Activated H-ras gene mutations in transitional cell carcinoma of urinary bladder in a Kashmiri population

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1 Tumori, 96: , 2010 Activated H-ras gene mutations in transitional cell carcinoma of urinary bladder in a Kashmiri population Arshad Ahmad Pandith 1, Zafar Shah 1, Roohi Rasool 1, Dil-Afroze 1, Adfar Yousuf 2, Nighat Parveen 2, Saleem Wani 3, and Mushtaq Siddiqi 1 Departments of 1 Immunology and Molecular Medicine, 2 Clinical Biochemistry, and 3 Urology, Sher-I-Kashmir Institute of Medical Sciences, Soura, Srinagar, Kashmir, India ABSTRACT Aims and background. The primary aim of the study was to evaluate the incidence of H-ras specific point mutations among a group of Kashmiri patients diagnosed with bladder cancer. We also explored the correlation of clinic-pathological status of the illness with these mutations. Methods and study design. The DNA samples of both tumor and normal tissue were evaluated for the occurrence of H-ras activating mutations in exon 1 and 2 by PCR- SCCP and DNA sequencing. In addition, blood was also collected from all the cases to rule out any germ-line mutation. Results. Point mutations of activated H-ras identified in bladder cancer patients were 14.5% (7 of 48), including four transversions (two G T and two A T) and three transitions (A G). Of the mutations, 71.4% were detected in codon 61 and 28.6% in codon 12. The pattern of mutation in the study showed a significant association with smoking in bladder tumors (P <0.05). No correlation was found between tumor grade and/or stage and the presence of H-ras mutation. Conclusions. Activation of H-ras by mutation plays a less frequent role than other genetic events in the development of the most transitional cell tumors of the bladder in Kashmiri population. Free full text available at Introduction Bladder cancer is the fourth most incident cancer in males and ninth most in females. The America Cancer Society estimates that 70,980 adults will be diagnosed with bladder cancer, leading to 14,330 adult deaths in the United States in In the United States, over 67,000 new cases are diagnosed per year, and over 350,000 cases diagnosed worldwide 1. Men have a higher risk of bladder cancer than women transitional cell carcinoma (3:1). Urothelium cancers account for 5.6% of male and 1.8% of female cancers in India, with actual crude rate incidence of about 1 in 174 and 1 in 561 women, respectively 2. According to an epidemiological survey by Dhar et al. 3 in a leading hospital of Kashmir, bladder cancer in Kashmir has an annual incidence of 9.66 (2.46%), ranking 9th in all types of cancers. This was a partial presentation of bladder cancer incidence, as the survey was done in a single hospital catering to the needs of a third of the population. Bladder cancer is showing an alarming increase in Kashmir, as evident by the fact that we could record and collect the samples of more than 50 patients from early March in our research institute, which provides health care facilities to only a certain portion of the Kashmiri population. Our observational study revealed that bladder cancer has attained a moderate degree of incidence since the last decade among the Kashmiri population. Bladder cancer arises primarily from transitional cells of the bladder mucosal epithelium (90% of cases) and may present as a noninvasive, papillary tumor protrud- Key words: cystectomy, H-ras, Kashmir, PCR-SSCP, transitional cell carcinoma. Acknowledgments: Our thanks to Dr Mashoob Masoodi of the Department of Statistics of Kashmir University for her statistical help. We also thank Mr Nazir and the technical staff of the operation theater of the Department of Urology who helped us in the tissue procurement. Correspondence to: Prof Mushtaq A. Siddiqi, Professor & Chairman, Department of Immunology and Molecular Medicine, Sher-i-Kashmir Institute of Medical Sciences, Soura, Srinagar, Kashmir, India Tel PABX , ext 2262; fax ; cell ; arshaajiz@gmail.com Received November 6, 2009; accepted April 8, 2010.

