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膀胱癌組織微小衛星體變化的臨床重要性

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(5) KeywordsMicrosatellite, Loss of heterozygosity, Urothelial carcinogeneis, Prognosis Tumorigenesis is a multistep process that includes amplification of proto-oncogenes and inactivation of tumor suppressor genes (TSGs). Loss of heterozygosity (LOH) of microsatellites is an important manifestation of allelic deletion. Since this molecular alteration was suggested to have prognostic value in several human cancers, we performed this study on 40 cases of urinary tract cancer to examine the clinical implications of allelic deletions. LOH of microsatellite markers from 12 important chromosomal loci found that chromosome 9p and 9q are among the highest incidence (80%) of LOH in at least one of microsatellite markers, followed by 14q (74.3%), 10q (68.6%), 21q (60.0%) and 8p (57.1%). Significant association was observed in chromosome 4q loss with tumor stage, 17p loss with larger tumor, and loss of 9p or 17p with nodular gross shape (p < 0.05). Besides, there was a marginal significance of larger tumors with loss of 4q, and multiple tumors with 8p or 11p, respectively (p < 0.09). Univariate analysis revealed that only tumor stage was significant in predicting the risk of recurrence (p = 0.004), and patient survival (p = 0.002). Interestingly, tumors with loss of chromosomal 6 tends to have poor clinical outcome compared with LOH in other loci (p = 0.063). Otherwise, LOH in the remaining chromosomal loci did not have prognostic value for urinary tract cancer patients. Our data suggest that chromosome 4q, 9p, and 17p may harbor TSGs involved in the progression of urothelial carcinogenesis. TGS located in the chromosome 6 may contribute to the invasion/metastasis of bladder cancer. The information obtained in this pilot study may be of great value in future gene hunting..

(6) Introduction Tumorigenesis is a multistep process that develops through the accumul ation of genetic alterations in proto-oncogenes, tumor suppressor genes (TSGs), or both [1]. Accumulated evidences suggest that loss of TSG activity is a key event in the development of human bladder cancer [2,3]. Among these, deletion of TSGs located in chromosome 9q and 9p appears to be earlier events. Besides, fractional allelic loss in 3p, 4, 5q, 6p, 8p, 11p, 12p, 13q, 14q, 16p, 17p, 18q, 21q and 22q is more often detected in tumors with invasion of the lamina propia (T1) or muscle layer (T2-T4) [4-8]. For instance, loss of heterozygosity (LOH) of 17p and mutations of p53 tumor suppressor gene, located at chromosome 17p13, are more common in high-grade and/or invasive urinary bladder. On this base, elucidation of specific gene(s) involving in the initiation or progression of bladder cancer may provide potential targets for therapy or useful prognostic tools. Despite of the fact that exact TSGs located within the above-mentioned chromosomal regions are currently not known, many studies have reported a positive correlation between LOH of specific chromosome with clinical parameters, or even patients’ outcome [9-31]. As an example in breast cancer, higher incidence of metastasis was observed in tumors with LOH in chromosome 11q23.1 [9], 17p13.1, 13q12-13, 16q22.2-23.2 [10], or 11q13 [22]. In addition, LOH in 11q23.1,13q12-13 or combined loss of 13q, 16q, and 17p had significantly poor clinical outcome compared with other patterns of genetic alterations [9,10,22,30]. In terms of bladder cancer, LOH in chromosome 3p or 5p13-12 was associated with muscular invasion, suggesting involvement of TSGs within that region in the progression of bladder carcinogenesis [26, 27]. This pilot study thus was designed to examine the relationship between LOH patterns with clinicopathological parameters of bladder cancer, and possible prognostic value..

