Mutational analysis of field cancerization in bladder cancer

Abstract

31By applying whole exome sequencing and deep targeted sequencing on bladder 32 tumors, it was recently shown that tumors developed years apart in the same patients 33 share multiple mutations and hence are clonally related 1-3 . Furthermore, apparently 34 normal urothelium has been documented to contain mutations with low allele 35 frequencies (~3%) that are typically observed at high frequencies in tumors (clonal 36 mutations) 1-3 . Multiple studies have investigated genomic alterations in normal 37 appearing bladder tissue from cystectomy specimens, however using technologies 38 that do not allow detection of low-frequency mutations. The genomic alterations 39 observed in these studies include copy number alterations of chromosome 5, 9, 13, 40 16, and 17 as well as mutations or loss of RB1 and TP53 4-9 . These findings 41 corroborates the suggestions of the presence of field cancerization in the bladder. 42 Similar results have been reported in other tissue types, where studies have revealed 43 the presence of mutations in well-characterized cancer driver genes in apparently 44 healthy tissue and pre-cancer lesions 10-13 . 45 46 Bladder cancer (BC) is multifocal in almost half of the cases with primary tumour and 47 101 102 Analysis of field cancerization. Patients 1 and 2 presented with multifocal disease, 103 whereas patients 3 and 4 had unifocal disease. In patients 1 and 2, 39% (25/64) of the 104 mutations were N-Mutations, and 34% (22/64) were S-Mutations. Mutations called in 105 patients 3 and 4 were mainly T-Mutations, with only 5% (7/143) being N-Mutations and 106 13% (19/143) S-Mutations -indicating that uni-and multifocal patients may show 107 different levels of field cancerization. Mutations in known BC driver genes were 108 detected in both N-, S-and T-Mutation groups, most of them being among T-109 Mutations. However, in patient 1, two N-mutations were observed in bladder cancer 110 driver genes. Damaging mutations were present in all N-, S-and T-Mutation groups. 111 We detected the introduction of premature stop codons, mainly in the T-Mutation 112 group. However, for patient 1 premature stop codons were solely observed within the 113 N-and S-Mutations. Mutation allele frequencies (AFs) varied for the different 114 mutations detected but were generally low for N-Mutations and high for T-Mutations. 115 See Figure 1b and Table 1 for details. 116 117 131 132 Allele frequencies (median (min-max)) N-Mutations 0.042 (0.029-0.091) 0.067 (0.035-0.15) 0.070 (0.049-0.091) 0.059 (0.049-0.091) T-Mutations 0.23 (0.049-0.31) 0.13 (0.031-0.40) 0.17 (0.039-0.50) 0.16 (0.032-0.67) S-Mutations (Normal pool) 0.022 (0.0074-0.14) 0.033 (0.0064-0.063) 0.10 (0.014-0.13) 0.025 (0.0065-0.13) S-Mutations (Tumor pool) 0.20 (0.067-0.44) 0.077 (0.016-0.56) 0.13 (0.057-0.19) 0.15 (0.020-0.61)

Molecular changes during progression from nonmuscle invasive to advanced urothelial carcinoma

“…Such a genetic heterogeneity has been found in the premalignant lesions in Barrett's esophagus, 5 and has been linked to progression of breast tumors. 37 The most detailed genetic studies of the premalignant urothelium has been done in research groups of Dyrskjøt,8,9 and Czerniak. 6,7 Both groups have used multiregion sampling from whole organs to identify tumor-associated mutations in the noncancerous epithelium.…”

“…5 Studies on the genetic heterogeneity of the premalignant field in patients with urothelial bladder cancer (UBC) have given important insights into the processes behind tumor initiation and favor a clonal origin. [6][7][8][9] The shared clonal origin of nonmuscle invasive bladder cancer (NMIBC) recurrences has been repeatedly demonstrated [10][11][12] even though tumors usually occur in different locations within the bladder. 13 Taken together, a model is supported in which tumors develop semiindependently out of a premalignant field that is usually clonal in nature but has additional heterogeneity that manifests as genetic differences between recurrences.…”

“…If the site of tumor recurrence is different from that of the primary tumor, this is a manifestation of field cancerization, either due to single clone expansion or due to independent fields, that is, oligo‐clonal acquisition of tumor‐initiating capacity . Studies on the genetic heterogeneity of the premalignant field in patients with urothelial bladder cancer (UBC) have given important insights into the processes behind tumor initiation and favor a clonal origin . The shared clonal origin of nonmuscle invasive bladder cancer (NMIBC) recurrences has been repeatedly demonstrated even though tumors usually occur in different locations within the bladder .…”

Knockdown of UTX/KDM6A Enriches Precursor Cell Populations in Urothelial Cell Cultures and Cell Lines

“…Recent studies have indeed observed clonal expansion of mutant cells in morphologically normal urothelium. In one study on four patients, intriguingly, KMT2D, another COMPASS component, was frequently mutated in morphologically normal urothelial tissue from cancer-carrying bladders [34]. An analogous scenario is established in the development of acute myeloid leukemia, which is often preceded by clonal hematopoiesis elicited by mutations in various genes, most often encoding epigenetic regulators like DNMT3A and TET2 [35], which shift the balance between stem cells and differentiated progeny and displace normal with stem cells with mutants.…”