reported previously in this region include G:C to A:T and G:C to C:G mutations (21, 43). Also, double mutations Fig. 3. Distribution of mutation positive cases in the various smoking groups have been seen in bladder cancer and have been suggested as number of cases with a mutation/number of cases analyzed). C’S. current smoker: features associated with smoking (21, 43). In the present study. ES. exsnioker: NS, nonsmoker. two tumor samples, both from current smokers, exhibited two separate mutations. Although exposure to tobacco smoke is clearly associated grade 2 tumors (33%) had a mutation; the corresponding fre- with bladder cancer, and the highest frequency of mutations was quency among grade 3 tumors was 45% ( 13 of 29). Thus, the seen in tumors from smokers, the pattern of mutations we observed prevalence of mutations appeared to be slightly increased in was not typical of the pattern associated with smoking in other tumors of higher grade. but the association was not significant cancers. The most frequent type of mutation we observed was G:C (Table 5). There was no indication of that gender or age were to A:T transition. Fifty % of the sequenced mutations were of this associated with p53 mutation. type, and most of them had occurred at CpG sites. It is known that spontaneous and enzymatic deamination of 5-methylcytosine re- Discussion suit in G:C to A:T transitions, and the prevalence of these substi- Asbestos Exposure and Bladder Cancer. The epidemiolog- tutions has been used as an index for the contribution of sponta- ical part of our study suggested an almost 3-fold increase in the neous mutations to the overall mutational pattern (44). Many risk of bladder cancer, associated with definite previous expo- previous studies have similarly indicated G to A transitions as the sure to asbestos. as compared with no such exposure. Sixteen most prevalent type of mutation in bladder cancer, with approxi- patients had been definitely exposed to asbestos; 1 1 of them had mately one-half at CpG sites (22, 45). In comparison, non-small worked mainly in the construction trade. Therefore, it is pos- cell lung cancers from smokers demonstrate predominantly G:C to sible that the observed association with construction work T:A substitutions (24, 40, 44, 46). This is consistent with the data could reflect exposures to carcinogens other than asbestos. showing that metabolites of polycyclic aromatic hydrocarbons of Among the few studies on bladder cancer and exposure to the chemical components of tobacco smoke are the genotoxic asbestos, only one has linked asbestos exposure with bladder agents most likely responsible for cellular DNA damage (47). We cancer ( 19), whereas asbestos exposure has been associated have analyzed recently 99 NSCLC titmons for p53 mutations and with kidney cancer in several studies (4-1 1). observed that 34% of all base substitutions were G to T transver- p53 Mutations, Asbestos Exposure, and Tobacco Smoking. sions (27, 48). In the p53 tumor suppressor gene, an overall mutation frequency Forty-six % of the mutations detected in our TCC samples of4O% was observed in the patients with TCC of the bladder. This occurred in exon 6 of the p53 gene. We succeeded in sequenc- is consistent with the data in the literature (21, 30-33). The ing only 2 of the 1 1 samples showing altered migration of an statistical analyses performed did not support an association be- exon 6 fragment in DGGE. However, the distinct pattern of tween asbestos exposure and p.53 mutations. However. factors bands in DGGE allowed us to distinguish the polymorphism at stimulating urothelial cell proliferation. as well as the tissue darn- codon 2 1 3 (49) from mutations as documented here and also age associated with chronic inflammation and the activation of previously (50). Other studies have also reported mutations in macrophages, have been shown to be involved in the malignant exon 6 (codon 213) in bladder cancer (21).

Table 5 Crude (univariate) and adjusted ( multivariate) logistic regression m odds ofp53 mutation (p 53+) and selected determinants.

