The search for the genomic sequences involved in human cancers can be greatly facilitated by maps of genomic imbalances identifying the involved chromosomal regions, particularly those that participate in the development of occult preneoplastic conditions that progress to clinically aggressive invasive cancer. The integration of such regions with human genome sequence variation may provide valuable clues about their overall structure and gene content. By extension, such knowledge may help us understand the underlying genetic components involved in the initiation and progression of these cancers. We describe the development of a genome-wide map of human bladder cancer that tracks its progression from in situ precursor conditions to invasive disease. Testing for allelic losses using a genome-wide panel of 787 microsatellite markers was performed on multiple DNA samples, extracted from the entire mucosal surface of the bladder and corresponding to normal urothelium, in situ preneoplastic lesions, and invasive carcinoma. Using this approach, we matched the clonal allelic losses in distinct chromosomal regions to specific NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript phases of bladder neoplasia and produced a detailed genetic map of bladder cancer development. These analyses revealed three major waves of genetic changes associated with growth advantages of successive clones and reflecting a stepwise conversion of normal urothelial cells into cancer cells. The genetic changes map to six regions at 3q22-q24, 5q22-q31, 9q21-q22, 10q26, 13q14, and 17p13, which may represent critical hits driving the development of bladder cancer. Finally, we performed high-resolution mapping using single nucleotide polymorphism markers within one region on chromosome 13q14, containing the model tumor suppressor gene RB1, and defined a minimal deleted region associated with clonal expansion of in situ neoplasia. These analyses provided new insights on the involvement of several non-coding sequences mapping to the region and identified novel target genes, termed forerunner (FR) genes, involved in early phases of cancer development. Keywordsforerunner genes; whole-organ histologic and genetic mapping; high-resolution mapping with SNPs; dual-track pathway of bladder cancer development; apoptosisWe have developed a strategy to identify genetic hits across the entire mucosa of an affected organ relative to cancer progression, from in situ precursor conditions to invasive disease, on a total genomic scale. We used the approach, which we refer to as whole-organ histologic and genetic mapping (WOHGM), to identify clonal hits associated with growth advantage, thus tracking the development of human bladder cancer from occult in situ lesions. Human bladder carcinoma was used as a model of a common epithelial malignancy that develops by progression of microscopically recognizable intraurothelial preneoplastic conditions known as dysplasia and carcinoma in situ. 1 Carcinoma of the bladder is the fifth most frequent...

The predominant mode of either spontaneous or drug-induced death of cells in tumors is apoptosis. A flow cytometric method was developed in our laboratory to identify apoptotic cells, based on labeling DNA strand breaks, which appear as a result of extensive DNA cleavage by the apoptosis-associated endonuclease, with biotinylated dUTP in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The aim of this study was to reveal whether this methodology can be applied to human solid tumors sampled by fine-needle biopsy. Twentytwo tumors, consisting of 11 breast carcinomas; three metastatic anaplastic carcinomas; three adenocarcinomas of colon, endometrium, and lung; two metastatic lymph node squamous cell carcinomas of the larynx; and three malignant Iymphomas were examined. It was possible to identify cells with DNA strand breaks in all these tumors. Extremely high variability in the proportion of cells with DNA strand breaks was observed between the individual tumors. In diploid tumors (n = 12) the percentage of cells with DNA strand breaks varied from 1% to 43%, and the mean value was 19%. In aneuploid tumors this percentage varied from 15% to 51% and the mean value was 37%. In the latter tumors the presence of cells with DNA strand breaks was limited to the DNA aneuploid cell population; very few diploid, presumably tumor infiltrating or stromal cells, showed the presence of DNA strand breaks. No correlation was observed between the percent of cells in S phase and those with DNA strand breaks. The data indicate that apoptosis is more frequent in populations of tumor cells than among normal cells of the same organs. The higher percent of cells with DNA strand breaks compared to the percent of cells with morphology typical of apoptosis suggests that the early phase of apoptosis, characterized by the appearance of DNA strand breaks, which precedes morphological changes (chromatin condensation, nuclear fragmentation), may be very long in individual cells of the tumors studied. The present method may by applicable to estimate DNA strand breaks in solid tumors, either during spontaneous apoptosis or during apoptosis induced by chemo or radiotherapy, to evaluate tumor cell response early during treatment. 0 1994 Wiley-Liss, Inc.Key terms: Flow cytometry, terminal transferase, cell cycle, endonucleolysis Apoptosis denotes a characteristic mode of cell death, common to various cell types and often triggered by a plethora of environmental stimuli (6,20-22). Morphological changes during apoptosis are characterized by chromatin condensation (which starts at the nuclear periphery), nuclear fragmentation, and the formation and shedding of "apoptotic bodies," which consist of nuclear and cytoplasmic fragments enveloped in

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.

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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...