Allelotype of Human Bladder Cancer 1

To identify common regions of deletion in human bladder tumors, we have screened 83 cases of transitional cell carcinoma for loss of heterozygosity (LOH) on all autosomal chromosome arms. Seventy-two restriction fragment length polymorphism, variable number of tandem repeats, and minisatellite markers and 18 microsatellite markers were used to obtain a minimum of 50% informative results for each chromosome arm. A mean of 29.6 informative results per patient was obtained from 39 chromosome arms studied, representing information for 76% of chromosome arms. The most frequent losses were apparent monosomies of chromosome 9 (9p, 51%; 9q, 57%), Other frequent losses were on chromosomes l lp (32%), 17p (32%), 8p (23%), 4p (22%), and 13q (15%). LOH of 4p has not been reported previously in bladder carcinoma. The frequency of LOH on all other chromosome arms was <12%. LOH on chromosome 8p showed a significant association with both high tumor grade and stage, and LOH on 13q showed a significant association with high tumor grade. Fractional allelic loss was calculated for all tumors and had a mean of 0.125 and a median of 0.110. A significant association was found between increased fractional ailelic loss and higher tumor grade. An association was also found between LOH of chromosomes 8p and 9q and values for fractional allelic loss the median value. No associations were found between LOH on different pairs of chromosome arms. I N T R O D U C T I O N Studies of many types of tumor indicate that inactivation of multiple tumor suppressor genes is required for tumor development. Analyses of LOH 3 using restriction fragment length polymorphisms have identifed nonrandom LOH at specific sites, indicating the likely locations of tumor suppressor genes. LOH at some loci (e.g., p53 on chromosome 17p) is present in a large proportion of many tumor types, while other sites of loss appear specific to one or a few tumor types. Several allelotype studies have been reported (1-10) in which the frequency of LOH on all autosomal arms has been measured. Such aUelotypes represent the most rapid method of surveying the genome for likely suppressor loci, and in the cases reported to date, highly individual allelotypes have been described for tumors derived from different tissues. In addition to an overall frequency of allele loss on different chromosome arms, allelotypes provide information on deletions at multiple sites in individual tumors. It is likely that such individual genetic profiles will lead to the identification of common combinations of lesions in specific tumors and hence to an improved basis for the subclassification of tumors derived from a single tissue. This may have significant diagnostic and/or prognostic significance. Previous studies of bladder tumors have found LOH on chromosomes 11, 9, 17, and 13 (11-16). Cytogenetic studies of bladder tumors have identified monosomy 9, monosomy 8, isochromosomes of 5p, 11p deletions, and trisomy 7 as the most frequent cytogenetic alterations (17-25). All of these changes apart from trisomy 7 may Received 7/26/93; accepted 11/15/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The Institute of Cancer Research is funded by the Medical Research Council and the Cancer Research Campaign. z To whom requests for reprints should be addressed. 3 The abbreviations used are: LOH, loss of heterozygosity; RFLP, restriction fragment length polymorphism; FAL, fractional allelic loss. identify sites of putative suppressor genes. In order to identify the common regions of deletion in bladder cancer and to analyze the complexity and combinations of deletions present, we have studied all nonacrocentric chromosome arms in a large group of bladder tumors. M A T E R I A L S A N D M E T H O D S Tissues. Specimens of bladder tumors were obtained from patients at cystoscopy and were cut using diathermy or cold cup biopsy forceps. A representative sample was taken for histopathological assessment and the remainder of the tissue was frozen at -20~ Tumors were graded according to WHO recommendations (26) and staged according to the tumor-nodes-metastasis classification (27) (Table 1). A venous blood sample was collected from each patient as a source of normal leukocytes and frozen in EDTA tubes. DNA Extraction and Southern Blotting. High molecular weight DNA was prepared from blood leukocytes and tissue samples as described previously (28). Eight to 10/xg of DNA were digested with the appropriate restriction enzyme according to the manufacturers' instructions and electrophoresed in 0.8-1.2% agarose gels for 24-48 h. DNA was transferred to Hybond N or N+ nylon membranes (Amersham, Aylesbury, United Kingdom) and fixed by UV irradiation or in 0.4 N NaOH, respectively. Probes and Hybridization. The markers used are listed in Table 2. RFLP, variable number of tandem repeats, and minisatellite probes are as described in Human Gene Mapping 11 (29). Probes were labeled by random priming (30). Prehybridization and hybridization were carried out as described previously (28). Membranes were stripped in 0.5% sodium dodecyl sulfate at 100~ and were hybridized repeatedly. Analysis of LOH Using Microsatellite Polymorphisms. Microsatellite markers were used to identify LOH on several chromosome arms where available probes had low information content. The microsatellite markers at APOB (GZ-9, 10), LPL (GZ14, 15), CD4, and D20S59 are described in Human Gene Mapping 11 (29). Other microsatellite markers used were CD8A (31), D4S174, D4S194 (32), D6S89 (33), ANK1, D8S85, D8S88 (34), DllS554, DllS569 (35), D14S43 (36), p53 (37), CSTP1 (38), and D21Sll (39). Polymerase chain reaction was carried out in 25-/xl reactions using buffer supplied by NBL (Cramlington, Northumberland, United Kingdom); 200 /xra concentrations each of dATP, dGTP, dCTP, and dTrP; 4 pmol of each primer (one end-labeled with 32p); 10 ng template DNA; and 1 unit Taq DNA polymerase (NBL). Primers were end-labeled using T4 polynucleotide kinase and [~/_32p]_ dCTP (40). Polymerase chain reaction reactions were carried out in a Techne PHC-2 and consisted of 27 cycles each of 95~ for 1 rain, 55~ for i rain, and 72~ for 1.5 rain followed by a final elongation for 10 rain at 72~ Fifteen/xl of loading buffer (90% formamide, 10 m~ EDTA, 0.3% bromophenol blue, and 0.3% xylene cyanol) were added to each reaction, and following incubation at 80~ for 3 min, 3-4-~1 aliquots were electrophoresed through 6% denaturing polyacrylamide gels. Gels were dried and exposed to Fuji RX film for 15 rain-18 h. Allele sizes were determined by comparison with M13 sequencing ladders.