% with determinant Crude Adjusted Determinant NCD” in p53+ in p53- OR (95% CI) OR (95% Cl) OR (95% CI)

Gender (male) 18 81.8 84.6 0.80(0.19-3.40) 0.32(0.05-1.98) 0.47(0.06-3.80) Age (67-9)) yr) II 50.0 54.5 0.83 (0.28-2.46) 0.67 (0.16-2.84) 0.75 (0.17-3.26) Tobacco smoking (current or formert 17 77.3 62.5 2.04 (0.60-6.96) 3.09 (0.58-16.5) 3.28 (0.57-18.9) Asbestos exposure (yes)” 6 50.0 60.0 0.67 (0. 14-3.09) 0.72 (0.08-6.44) Asbestos exposure (unknown)” 10 50.0 50.0 1.00 0.14 (0.01-3.16) Tumor differentiation (grade 3)’” 3 59.0 53.3 1.26 (0.42-3.84) 0.72 (0.15-3.41 ) 0.66 (0.18-39.3) Tumor stage IT2. T3, or T4Y 12 70.6 46.4 2.77 (0.77-9.97) 3.75 (0.71-20.0) 4.90 (0.80-30.1)

‘, NCD. number of p53+ cases with determinant.

,, Women dismissed from analysis (none exposed).

‘ Histological grading according to WHO recommendations (25).

‘I No grade 4 in the data.

( Pathological staging according to the tumor-node-metastasis classification (26).

PS3 Mutations and Exposure to Bladder Carcinogens. sure and the prevalence of p53 mutations in bladder cancer Human exposure to aromatic amines comes mainly from to- patients. We observed an increasing occurrence of p53 mu- bacco smoking and occupational exposure. This group of chem- tations in tumors from nonsmokers, former smokers, and icals includes 4-ABP and 2-naphthylamine known as potential current smokers, in that order; however, the correlation human bladder carcinogens (3, 5 1). Consequently, DNA-bind- remained statistically nonsignificant. ing products of 4-ABP have been found as a major adduct in biopsies from bladder cancer patients who were smokers (52) Acknowledgments and from DNA isolated from exfoliated urothelial cells in urine We wish to acknowledge Anneli Ojajarvi. M.Sc., for statistical analyses, Tuula of smokers (53). 4-ABP has been shown to induce primarily Suitiala for excellent technical assistance, and Anne Hand, B.Sc.. for language G:C to T:A substitutions in experimental situations (54). revision. Information about p.53 mutations in bladder cancer from patients with an occupational history of exposure to aromatic References amines is limited. A recent study (42) looked into associations I. Coleman, M. P., Esteve, J., Damiecki, P., Arslan, A., and Renard, H. (eds.). among occupations with exposure to aromatic amines, tobacco Trends in cancer incidence and mortality. IARC Sci. PubI. No. 121, pp. 543-576. Lyon. France: IARC, 1993. smoking, and nuclear overexpression of p53 protein in bladder 2. Cancer Society ofFinland. Cancer Incidence in Finland 1992. Cancer Statistics cancer patients with superficial TCC. Elevated, but statistically of the National Research and Development Centre for Welfare and Health. PubI. nonsignificant. ORs for association with dye-Ttnk-related and No. 54. Helsinki, Finland: Cancer Society of Finland. 1994. cooking-related occupations were found. Another study (45) ex- 3. Tomatis, L.. Aitio. A., Day. N. E.. Heseltine, E.. Kaldor, J., Miller A. B., amined mutations in p53 in TCC from workers exposed to 4-ABP, Parkin, D. M.. and Riboli, E. (eds.). Cancer: causes, occurrence and control. IARC 2-naphthylamine. and benzidine in the dye industry. The missense Sci. PubI. No. 100. Lyon, France: IARC, 1990. mutations identified included one G to T transversion, two G to A 4. Selikoff, I. J., Hammond, E. C., and Seidman. H. Mortality experience of insulation workers in the United States and Canada, l943-1976. Ann. NY Acad. transitions, and one C to T transition. However, the number of Sci., 330: 91-1 16, 1979. cases reported from the on-going study was relatively small. 