(7) Materials and methods Tissue sample and histopathology A total of 40 cases of transitional cell carcinoma were retrieved from the tissue bank. The tumors had been frozen immediately received from the operation room. For patients with cancers of the kidney and ureter, unilateral nephroureterectomy was performed together with bladder cuff resection. As for bladder cancer, decision of treatment modality was determined according to the clinical status of the tumor. Each case was reviewed for histological grading, tumor stage at diagnosis, and other biological indicators without knowledge of LOH analysis. Both recurrence and survival status were confirmed by reviewing of medical records and telecommunication with patients or their families. The patients have been followed up from 13 to 114 months, with means at 66.5 months. Tissue preparation and DNA extraction For each case, neoplastic lesions were dissected for LOH analysis. The process of tissue preparation was described in detail previously [32]. Briefly, tissue fragmens were treated with 1% SDS/ proteinase K for overnight digestion. The digested tissues then were subjected to phenol-chloroform extraction and ethanol precipitation. Normal control DNA was obtained from non-tumor stromal tissue in the same section or non-urinary tissue from prior surgery. Microsatellite analysis Microsatellite markers suitable for PCR analysis were obtained from Research Genetics (Huntsville, AL). The marker pairs included 4q (FGA), 6 (ACTBP2), 8p (D8S288, D8S261, LPL), 9p (D9S162, D9S171, D9S157), 9q (GSN, D9S12, D9S176), 10q (D10S1744, D10S581), 11p (D11S907, D11S488), 13q (D13S133, D13S272), 14q (D14S267), 17p(TP53), 18q (D18S67), 21q (D21S1899, D21S1800). Prior to amplification, one primer from each pair was end labeled with [γ-32P]ATP (20 mCi; Amersham) and T4 kinase (New England Biolabs) [33]. PCR amplification for each.

(8) primer set was performed for 35 cycles consisting of denaturation at 95°C for 30 seconds, annealing at 50 - 60°C for one minute, and extension at 70°C for one minute. One third of the PCR products were separated on 7% urea-formamide-polyacrylamide gels and exposed from 4 to 24 hours. For informative cases, LOH was scored if the intensity of tumor allele demonstrated a greater than 30% reduction in intensity compared to the corresponding allele in normal control DNA [34]. Allelic loss was confirmed by observation of LOH in one of informative markers mapped to the same chromosomal region.. Results The pertinent data of the study cases were shown in table 1. There were 23 males and 17 females, with ages ranging from 37 – 83 years old (means at 63 years old). The tumors included 10 cases of kidney cancer, 11 cases of ureter cancer, and 19 cases bladder cancer. Representative results of LOH analysis could be retrieved from the attached file. The data were obtained from the markers of D9S12, GSN, D14S267, TP53 and D14S28. As seen in table 2, chromosome 9p and 9q are among the highest incidence (80%) of LOH in at least one of microsatellite markers, followed by 14q (74.3%), 10q (68.6%), 21q (60.0%) and 8p (57.1%) etc.. Then LOH patterns were correlated with biological parameters and patient prognosis. Significant association was observed in loss of chromosome 4q with tumor stage, 17p with larger tumor, and 9p or 17p with nodular gross shape (p < 0.05). Besides, there was a marginal significance of larger tumors with loss of 4q, multiple tumors with 8p and 11p, respectively (p < 0.09). All biological indicators and LOH data were compared with the tumor recurrence and clinical outcome (table 3). Univariate analysis revealed that only tumor stage was significant in predicting the risk of recurrence (p = 0.004), and patient survival (p = 0.002). Interestingly, tumors with loss of chromosomal 6 tends to have poor clinical.

(9) outcome compared with LOH in other loci (p = 0.063). Otherwise, LOH in the remaining chromosomal loci did not have prognostic value for urinary tract cancer patients. A multivariate analysis is underway to identify the most effective parameter in predicting clinical outcome.. Discussion The genetic basis for neoplastic progression is now widely accepted. The contemporary concept suggests that loss of genetic material on multiple chromosomal loci is required for transformation from normal epithelium to malignacy [35]. In terms of bladder cancer, molecular changes involved in the progression of papillary tumor to invasive/metastatic bladder cancer have been investigated in the last 10 years [36]. The higher incidence of LOH in chromosomes 9p and 9q in our series is essentially consistent with previous works, suggesting that TSGs located within the two loci are among the earliest events in the development of bladder cancer. The association of 9p loss with nodular tumor shape implies that TSG in this region appears to occur somewhat later in tumor progression. Likewise, TSGs in the 4q, 17p, and 18q may further contribute to progression of urothelial carcinogenesis. If these observations could be verified in a large cohort, a thorough investigation for gene cloning in these loci is warranted. It is interesting to note that loss of chromosome 6 appears to have value in predicting patient survival, an observation not reported before. The result indicates that TSG located in this region may involve in the invasive/metastatic progression of bladder cancer. Further verification is underway to expand our study series and analyzed by multivariate analysis. Future work will also include examination of combined LOH patterns in correlation with biological indicators and clinical outcome..

