Genetic mapping and DNA sequence-based analysis of deleted regions on chromosome 16 involved in progression of bladder cancer from occult preneoplastic conditions to invasive disease

Abstract

Histologic and genetic mapping with 30 hypervariable markers mapped to chromosome 16 were performed on 234 DNA samples of ®ve cystectomy specimens from patients with invasive bladder cancer. Allelic losses of individual markers were related to microscopically identi®ed precursor conditions in the entire bladder mucosa and invasive cancer. Their signi®cance for the development and progression of neoplasia from in situ preneoplastic conditions to invasive disease was analysed by the nearest neighbor algorithm and binomial maximum likelihood analysis. Using this approach we identi®ed ®ve distinct regions of allelic losses de®ned by their¯anking markers and predicted size as follows. p13.3(D16S418-D16S406, 1.2 cM), p13.1(D16S748-D16S287, 12.9 cM), q12 1(D16S409-D16S514, 24.0 cM), q22.1 (D16S496-D16S515, 5.4 cM), and q24 (D16S507-D16S511, 5.9 cM and D16S402-D16S413, 17.4 cM). The regions mapping to p13.1 and q24 were involved in early intraurothelial phases of bladder neoplasia such as mild to moderate dysplasia. On the other hand the deleted region mapping to p13.3 was involved in progression of severe dysplasia/carcinoma in situ to invasive bladder cancer. Testing of markers that exhibited statistically signi®cant LOH in relation to progression of neoplasia from precursor conditions to invasive cancer on 28 tumors and voided urine samples from 25 patients with bladder cancer revealed that q12.1 showed LOH in 46.4% of tumor and 32.0% of voided urine samples. The LOH of a single marker D16S541 could be detected in approximately 28% of tumors and 20% of voided urine samples of patients with bladder cancer. These data imply that the deleted region centered around marker D16S541 spanning approximately 10 cM and¯anked by D16S409 and D16S415 contains a novel putative tumor suppressor gene or genes playing an important role in the development of human bladder cancer. To facilitate more precise positional mapping and identi®cation of pathogenetically relevent genes, we analysed of human genome contig and sequence databases spanning the deleted regions. Multiple known candidate genes and several smaller gene-rich areas mapping to the target regions of chromosome 16 were identi®ed Oncogene (2001) 20, 5005 ± 5014.

Evidence for alternative candidate genes near RB1 involved in clonal expansion of in situ urothelial neoplasia

“…[6][7][8][9][10] In brief, each fresh cystectomy specimen was opened longitudinally along the anterior wall of the bladder and pinned down to a paraffin block. The entire mucosa was divided into 1 Â 2 cm rectangular samples and evaluated microscopically on frozen sections.…”

“…The precursor in situ lesions and tumor samples were microscopically classified as described previously. [5][6][7][8][9][10] In brief, the intraurothelial precancerous changes were classified as mild, moderate, or severe dysplasia or as carcinoma in situ. For the purpose of statistical analyses, the intraurothelial precancerous changes were classified into two major groups: lowgrade intraurothelial neoplasia (LGIN, mild and moderate dysplasia) and high-grade intraurothelial neoplasia (HGIN, severe dysplasia and carcinoma in situ) as described previously.…”

“…9,10 In brief, a set of primers for 40 microsatellite markers on chromosome 13 based on the sex-averaged microsatellite maps from Genethon and Marshfield using reference families from the Cooperative Human Linkage Center was obtained from Research Genetics (Huntsville, AL, USA; now Invitrogen, CA, USA). The allelic patterns of markers were resolved on 6% polyacrylamide gels after polymerase chain reaction (PCR) amplification.…”

“…18 DNA was extracted from individual bladder tumors and sediments of voided urine samples as described previously. 9 For controls, DNA was also extracted from the peripheral blood lymphocytes and/or normal nonbladder tissue in the resected specimens from each patient.…”

“…[6][7][8][9][10] These studies utilized a whole-organ histologic and genetic mapping approach with microsatellite markers and low-resolution recombination-based chromosomal maps. More recently, we have reported on whole-organ histologic and genetic mapping of tumor suppressor gene loci using single-nucleotide polymorphic sites (SNPs).…”