5. Maclure, M. Asbestos and renal adenocarcinoma: a case-control study. Envi. Clinical Features and p5.3 Mutations. Tumor stage and grade ron. Res., 42: 353-361, 1987. are considered to be significant factors in disease progression to 6. Enterline, P. E., Hartley. J.. and Henderson, V. Asbestos and cancer: a cohort muscle invasive bladder cancer (55, 56). Of superficial carci- followed up to death. Br. J. Ind. Med.. 44: 396-401. 1987. nomas, T, tumors (confined to the bladder epithelium) have a 7. Morrison, A. S. Epidemiology and environmental factors in urologic cancer. progression rate of 3%, and T1 tumors (invading the lamina Cancer (Phila.), 60: 632-634, 1987. 8. Smith, A. H., Sheam, V. I., and Wood, R. Asbestos and kidney cancer: the propria) have a rate of 25% during the 2 years after diagnosis. evidence supports a causal association. Am. J. md. Med., 16: 159-166, 1989. About 50% of muscle invasive tumors (T2-T4) progress and 9. Enterline, P. E., and Henderson, V. Asbestos and kidney cancer. Am. J. Ind. metastasize (57, 58). In previous studies, mutations ofp53 have Med., 17: 645-646, 1990. been linked with high-stage and high-grade bladder tumors, 10. Edelman, D. A. Does asbestos exposure increase the risk of urogenital whereas the lowest frequencies were observed in noninvasive, cancer? Int. Arch. Occup. Environ. Health, 63: 469-475, 1992. low-grade tumors (30, 32, 56, 59). In our study, the p53 mu- I 1 . Selikoff, I. J., and Seidman, H. Use of death certificates in epidemiological tations appeared to be equally common in low-grade and high- studies including occupational hazards: variations in discordance of different asbestos-associated diseases on best evidence ascertainment. Am. J. Ind. Med., grade bladder cancers (OR 1.3; 95% CI = 0.4-3.8). but the 22: 481-492, 1992. prevalence ofp53 mutations was greatest in stage T3-T4 tumors 12. Auerbach, 0., Conston, A. S., Garfinkel, L., Parks, V. R., Kaslow, H. D., and with an OR of 3-5, depending for which covariates the analysis Hammonmd, E. C. Presence of asbestos bodies in organs other than the lung. was adjusted. These data are consistent with many recent im- Chest, 77: 133-137, 1980. munohistochemical and molecular analyses, which suggest that 13. Huang. J.. Hisanagu. N., Sakai, K., Iwata, M., Ono, Y., Shibata, E., and there is no clear association between p53 nuclear overexpres- Takeuchi, Y. Asbestos fibers in human pulmonary and extrapulmonary tissues. sion and either stage or grade of the primary tumor (60-62). Am. J. Ind. Med., /4: 331-339, 1988. Accumulation of nuclear p53 has been suggested recently to be 14. Tossavainen, A., Karjalainen, A., and Karhunen, P. J. Retention of asbestos fibers in the human body. Environ. Health Perspect., 102: 253-255, 1994. an independent prognostic marker in bladder cancer (63). 15. Finn, M. A., and Hallenbeck, W. H. Detection of chrysotile asbestos in In summary, the overall frequency of mutations among workers’ urine. Am. md. Hyg. Assoc. J., 46: 162-169, 1985. the 55 cases ofTCC studied was 40%. We failed in this study 16. Guilleman, M. P., Litzistorf, G., and Buffat, P. A. Urinary fibres in occupa- to find an association between work-related asbestos expo- tional exposure to asbestos. Ann. Occup. Hyg.. 33: 219-233, 1989.

