Mapping and Genome Sequence Analysis of Chromosome 5 Regions Involved in Bladder Cancer Progression

Abstract

SUMMARY:We studied the evolution of allelic losses on chromosome 5 by whole-organ histologic and genetic mapping in 234 mucosal DNA samples of 5 cystectomy specimens with invasive bladder cancer and preneoplastic changes in adjacent urothelium. The frequency of alterations in individual loci was verified on 32 tumors and 29 voided urine samples from patients with bladder cancer. Finally, deleted regions on chromosome 5 were integrated with the human genome contigs and sequence-based databases. Deleted regions on chromosome 5 involved in intraurothelial phases of bladder neoplasia defined by their nearest flanking markers and predicted size were identified as follows: q13.3-q22 (D5S424-D5S656; 38.8 centimorgan [cM]); q22-q31.1 (D5S656-D5S808; 19.2 cM), q31.1-q32 (D5S816-SPARC; 11.5 cM), and q34 (GABRA1-D5S415; 6.4 cM). The two most frequently deleted neighbor markers (D5S2055 and D5S818) mapping to q22-q31.1 defined a 9 cM region, which may contain genes that play an important role in early phases of urinary bladder carcinogenesis. Human genome database analysis provided an accurate map of deleted regions with positions of 138 known genes and revealed several smaller gene-rich areas representing putative targets for further mapping. The strategic approach presented here, which combines whole-organ histologic and genetic mapping with analysis of the rapidly emerging human genome sequence database, facilitates identification of genes potentially involved in early phases of bladder carcinogenesis. (Lab Invest 2001, 81:1039 -1048.C ancer develops via multiple, cumulative steps, many of which precede the development of clinically and even microscopically recognizable disease. Mapping and human genome sequence analysis of chromosomal regions involved in clinically occult preinvasive phases of neoplasia may provide valuable clues for more specific studies of early events in human carcinogenesis and could lead to the development of novel early detection markers as well as preventive strategies.We have previously reported the identification of several putative tumor suppressor gene loci involved in early preinvasive phases of human urinary bladder carcinogenesis (Chaturvedi et al, 1997;Czerniak et al, 1999Czerniak et al, , 2000. Bladder tumors were used as a common model of human cancer, which develops by progression of microscopically recognizable in situ precursor conditions, and are easily accessible by various minimally invasive or noninvasive techniques (Gazdar and Czerniak, 2001;Greenlee et al, 2000). The entire mucosal surface of the bladder can be examined by cystoscopy and biopsies with minimal risk for the patient, and exfoliated urothelial cells can be repeatedly tested for various alterations in voided urine at no risk at all (Gazdar and Czerniak, 2001). Moreover, the simple anatomy and appropriate size of the bladder permit the histologic and genetic mapping studies of invasive cancer and preneoplastic lesions in the entire mucosa of cystectomy specimens. The whole-organ histologic and genetic mapping combine...

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

“…10 In brief, the positions of all currently defined SNPs mapping within the 13q14 region from the SNP database of NCBI (http:// www.ncbi.nlm.nih.gov/SNP) were integrated with the genome sequence map containing genes and gene predictions. The integrated gene and SNP map of the region was used to select 79 SNPs mapping within the approximately 800 kb segment around RB1, which included 30 SNPs located within the RB gene.…”

“…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.…”

“…[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.…”

“…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.…”

“…[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).…”