Figure 10

Integration of LOH and LOP patterns identified in the 13q14 region with RB1 sequencing data and RB protein expression implicating the involvement of FR genes in the intraurothelial expansion of a neoplastic clone. (a) Regions of LOP associated with early clonal expansion identified by WOHGM with SNPs in five cystectomy specimens related to the status of RB1 sequence, RB1(S), and RB protein expression revealed by immunohistochemistry, RB(IH), are illustrated. The results of RB1 sequencing and immunohistochemical studies for RB protein expression are tabulated below the maps of individual bladders. W, wild-type RB1; M, mutant RB1. The mutation in map 2 involved codon 556 of exon 17 consisting of CGA → TGA and resulting in the change of Arg to a stop codon. The presence of immunohistochemically detectable RB protein is designated by +. The absence of RB protein expression is designated by −, and its distribution pattern is shown in the lower panel of (b). The genome sequence map in which the positions of hypervariable markers as well as known genes are designated by the bars on the left side of map. The regions of LOP in five cystectomies (maps 1–5) are depicted by the blue solid bars. The shadowed areas labeled delA and delB designate the regions of LOP flanking RB1 involved in the incipient expansion of a neoplastic clone. The shaded area labeled delRB1 designates the segment of LOP corresponding to the position of RB1 on the sequence genome map. (b) The distribution of clonal LOP involving RB1 and the same regions shown in (a) for map 5 (upper panel) is depicted. The lower panel shows the distribution of the segment with LOP in map 2 depicted in (a). The code for histologic map is shown in Figure 3. (c) Region of clonal LOP associated with growth advantage of in situ neoplasia identified by SNP-based mapping. (d) The immunohistochemical pattern of RB protein expression in representative mucosal samples of map 5 illustrated in (b) as the upper panel and corresponding to NU, LGIN, HGIN, and TCC is shown. The presence of RB protein in all mucosal samples correlated with the sequencing data, which indicated that the remaining wild-type RB1 allele was retained in this case. (e) The immunohistochemical pattern of RB protein expression in representative mucosal samples of map 2 illustrated in the lower panel of (b). Positive nuclear staining for RB1 protein in stromal endothelial cells serves as an internal positive control (arrows). Note the absence of RB protein expression in HGIN and TCC corresponding to an area containing a mutant RB1 allele. Solid black bars within photomicrographs indicate 50 μm. (Reprinted with permission from Lee S, Jeong J, Majewski T, et al. Proc Natl Acad Sci USA 2007;104:13732–13737.)