Figure 9

An example of high-resolution whole-organ mapping by allelotyping of SNPs and the assembly of LOH distribution patterns within RB1-containing region in a single cystectomy specimen. (a) The region containing a cluster of SNPs with allelic loss flanked by markers D13S328 and D13S155 is shown. The bars on the left side indicate the positions of all known and computationally predicted genes. The blue bars on the right side designate the positions of informative polymorphic SNPs. The solid black dots and bars designate SNPs with allelic loss. (b) The genomic map of RB1 is expanded and shows the positions of the five polymorphic SNPs with allelic loss as well as the positions of two polymorphic DNA markers (RB1.2 and RB1.20) with allelic loss. (c) The distribution of clonal allelic losses as they relate to precursor in situ lesions and invasive TCC shown as a histologic map at the bottom is demonstrated. The blocks depict the distribution of clonal allelic losses identified by the hypervariable DNA markers (red blocks) and SNPs (gray blocks). The allelic patterns of SNP markers in mucosal samples were compared to their patterns in peripheral blood lymphocytes of the same patient. The code for the histologic map is shown in Figure 3. The hypervariable DNA markers and SNPs with allelic loss associated with plaque-like clonal expansion involving large areas of bladder mucosa were clustered within and around RB1 and involved approximately 7 Mb. These defined several discontinuous regions of allelic losses associated with early clonal expansion of urothelial cells that ranged in size from approximately 0.27–1.11 Mb and are indicated by the vertical blue bars and gray-shaded areas in (a). The borders and predicted size of these regions were defined by the nearest flanking SNPs or microsatellite markers that retained polymorphism. The blue numbers indicate the predicted size of the deleted regions. (d) An example of clonal loss of a G/A polymorphism in SNP 6 located within intron 12 of RB1 is illustrated. Non-tumor DNA of peripheral blood lymphocytes of the same patient (PBDNA) shows G/A polymorphism of SNP 6, while samples corresponding to NU, LGIN, HGIN, and TCC show clonal loss of G. Retention of polymorphism in two SNPs flanking a segment of allelic loss that involves the RB1 gene is also shown. Overall, these data imply that several discontinuous losses of genetic material, which included RB1 and its flanking regions, occurred in early phases of bladder neoplasia and were associated with in situ expansion of a dominant neoplastic clone. (Modified and reprinted with permission from Lee S, Jeong J, Majewski T, et al. Proc Natl Acad Sci USA 2007;104:13732–13737; (c) and (d) represent new data.)