2 994 AA PANDITH, Z SHAH, R RASOOL ET AL ing from the mucosal surface, which is readily resectable. This stark difference in morphology and survival implicates separate oncogenic pathways for noninvasive compared to muscle-invasive cancer 4. The incidence of bladder cancer was strongly associated with occupational exposure to aromatic amines used in the dye industry, before their potent carcinogenicity to the bladder was demonstrated. With reduction of such workplace exposure, active smoking is now the strongest environmental risk for bladder cancer, contributing to more than 50% of cases 5. The four cellular Ras genes encode four highly homologous 21 kda proteins: HRAS, NRAS, KRAS4A and KRAS4B. Activating Ras mutations occur in ~30% of human cancers. H-ras mutations predominate in bladder cancer 6. In most cases including bladder cancer, the somatic missense Ras mutations found in cancer cells introduce amino acid substitutions at positions 12, 13 and 61. These changes impair the intrinsic GTPase activity and confer resistance to GAPs, thereby causing cancerassociated mutant Ras proteins to accumulate in the active, GTP bound conformation 7. The three-dimensional structure of H-ras bound to the catalytic domain of p120 GAP, known as the GAP-related domain, has been solved and provides a structural understanding of the biochemical activation of oncogenic Ras proteins 8. Many studies have examined H-ras for mutations in bladder cancer 9,10. Many studies have examined only HRAS and have reported a wide range of mutation frequencies (0-70%) that may reflect true differences in the tumors examined or technical differences between assays. Currently, there is agreement from several studies that the frequency for H-ras is in the range of 10-20%. Therefore, the aim of our study was to assess the contribution of H-ras gene mutations TO the incidence and/or development of transitional cell carcinoma of urinary bladder in patients from the Kashmir valley, as no such study has been reported from this region. bladder cancers. Venous blood was collected from each patient in EDTA vials to rule out germ line mutation. DNA extraction Tumor samples (both tumor and adjacent normal tissue) collected after transurethral resection of bladder tumors were immediately snap-frozen and stored at -70 C. DNA from tissues as well as blood was extracted using a DNA easy tissue kit (Qiagen GmbH, Hilden, Germany) according to the enclosed protocol. Polymerase chain reaction (PCR) Exons 1 and 2 of the H-ras containing hotspot codons 12, 13, and 61 were amplified using the previously described specific primers 11 (exon 1F, 5 -CAGGAGACCCT- GTAGGAGGA-3 ; R 5 -TCG TCCACAAAATGGTTCTG-3, exon 2 F 5 -TCCTGCAGGATTCCTACCGG-3 ; R-5 - GGTTCACCT GTACTGGTGGA-3 ). PCR amplification was carried out in a 50 µl volume containing 50 ng of genomic DNA, 1X PCR buffer containing 15 mm MgCl 2, 100 µm each of datp, dgtp, dttp and dctp, 1.5 U of Taq DNA polymerase (Biotool, Spain), and 1 µm of forward and reverse primers. After an initial denaturation at 95 C for 5 min, 35 cycles at 94 C for 1 min, a specific annealing temperature (H-ras exon 1, 60 C; H-ras exon 2, 55 C) for 1 min, and 72 C for 80 s were performed. PCR products were run on 2% agarose gel and analyzed under a UV illuminator (Figure 1 and 2). Single- M Materials and methods Patients and controls Forty-eight consecutive patients who underwent transurethral resection of bladder tumors in the Surgery Department at the Sher-i-Kashmir Institute of Medical Sciences between 2008 and 2009 were included in the study. Every patient signed a written informed consent after approval from the Institutional Ethics Committee. Median age of the patients at the time of diagnosis was 59 years (range, 38-80) with a male-to-female ratio of 6:1. Diagnostic slides were reviewed by a panel of two expert pathologists to confirm the diagnosis and ensure uniformity of classification criteria. All the samples resected by transurethral resection of bladder tumors by a urological surgeon were confirmed histologically to be Figure 1 - Amplified DNA fragments of exon 1 of H-ras (194 bp amplicon) gene.

ACTIVATED H-ras GENE MUTATIONS IN BLADDER CANCER 995 M 1 2 3 4 5 6 Figure 2 - Amplified DNA fragments of exon 2 of H-ras (162 bp amplicon) gene.