(10) References 1.. Bishop JM: Molecular themes in oncogenesis. Cell 64: 235-248, 1991.. 2.. Knowles MA, Elder PA, Williamson M, Cairns JP, Shaw ME, Law MG: Allelotype of human bladder cancer. Cancer Res 54: 531-538, 1994.. 3.. Cairns P, Tokino K, Eby Y, Sidransky D: Homozygous deletions of 9p21 in primary human bladder tumors detected by comparative multiplex polymerase chain reaction. Cancer Res 54: 1422-1424, 1994.. 4.. Olumi AF, Tsai YC, Nichols PW, Skinner DG, Cain DR, Bender LI, Jones PA: Allelic loss of chromosome 17p distinguishes high grade from low grade transitional cell carcinomas of the bladder. Cancer Res 50:7081-7083, 1990.. 5.. Uchida T, Wada C, Ishida H, Wang C, Egawa S, Yokoyama E, Kameya T, Koshiba K: p53 mutations and prognosis in bladder tumors. J Urol 153: 1097-1104, 1995.. 6.. Saran KK, Gould D, Godec CJ, Verma RS: Genetics of bladder cancer. J Mol Med 74: 441-445, 1996.. 7.. Chaturvedi V, Li L, Hodges S, Johnston D, Ro JY, Logothetis C, von Eschenbach AC, Batsakis JG, Czerniak B: Superimposed histologic and genetic mapping of chromosome 17 alterations in human urinary bladder neoplasia. Oncogene 14: 205970, 1997.. 8.. Brewster SF, Gingell JC, Browne S, Brown KW. Loss of heterozygosity on chromosome 18q is associated with muscle-invasive transitional cell carcinoma of the bladder. British Journal of Cancer. 70(4):697-700, 1994.. 9.. Laake K, Launonen V, Niederacher D, Gudlaugsdottir S, Seitz S, Rio P, Champeme MH, Bieche I, Birnbaum D, White G, Sztan M, Sever N, Plummer S, Osorio A, Broeks A, Huusko P, Spurr N, Borg A, Cleton-Jansen AM, van't Veer L, Benitez J, Casey G, Peterlin B, Olah E, Borresen-Dale AL, et al. Loss of heterozygosity at 11q23.1 and survival in breast cancer: results of a large European study. Breast Cancer Somatic Genetics Consortium. Genes Chrom Cancer. 25:212-21, 1999..