17. La Vecchia, C.. Negri. E., D’Avanzo. B.. and Franceschi, S. Occupation and cigarette smoking and mutation of the p53 gene in squamous-cell carcinoma of the risk of bladder cancer. Int. J. Epidemiol.. 19: 264-268, 1990. the head and neck. N. EngI. J. Med., 332: 712-717, 1995. 18. Molinini, R.. Paoletti, L.. Albrizio, M., Penneella, A., Nardulli, F., and 42. Thang. Z., Sarkis, A. S., Cordon-Cardo, C.. Dalbagni, G., Melamed, J., Aprikian, Caruso. G. Occupational exposure to asbestos and urinary bladder cancer. Envi- A., Pollack, D., Sheinfeld, J., Herr, H. W, Fair, W. R., Reuter. V. E., and Begg, C. ron. Res., 58: 176-183, 1992. Tobacco smoking, occupation, and p53 nuclear overexpression in early stage bladder 19. Bravo, M. P., Del Rey-Calero. J., and Conde, M. Bladder cancer and asbestos cancer. Cancer Epidemiol., Biomarkers & Prey., 3: 19-24, 1994. in Spain. Rev. Epidemiol. Sante Publique. 36: 10-14, 1988. 43. Kusser, W. J., Mioa, X., Glickman, B. W., Friedland, J. M., Rothman, N., 20. Koskinen. H., Hemberg, S., HeikkilS, P., Kauppinen, T., Kurppa, K., Nurminen. Hemstreet, G. P., Mellot, J., Swan, D. C., Schulte, P. A., and Hayes, B. R. p53 M., Partanen T., Tossavainen, A., Liippo, K., Niinikoski, J., Tala, E., and Lahdensuo, mutations in human bladder cancer. Environ. Mol. Mutagen., 24: 156-160. 1994. A. Occupational causes oflung cancer an epidemiologic pilot study (Final Report; In 44. Harris, C. C., and Hollstein, M. Clinical implications of the p53 tumor- Finnish). Helsinki, Finland: Finnish Work Protection Fund, 1989. suppressor gene. N. Engl. J. Med.. 329: 1318-1327, 1993. 21. Spruck. C. H.. III. Rideout, W. M., III, Olumi, A. F., Ohneseit, P. F., Yang, 45. Esteve, A., S#{216}rlie,T., Martel-Planche, G., Hollstein, M., Kusters, I., A. S.. Tsai, Y. C., Nichols, P. W., Hom, T., Hermann, G. G., Steven, K., Ross, Lewalter, J., Vineis, P., and Montesano, R. Screening for p53 gene mutations in R. K., Yu. M. C.. and Jones, P. A. Distinct pattern of p53 mutations in bladder archived tumors of workers occupationally exposed to carcinogens: examples cancer: relationship to tobacco usage. Cancer Res.. 53: 1 162-1 166, 1993. from analysis of bladder tumors. J. Occup. Environ. Med., 37: 59-68, 1995. 22. Williamson, M. P., Elder, P. A., and Knowles, M. A. The spectrum of TP53 46. Hollstein, M., Rice, K., Greenblatt, M. S., Soussi, T., Fuchs, R., S#{248}rlie,T., mutations in bladder carcinoma Genes, Chromosomes & Cancer, 9: 108-1 18, 1994. Hovig. E., Smith-S#{248}rensen, B., Montesano, R., and Harris, C. C. Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res., 23. Hollstein, M., Sidransky. D.. Vogelstein, B., and Harris, C. C. p53 mutations 22: 3551-3555, 1994. in human cancers. Science (Washington DC). 253: 49-53. 1991. 47. Phillips. D. H.. Hewer, A., Martin, C. N., Garner, R. C., and King, M. M. 24. Puisieux, A., Lim, S., Groopman, J., and Ozturk, M. Selective targeting of Correlation of DNA adduct level in the human lung with cigarette smoking. p53 gene mutational hotspots in human cancers by etiologically defined carcin- Nature (Land.), 336: 790-792, 1988. ogens. Cancer Res.. 51: 6185-6189. 1991. 48. Husgafvel-Pursiainen, K., Ridanp#{228}#{228},M.,Anttila, S., and Vainio, H. p53 and 25. Mostofi, F. K., Sobin, L. H., and Torloni, H. Histological typing of urinary ras gene mutations in lung cancer: implications for smoking and occupational bladder tumours. In: WHO Intemational Histological Classification of Tumours, exposures. J. Occup. Environ. Med., 37: 69-76, 1995. No. 10, pp. 15-19. Geneva, Switzerland: WHO, 1973. 