3 ACTIVATED H-ras GENE MUTATIONS IN BLADDER CANCER 995 M Figure 2 - Amplified DNA fragments of exon 2 of H-ras (162 bp amplicon) gene. strand conformation polymorphism (SSCP) analysis of the amplicons of exon 1 and 2 of H-ras was performed on 6% nondenaturing polyacrylamide gel (PAGE) utilizing a non-radioactive silver staining procedure 12. The purified PCR amplicons of the tumor samples showing mobility shift on SSCP analysis were confirmed by direct sequencing (Figures 3 and 4). In order to rule out germ line mutation, DNA from the blood samples of the patients showing mutations were subjected to direct DNA sequencing, using the automated DNA sequencer ABI prism 310. For statistical analysis of the data, the chi-square test was used to evaluate the association between clinicopathological variables. P values <0.05 were considered significant. Results The mean age of the patients was 59 years (range, 38-80); but 75% of them were older than 60 years. Fortythree patients were men (89.6 %) and 35 were smokers. Of 35 smokers, 4 were former smokers (had quit smoking 5-10 years before). Almost all the patients had attended the hospital with a clinical presentation of hematuria, a hallmark of bladder cancer. Information on clinicopathological grade was available for 46 and staging for 48 patient samples. There were five grade 1 tumors, 14 Figure 3 - Partial nucleotide sequences (forward) of the normal (above) and mutants in exon 2 of the H- ras oncogene (See Table 1). grade 2, 16 grade 3, 10 grade 4 and one was intermediate grade (Table 1). In total, 17 were pta, 13 pt1 and 17 pt2, whereas one sample was carcinoma in situ. We screened 48 confirmed transitional cell carcinoma samples for mutations in H-ras by SSCP analysis to screen for mutations in exons 1 and 2 followed by direct sequencing of the samples showing a mobility shift in SSCP. The overall mutations in exon 1 and 2 of H-ras identified in the study aggregated to 14.6%. In all, there were 7 mutations (3 transitions and 4 transversions), 3 were A G transitions, 2 G T transversions, and 2 A T transversion, out of which 2 mutations affected codons 12 (28.6%) and 5 mutations affected codon 61 (71.4%) (Table 1). No mutation was detected in codon 13 in this study. Among the samples harboring mutations, there were three Q61R (CAG >CGG), two G12C (GGC >TGC), and two Q61L (CAG >CTG). Among all 7 mutations detected, most of the H-ras mutations were similar basepair transitions, occurring predominantly at the second

4 996 AA PANDITH, Z SHAH, R RASOOL ET AL Discussion Figure 4 - Partial nucleotide sequences (forward) of the normal (above) and mutant in exon 1 of the H- ras oncogene (See Table 1). Table 1 - Details and nature of H-ras mutations in bladder cancer patients from Kashmir valley Case Age/ Grade/ Smoking Exon Codon Base Aminoacid Sex Stage status change change AT1 70/M G3/pT2 SK 1 12 GGC>TCC Gly>Cys AT18 55/M G3/pT2 SK 1 12 GGC>TGC Gly>Cys AT19 40/M G2/pTa SK 2 61 CAG>CGG Gln>Arg AT26 50/M G4/pT1 SK 2 61 CAG>CGG Gln>Arg AT16 70/M G2/pTa SK 2 61 CAG>CGG Gln>Arg AT32 75/M G3/pT1 SK 2 61 CAG>CTG Gln>Leu AT35 60/M G4/pT2 FSK 2 61 CAG>CTG Gln>Leu Smoking status: Sk, smoker; FSK, former smoker. bases of codons 61 (A G) (Table 2). The mutations identified were mostly seen in current smokers (6/31) and one in a former smoker (1/4). No mutation was detected in bladder tumor of non-smokers. There was no significant association between tumor grade or stage and the mutations detected. Seven tumor samples with mutation detected in the study included, 3 grade 2 and 4 grade 3 tumors. However, on pathological staging, 2 were classified as pta, 2 as pt1, and 3 samples as pt2 tumors. We found no association with age, tumor location, or tumor size. The matched constitutional DNA (blood cell DNA) contained the wild-type sequence for H-ras in every case, demonstrating the somatic nature of these mutations in (bladder) cancer. The H-ras mutation was first detected in the human bladder cancer cell line T Subsequent studies demonstrated that H-ras mutations were more frequently observed in urinary tract tumors than the K-ras or N-ras genes 14. This initial expectation has been realized, since later analysis of bladder tumor tissues showed that only about 10% of the samples contained a mutated H-ras 15. However, later reports showed higher frequencies of mutations in the H-ras gene. Whereas Fitzgerald et al. 16 reported mutations in the H-ras gene in 44% of urine sediments from bladder cancer patients, Czerniak et al. 17 observed H-ras mutation specifically in connection to codon 12 in 45% of bladder cancers. In a recent study by Jebar et al. 18 on 98 bladder tumors and 31 bladder cell lines, RAS mutation was also detected in 13% of both types of samples. In total, there were 10 mutations in H-ras (4 in codon 61 and 2 each in codon 12 and 13), 4 in K-ras, and 4 in N-ras. Our study detected H-ras mutations up to the frequency of 14.6%, which is consistent with most of earlier studies 15,18. Of these, 10.4% were in codon 61, 4.16% in codon 12, and no mutation was found in codon 13. All the mutations identified in the present study were of missense nature. The rate of transversion (57.2%) was found to be higher than transition (42.8%). Based on previous studies, it was expected that codon 12 mutations would predominate, but this was not the case in our study. Five mutations in codon 61 and only 2 mutations in codon 12 were identified. Most of the mutations found in H-ras in human tumors involve these two codons, coding for glycine and glutamine residues, which play an important role in the catalytic site of RAS. These substitutions alter its GTPase activity to a different extent and/or its ability to interact with its regulators, depending on the substituted amino acid residue. It was also predicted that the most frequent substitution in codon 12 would be G A at the second position (Val), but the mutation was not detected in any tumor. The mutations detected in the present study were diverse, comprising of 4 transversions (three G:C T:A and one A:T T:A) and 3 transitions (A: T G: C). A similar spectrum of substitutions is characteristic of p53 mutations in bladder tumors 19. This may reflect the known association of increased bladder cancer risk with cigarette smoking 20. A G T transversion is not only the most frequently observed type of mutation but has also been linked to smoking 21. Habuchi et al. 22 found A:T G:C transitions only in smokers 22. A similar pattern of mutation was identified in the present study, consistent with above data, with almost all mutations in conformation with three G T and three A G correlating with smoking status. Interestingly, in our study, all these mutations were traced in male subjects who were ever smokers except for one former smoker, and the mutation pattern showed a significant association