(11) 10. Hampl M, Hampl JA, Reiss G, Schackert G, Saeger HD, Schackert HK: Loss of heterozygosity accumulation in primary breast carcinomas and additionally in corresponding distant metastases is associated with poor outcome. Clin Cancer Res 5: 1417-1425, 1999. 11. Jernvall P. Makinen MJ. Karttunen TJ. Makela J. Vihko P. Loss of heterozygosity at 18q21 is indicative of recurrence and therefore poor prognosis in a subset of colorectal cancers. Br J Cancer. 79: 903-8, 1999 Feb. 12. Jen J, Kim H, Piantadosi S, Liu ZF, Levitt RC, Sistonen P, Kinzler KW, Vogelstein B, Hamilton SR: Allelic loss of chromosome 18q and prognosis in colorectal cancer. N Engl J Med 331: 213-21, 1994. 13. Pearlstein RP, Benninger MS, Carey TE, Zarbo RJ, Torres FX, Rybicki BA, Dyke DL: Loss of 18q predicts poor survival of patients with squamous cell carcinoma of the head and neck. Genes Chrom Cancer. 21: 333-9, 1998. 14. Jenkins R, Takahashi S, DeLacey K, Bergstralh E, Lieber M: Prognostic significance of allelic imbalance of chromosome arms 7q, 8p, 16q, and 18q in stage T3N0M0 prostate cancer. Genes Chrom Cancer 21: 131-43, 1998. 15. Gabra H, Watson JE, Taylor KJ, Mackay J, Leonard RC, Steel CM, Porteous DJ, Smyth JF: Definition and refinement of a region of loss of heterozygosity at 11q23.3q24.3 in epithelial ovarian cancer associated with poor prognosis. Cancer Res 56: 950-4, 1996. 16. Kihana T, Yano N, Murao S, Iketani H, Hamada K, Yano J, Murao S, Iketani H, Hamada K, Yano J, Matsuura S: Allelic loss of chromosome 16q in endometrial cancer: correlation with poor prognosis of patients and less differentiated histology. Jap J Cancer Res 87: 1184-90, 1996. 17. Beroud C, Fournet JC, Jeanpierre C, Droz D, Bouvier R, Froger D, Chretien Y, Marechal JM, Weissenbach J, Junien C: Correlations of allelic imbalance of chromosome 14 with adverse prognostic parameters in 148 renal cell carcinomas..

(12) Genes Chrom Cancer. 17: 215-24, 1996. 18. Gleich LL, Li YQ, Biddinger PW, Gartside PS, Stambrook PJ, Pavelic ZP, Gluckman JL: The loss of heterozygosity in retinoblastoma and p53 suppressor genes as a prognostic indicator for head and neck cancer Laryngoscope 106: 1378-81, 1996. 19. Maruno M, Yoshimine T, Muhammad AK, Tokiyoshi K, Hayakawa T: Loss of heterozygosity of microsatellite loci on chromosome 9p in astrocytic tumors and its rognostic implications. J Neuro-Oncol 30: 19-24, 1996. 20. Scholnick SB, Haughey BH, Sunwoo JB, el-Mofty SK, Baty JD, Piccirillo JF, Zequeira MR: Chromosome 8 allelic loss and the outcome of patients with squamous cell carcinoma of the supraglottic larynx. J Natl Cancer Inst 88: 1676-82, 1996. 21. Eiriksdottir G, Sigurdsson A, Jonasson JG, Agnarsson BA, Sigurdsson H, Gudmundsson J, Bergthorsson JT, Barkardottir RB, Egilsson V, Ingvarsson S: Loss of heterozygosity on chromosome 9 in human breast cancer: association with clinical variables and genetic changes at other chromosome regions. Int J Cancer. 64: 378-82, 1995. 22. Gudmundsson J, Barkardottir RB, Eiriksdottir G, Baldursson T, Arason A, Egilsson V, Ingvarsson S: Loss of heterozygosity at chromosome 11 in breast cancer: association of prognostic factors with genetic alterations. Br J Cancer. 72: 696-701, 1995. 23. Kelemen PR, Yaremko ML, Kim AH, Montag A, Michelassi F, Westbrook CA: Loss of heterozygosity in 8p is associated with microinvasion in colorectal carcinoma. Genes Chrom Cancer. 11: 195-8, 1994. 24. Sanchez-Cespedes M, Rosell R, Pifarre A, Lopez-Cabrerizo MP, Barnadas A, Sanchez JJ, Lorenzo JC, Abad A, Monzo M, Navas-Palacios JJ: Microsatellite alterations at 5q21, 11p13, and 11p15.5 do not predict survival in non-small cell lung cancer. Clin Cancer Res 3: 1229-35, 1997. 25. Yaremko ML, Kutza C, Lyzak J, Mick R, Recant WM, Westbrook CA: Loss of.

