Mutational Analysis of Field Cancerization in Bladder Cancer

Abstract

BACKGROUND: Morphologically normal tissue, adjacent to tumors, contains multiple molecular changes, the so-called field cancerization. The multifocal and recurrent nature of bladder cancer has been hypothesized to originate from this. However, further studies are required to explore the mutational composition of normal tissue adjacent to tumors. OBJECTIVE: To analyze field cancerization in bladder cancer patients using a non-tumor guided approach. METHODS: We investigated the mutational landscape of normal appearing urothelium and paired bladder tumors from four patients by applying deep-targeted sequencing. RESULTS: Sequencing of 509 cancer driver genes revealed the presence of 2– 13 mutations exclusively localized in normal tissue (average target read depth 634×). Furthermore, 6– 13 mutations were shared between tumor and normal samples and 8– 75 mutations were exclusively detected in tumor samples. More mutations were observed in normal samples from patients with multifocal disease compared to patients with unifocal disease. Mutations in normal samples had lower variant allele fractions (VAF) compared to tumor mutations (p < 2.2*10–16). Furthermore, significant differences in the type of nucleotide changes between tumor, normal and shared mutations (p = 2.2*10–5) were observed, and mutations in APOBEC context were observed primarily among tumor mutations (p = 0.02). No differences in functional impact between normal, shared and tumor mutations were observed (p = 0.61). CONCLUSION: Overall, these findings support the presence of more than one field in the bladder, and document non-tumor specific driver mutations to be present in normal appearing bladder tissue.

Recurring urothelial carcinomas show genomic rearrangements incompatible with a direct relationship

“…Similar detailed molecular analyses of pathologically normal sections of the bladder from patients with NMI tumors remains scarce, as these patients rarely are subjected to cystectomy. However, Thomsen et al 9 and Strandgaard et al 10 approached the problem by multiple sampling of tumor free regions of the urothelium from bladders with non-muscle invasive tumors. These studies revealed, although with less substantial data, the presence of genetic and genomic alterations in the pathologically normal urothelium of patients also with NMI tumors.…”

Many are called but few are chosen – Multiple clonal origins greatly elevate the functional heterogeneity of tumors

“…Therefore, the architecture appears to predispose tissues to multiple origins of tumors. However, DNA sequence data usually suggest a process of single origin (Parsons 2008;Thirlwell, et al 2010;Lindberg, et al 2013;Ma, et al 2017;Wei, et al 2017;Cross, et al 2018;Parsons 2018;Strandgaard, et al 2019). This study aims to resolve this apparent contradiction.…”

“…Given l = n x e < 0.1, e is extremely small, determined by the gradual accumulation of advantageous mutations that drive tumorigenesis. The probability of having more than 2 independent tumors should thus be negligible (Parsons 2008;Thirlwell, et al 2010;Lindberg, et al 2013;Ma, et al 2017;Wei, et al 2017;Parsons 2018;Strandgaard, et al 2019). This is true even when multiple nodules are observed as they usually result from metastasis (Cresswell, et al 2020;Hu, et al 2020).…”

Mutations in Non-Tumoral Human Urothelium: Disease Prelude or Epilogue?

“…Next generation sequencing technologies are allowing the detection of mutations present in small areas of tissue at an unprecedented level of resolution, thus being suitable tools to characterize field changes in the urothelium. In this issue, Strandgaard et al [3] perform deep-targeted amplicon sequencing of paired samples of tumor and histologically normal-appearing urothelium from four patients with advanced bladder cancer treated with radical cystectomy. The small cohort comprises two individuals with multifocal disease and two with unifocal disease.…”

“…Three types of mutations are identified by Strandgaard and colleagues [3]: T-mutations, found exclusively in the tumor; S-mutations, found both in tumor and non-neoplastic urothelium; and Nmutations, found exclusively in non-neoplastic samples (hereafter referred to as normal tissue). The two former types are more abundant.…”