5 ACTIVATED H-ras GENE MUTATIONS IN BLADDER CANCER 997 Table 2 - Relationship of specific H-ras mutations to the clinicopathological variables in 48 bladder cancer patients Variables Cases Wild type Mutation in Codon 12 Mutation in Codon 61 P Total Mean age >0.05 (yr) Sex Male (85.7) 2 (4.76) 4 (9.5) Female 6 5 (83.3) 0 1 (16.6) >0.05 Tumor size (cm) < (86) 1 (3.4) 3 (10.6) > (84.2) 1 (5.2) 2 (10.5) >0.05 Smoking Current (87) 2 (6.4) 2 (6.4) Former 4 1 (25) 2 (50) 1 (25) <0.05 Never (100) 0 0 Stage Ta (88.2) 1 (5.8) 1 (5.8) T (54.6) 0 2 (15.4) >0.05 T2 or higher (82.3) 1 (5.8) 2 (11.76) Grade (100) (85.7) 1 (7.14) 1 (7.14) > (18.75) 1 (6.25) 2 (12.5) (80) 0 2 (20) In parenthesis, percentage. H-ras with smoking (P <0.05). As Kashmir is not an industrialized region, exposure to chemicals is rare, particularly aromatic (aryl)-amines such as naphthalene, benzidine, aniline dyes, and 4-aminobiphenyl, which are known bladder carcinogens 23. With moderate reduction of such workplace exposure, active smoking seems the strongest environmental risk contributing to all bladder cancer cases in the Kashmiri population in the study. In Kashmir, besides cigarette smoking, hookah smoking (smoking through a water filter) is also a popular form. However, smoking is mostly practiced by males, and women are seldom involved in this practice. Furthermore, the population under study is a conservative society where females generally hide their sickness, especially related to the genito-urinary system. In our opinion, these two factors seem to be contributing to a high male:female ratio (6:1) of bladder cancer in our population. Ras mutations do not seem to be associated with stage or grade 24. Ras genes are mutated in some invasive urothelial cell carcinomas, and there is no clear association with invasive rather than superficial disease. In vitro experiments on human tumor cells indicate that HRAS can up-regulate EGFR expression and induce an invasive phenotype 25. However, in transgenic mice engineered to express mutant HRAS leads to the development of superficial papillary tumors 26. In our study, we could not detect any significant correlation of H-ras mutation with different grades and stages of bladder cancer, neither between superficial and invasive cancer, which is in clear agreement with published data. Low-grade cases were reported as harboring 28.5% mutations and high-grade cases around 42.8% in grade III and 28.5% in grade IV, whereas 43% mutations were found in high-stage (pt2) and 28% each in low-stage pta and pt1 disease. This even distribution of mutations implies that the H-ras mutation is involved through the early event to late-stage disease progression, but evaluation on a large cohort of samples is necessary this. Furthermore, no association was found with age, tumor location, or tumor size. Conclusion We conclude that H-ras mutation in bladder tumorigenesis plays a less frequent but significant role compared to other genetic events. Taken together, the results in all grades and stages of transitional cell carcinoma clearly suggest that the oncogene contributes to bladder tumorigenesis but represents neither an early nor a late event. Although smoking showed a significant correlation, the group under investigation was too small to be considered for epidemiological conclusions. Moreover, the wild type samples reflect the involvement of different etiological factors, but this needs to be evaluated in further studies in bladder cancer patients of the ethnic Kashmiri population.