(13) heterozygosity from the short arm of chromosome 8 is associated with invasive behavior in breast cancer. Genes Chrom Cancer. 16: 189-95, 1996. 26. Li M, Zhang ZF, Reuter VE, Cordon-Cardo C: Chromosome 3 allelic losses and microsatellite alterations in transitional cell carcinoma of the urinary bladder. Am J Pathol 149: 229-35, 1996. 27. Bohm M, Kirch H, Otto T, Rubben H, Wieland I: Deletion analysis at the DEL-27, APC and MTS1 loci in bladder cancer: LOH at the DEL-27 locus on 5p13-12 is a prognostic marker of tumor progression. Int J Cancer. 74: 291-5, 1997. 28. Lee DJ, Koch WM, Yoo G, Lango M, Reed A, Califano J, Brennan JA, Westra WH, Zahurak M, Sidransky D: Impact of chromosome 14q loss on survival in primary head and neck squamous cell carcinoma. Clin Cancer Res 3: 501-5, 1997. 29. Chung DC, Smith AP, Louis DN, Graeme-Cook F, Warshaw AL, Arnold A: A novel pancreatic endocrine tumor suppressor gene locus on chromosome 3p with clinical prognostic implications. J Clin Invest 100: 404-10, 1997. 30. van den Berg J, Johannsson O, Hakansson S, Olsson H, Borg A: Allelic loss at chromosome 13q12-q13 is associated with poor prognosis in familial and sporadic breast cancer. Br J Cancer. 74: 1615-9, 1996. 31. Inoue T, Uchino S, Shiraishi N, Adachi Y, Kitano S: Loss of heterozygosity on chromosome 18q in cohesive-type gastric cancer is associated with tumor progression and poor prognosis. Clin Cancer Res 4: 973-7, 1998. 32. Califano J, van der Riet P, Westra W, Nawroz H, Clayman G, Piantadosi S, Corio R, Lee D, Greenberg B, Koch W, Sidransky, D: Genetic progression model for head and neck cancer: Implications for field cancerization. Cancer Res 56: 2488-2492, 1996. 33. Mao L, Schoenberg MP, Scicchitano M, Erozan YS, Merlo A, Schwab D, Sidransky D: Molecular Detection of Primary Bladder Cancer by Microsatellite Analysis. Science 271: 659-662, 1996. 34. Steiner G, Schoenberg MP, Linn JF, Mao L, Sidransky D: Detection of bladder cancer.

(14) recurrence by microsatellite analysis of urine. Nature Med 3: 621-624, 1997. 35. Rosin MP, Cairns P, Epstein JI, Schoenberg MP, and Sidransky D: Partial allelotype of carcinoma in situ of the human bladder. Cancer Res 55: 5213-6, 1995. 36. Fearon ER, Volgestein B: A genetic model for colorectal tumorigenesis. Cell 61: 759767, 1990..

(15) Table 1. Clinicopathological characteristics of study cases Factors Data Sex (M/F) 23/17 Age range (mean) 37 – 83 (63) years of age Location (kidney/ureter/bladder) 10/11/19 Grade (1/2/3) 1/20/19 Stage (O/A/B/C/D) 7/8/9/9/7 Size 1.5 – 12 cm Shape (papillary/nodular) 24/16 Number (single/multiple) 27/13 Follow-up range (mean) 13 – 114 (66.5) months.

(16) Table 2. Association of microsatellite allelotyping with biological indicators of bladder cancer Chromosome LOH Grade Stage Size Shape Number Death 4q 31.4% * # 6 25.7% # 8p 57.1% # 9p 80.0% * 9q 80.0% 10q 68.6% 11p 17.1% # 13q 51.4% 14q 74.3% 17p 45.7% * 18q 37.1% * 21q 60.0% * p < 0.05; # p < 0.09.

(17) Table 3. Prognostic significance of microsatellite allelotyping and clinicopathological indicators of bladder cancer (logistic regression model) Univariate Multivariate Factors __________________________ __________________________ Recurrence Survival Recurrence Survival Age 0.255 0.345 Sex. 0.591. 0.963. ?. Grade. 0.628. 0.225. ?. Stage. 0.004*. 0.002*. ?. Size. 0.363. 0.704. ?. Shape. 0.617. 0.427. ?. Multiplicity. 0.138. 0.593. ?. LOH (Ch.6) * p < 0.05. 0.833. 0.063. ?.

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