49. Carbone, D.. Chiba, I., and Mitsudomi, T. Polymorphism at codon 213 within 26. Union Intemationale Contre Ic Cancer. ThM Classification of Malignant the p53 gene. Oncogene. 6: 1691-1692, 1991. Tumours, Bladder, Ed. 3, pp. 1 13-117. Geneva, Switzerland: Union Intematio- nale Contre Ic Cancer. 1978. 50. Wild, C. P., RidanpS#{228},M., Anttila, S., Lubin, R., Soussi, T., Husgafvel-Pursi- amen, K., and Vainio H. p53 antibodies in the set’s of lung cancer patients: compar- 27. Ridanpaa. M.. Karjalainen. A., Anttila, S.. Vainio, H., and Husgafvel- ison with p53 mutation in the tumour tissue. tnt. J. Cancer, 64: 176-181, 1995. Pursiainen, K. Genetic alterations in p53 and K-ras in lung cancer in relation to histopathology of the tumor and smoking history of the patient. Int. J. Oncol., 5: 51. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals 1109-1117, 1994. to Humans Tobacco Smoking. Vol. 38. Lyon, France: IARC. 1986. 28. Beck, J. S.. Kwitek, A. E., Cogen. P. H., Metzger, A. K., Duyk, G. M.. and 52. Talaska, G., Al-juburi. A. Z. S. S., and Kadlubar, F. Smoking related Sheffield, V. C. A denaturing gradient gel electrophoresis assay for sensitive carcinogen-DNA adducts in biopsy samples of human urinary bladder: identifi- detection ofp53 mutations. Hum. Genet., 91: 25-30, 1993. cation ofN-(deoxyguanosin-8-yl)-4-aminobiphenyl as a major adduct. Proc. NatI. Acad. Sci. USA, 88: 5350-5354, 1991. 29. Ridanpaa. M.. and Husgafvel-Pursiainen. K. Denaturing gradient gel dcc- trophoresis (DGGE) assay for K-ras and N-ras genes: detection of K-ras point 53. Bartsch, H., Malaveille, C., Friesen, M., Kadlubar, F. F., and Vineis, P. Black mutations in human lung tumour DNA. Hum. Mol. Genet., 2: 639-644, 1993. (air-cured) and blond (flue-cured) tobacco cancer risk IV: molecular dosimetry studies implicate aromatic amines as bladder carcinogens. Eur. J. Cancer, 29A: 30. Sidransky. D., von Eschenbach, A., Tsai, Y. C., Jones, P., Summmerhayes, 1199-1207, 1993. I., Marshall, F.. Paul, M., Green. P., Hamilton, S. R., Frost, P., and Vogelstein, B. Identification of p53 gene mutations in bladder cancers and urine samples. 54. Lasko, D. D., Harvey. S. C.. Malaikal, S. B., Kadlubar, F. F., and Essigman. Science (Washington DC). 252: 706-709, 1991. J. M. Specificity of mutagenesis by 4-aminobiphenyl. J. Biol. Chem.. 263: 15429-15435, 1988. 31 . Cordon-Cardo, C., Dalbagni. G.. Saez, G. T.. Oliva. M. R., Zhang, Z.. Rosai, J.. Reuter, V. E.. and Pellicer, A. p53 mutations in human bladder cancer: 55. Heney, N. M., Ahmed, S., Flanagan. M. J.. Frable, W., Corder, M. P., Hafermann, M. D., and Hawkins, I. R. Superficial bladder cancer: progression and genotypic versus phenotypic pattems. Int. J. Cancer, 56: 347-353, 1994. recurrence. J. Urol., 130: 1083-1086, 1983. 32. Fujimoto, K.. Yukishige, Y., Okajima, E.. Kakizoe, T., Sasaki, H., Sugimura, 56. Kaubisch, S., Lum, B. L., Reese, J., Freiha, F., and Torti, F. M. Stage TI T., and Terada. M. Frequent association ofp53 gene mutation in invasive bladder bladder cancer: grade is the primary determinant for risk of muscle invasion. cancer. Cancer Res., 52: 1393-1398, 1992. J. Urol., 146: 28-31, 1991. 33. Petersen, I.. Ohgaki. H., Ludeke, B. I., and Kleihues, P. p53 mutations in 57. Anderstr#{246}m, C., Johansson, S., and Nilsson, S. The significance of lamina phenacetin-associated human urothelial carcinomas. Carcinogenesis (Land.), 14: propna invasion on the prognosis of patients with bladder tumours. J. Urol., 124: 2119-2122, 1993. 23-26, 1980. 34. Cohen, S. M., and Ellwein, L. B. Cell proliferation in carcinogenesis. Science 58. Cutler, S. J., Heney, N. M., and Friedell, C. H. Longitudinal study of patients (Washington DC). 