6 998 AA PANDITH, Z SHAH, R RASOOL ET AL References 1. Jemal A, Siegel R, Ward E: Cancer statistics CA Cancer J Clin, 57: 43-66, Kamarana NM, Kamat MR, Kurkure AP: National Cancer registry project, ICMR; 2000 published in Dhar GM, Shah GN, Naheed B, Hafiza: Epidemiological trend in the distribution of cancer in Kashmir Valley. J Epidemiol Commun Health, 47: , Dinney CP, McConkey DJ, Millikan RE: Focus on bladder cancer. Cancer Cell, 6: , Clavel J: Progress in the epidemiological understanding of gene environment interactions in major diseases: cancer. C R Biol, 330: , Bos JL: ras oncogenes in human cancer: a review. Cancer Res, 49: , Trahey M, McCormick F: A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science, 238: , Scheffzek K, Ahmadian MR, Kabsch W, Wiesmuller L, Lautwein A, Schmitz F, Wittinghofer A: The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science, 277: , Burchill SA, Neal DE, Lunec J: Frequency of H-ras mutations in human bladder cancer detected by direct sequencing. Br J Urol, 73: , Czerniak B, Deitch D, Simmons H, Etkind P, Herz F, Koss LG: Ha-Ras gene mutation and DNA ploidy in urinary bladder carcinoma. Br J Cancer, 62: , Adel H, Jebar AH, Carolyn DH, Darren C, Tomlinson I, Colin J, Claire F, Knowles MA: FGFR3 and Ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma. Oncogene, 24: , Bosari S, Marchetti A, Buttitta F, Graziani D, Borsani G, Loda M: Detection of p53 mutations by single strand conformation polymorphism (SSCP) gel electrophoresis: a comparative study of radioactive and non-radioactive silver-stained SSCP analysis. Diagn Mol Pathol, 4: , Capon DJ, Chen EY, Levinson AD, Seeburg PH, Goeddel DV: Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue. Nature, 302: 33-37, Rabbani F, Cordon-Cardo C: Mutation of cell cycle regulators and their impact on superficial bladder cancer. Urol Clin North Am, 27: , Saito S, Hata M, Fukuyama R: Screening of H-ras gene point mutations in 50 cases of bladder carcinoma. Int J Urol, 4: , Fitzgerald JM, Ramchurren N, Rieger K: Identification of H- ras mutations in urine sediments complements cytology in the detection of bladder tumors. J Natl Cancer Inst, 87: , Czerniak B, Cohen GL, Etkind P: Concurrent mutations of coding and regulatory sequences of the Ha-ras gene in urinary bladder carcinomas. Hum Pathol, 23: , Jebar AH, Hurst CD, Tomlinson DC, Johnston C, Taylor CF, Knowles MA: FGFR3 and Ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma. Oncogene, 24: , Sidransky D, Von Eschenbach, A, Tsai YC, Jones P, Summerhayes I, Marshall F, Paul M, GreenP, Hamilton SR, Frost P, Vogelstein B: Identification of p53 gene mutations in bladder cancers and urine samples. Science, 252: , Hartge P, Silverman D, Hoover R, Schairer C, Altman R, Austin D, Cantor K, Child M, Key C, Marren LD, Mason TJ, Meigs JW, Myers MH, Narayana A, Sullivan JW, Swanson GM, Thomas D, West D: Changing cigarette habits and bladder cancer risk; a case-control study. J Nati Cancer Inst, 78: , Slebos RJ, Hruban RH, Dalesio O, Mooi WJ, Offerhaus GJ, Rodenhuis S: K-ras oncogene activation and smoking in adenocarcinoma of the human lung. J Natl Cancer Inst, 83: , Habuchi T, Takahashi R, Yamada H: Influence of cigarette smoking and schistosomiasis on p53 gene mutation in urothelial cancer. Cancer Res, 53: , Clavel J: Progress in the epidemiological understanding of gene environment interactions in major diseases: cancer. C R Biol, 330: , Jebar AH, Hurst CD, Tomlinson DC: FGFR3 and ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma. Oncogene, 24: , Theodorescu D, Cornil I, Sheehan C, Man MS, Kerbel RS: Ha-ras induction of the invasive phenotype results in upregulation of epidermal growth factor receptors and altered responsiveness to epidermal growth factor in human papillary transitional cell carcinoma cells. Cancer Res, 51: , Zhang ZT, Pak J, Huang HY, Shapiro E, Sun TT, Pellicer A, Wu XR: Role of Ha-ras activation in superficial papillary pathway of urothelial tumor formation. Oncogene, 20: , 1991.

Urologic Oncology: Seminars and Original Investigations 31 (2013)

Urologic Oncology: Seminars and Original Investigations 31 (2013) 487 492 Original article HRAS T81C polymorphism modulates risk of urinary bladder cancer and predicts advanced tumors in ethnic Kashmiri