249: 1007-101 1. 1990. with bladder cancer. factors associated with disease recurrence and progression. In: 35. Ngyen. T., Brunson, D.. Crespi. C. L., Penman, B. W., Wishnok, J. S., and W. W. Barney and 0. R. Prout, Jr. (cdx). American Urological Association Mono- Tannenbaum, S. R. DNA damage and mutation in human cells exposed to nitric graph Bladder Cancer, Vol. 1. p. 35. Baltimore, MD: Williams & Wilkins, 1982. oxide in vitro. Proc. NatI. Acad. Sci. USA. 89: 3030-3034, 1992. 59. Esng, D., Spruck, C. H., III, Nichols, P. W., Chaiwun, B., Steven, K., 36. Badawi, A. F., Mostafa, M. H., and O’Connor, P. J. Involvement of alkylating Groshen, S., Chen, S. H., Skinner, D. G., Jones, P. A., and Cote, R. J. p53 nuclear agents in schistosome-associated bladder cancer: the possible basic mechanism of protein accumulation correlates with mutations in the p53 gene. tumor grade, and induction. Cancer Lett., 63: 171-188, 1992. stage in bladder cancer. Am. J. Pathol., 143: 1389-1397. 1993. 37. Walker, C., Everitt. J., and Barrett, J. C. Possible cellular and molecular 60. Hamey, J., Murphy, D. M.. Jones, M.. and Mothersill, C. Expression of p53 mechanisms for asbestos carcinogenicity. Am. J. Ind. Med., 21: 253-273, 1992. in urothelial cell cultures from tumour-bearing and tumour-free patients. Br. J. 38. Nuorva, K., MSkitaro, R., Huhti, E., Kamel, D., Vahakangas. K.. Bloigu. R.. Cancer, 71: 25-29, 1995. Soini, Y., and PSSkkb, P. p53 protein accumulation in lung carcinomas of patients 61. Schmitz-Drager, B. J., van Roeyen, C. R. C.. Grimm, M-O., Gerharz, C-D., exposed to asbestos and tobacco smoke. Am. J. Respir. Crit. Care Med.. 150: Decken, K., Schulz, W. A., Bultel, H., Makri, D., Ebert, T., and Ackermann, R. 528-533. 1994. p53 accumulation in precursor lesions and early stages of bladder cancer. World 39. Hollstein, M., Peri, L., Mandard, A. M., Welsh, J. A., Montesano, R., J. Urol., 12: 79-83, 1994. Metcalf, R. A., Bak, M., and Harris, C. C. Genetic analysis of human esophageal 62. Serth. J., Kuczyk, M. A., Bokemeyer. C., Hervatin, C., Nafe, R., Tan, H. K., and tumors from two high incidence geographic areas: frequent p53 base substitutions Jonas, U. p53 immunohistochemistry as an independent prognostic factor for super- and absence of ras mutations. Cancer Res., 51: 4102-4106, 1991. ficial transitional cell carcinoma of the bladder. Br. J. Cancer, 71: 201-205, 1995. 40. Soussi, T., Legros. Y., Lubin, R.. Ory, K., and Schlichtholz, B. Multifactorial 63. Esrig, D., Elmajian. D., Groshen, S., Freeman, J. A., Stein, J. P., Chen, S-C., analysis ofp53 alteration in human cancer: a review. Int. J. Cancer. 57: 1-9, 1994. Nichols, P. W., Skinner, D. G.. Jones, P. A.. and Cote, R. J. Accumulation of 41. Brennan. J. A., Boyle. J. 0.. Koch. W. M.. Goodman, S. N., Hruban, R. H., nuclear p53 and tumor progression in bladder cancer. N. EngI. J. Med.. 331: Eby. Y. J.. Couch. M. J.. Forastiere, A. A., and Sidransky. D. Association between 1259-1264, 1994.

Downloaded from cebp.aacrjournals.org on September 30, 2021. © 1996 American Association for Cancer Research. A molecular and epidemiological study on bladder cancer: p53 mutations, tobacco smoking, and occupational exposure to asbestos.

A Kannio, M Ridanpää, H Koskinen, et al.

Cancer Epidemiol Biomarkers Prev 1996;5:33-39.

Updated version Access the most recent version of this article at: http://cebp.aacrjournals.org/content/5/1/33

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cebp.aacrjournals.org/content/5/1/33. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.