Mutational Heterogeneity in APC and KRAS Arises at the Crypt Level and Leads to Polyclonality in Early Colorectal Tumorigenesis

Abstract

Purpose The majority of genomic alterations causing intratumoral heterogeneity (ITH) in colorectal cancer (CRC) are thought to arise during early stages of carcinogenesis as a burst but only after truncal mutations in APC have expanded a single founder clone. We have investigated if the initial source of ITH is consequent to multiple independent lineages derived from different crypts harboring distinct truncal APC and driver KRAS mutations, thus challenging the prevailing monoclonal monocryptal model. Experimental design High-depth next-generation sequencing and SNP arrays were performed in whole lesion extracts of 37 FAP colorectal adenomas. Also, ultra-sensitive genotyping of hotspot mutations of APC and KRAS was performed using nanofluidic PCRs in matched bulk biopsies (n=59) and crypts (n=591) from 18 adenomas and 7 carcinomas and adjacent normal tissues. Results Multiple co-occurring truncal APC and driver KRAS alterations were uncovered in whole lesion extracts from adenomas and subsequently confirmed to belong to multiple clones. Ultra-sensitive genotyping of bulk biopsies and crypts revealed novel undetected APC mutations that were prominent among carcinomas, whereas abundant wild-type APC crypts were detected in adenomas. KRAS mutational heterogeneity within crypts was evident in both adenomas and carcinomas with a higher degree of concordance between biopsy and crypt genotyping in carcinomas. Non-random heterogeneity among crypts was also observed. Conclusions The striking degree of non-random intercrypt heterogeneity in truncal and driver gene mutations observed in adenomas and carcinomas is consistent with a polycryptal model derived from multiple independent initiation linages as the source of early ITH in colorectal carcinogenesis.

Genomic and transcriptomic profiling of carcinogenesis in patients with familial adenomatous polyposis

“…In addition to somatic mutations, Car R3 also had distinct CNAs and loss of heterogeneity mutations compared with the other three regions (figure 2D and online supplementary figure 9B,C). 29 Clonal architecture analysis of other multiregionally sampled lesions from FAP4 and FAP5 supported the punctuated evolution model; most of the potential driver mutations and recurrent CNAs were clonal (online supplementary figures 6 and 7). 30…”

“…52 Gausachs et al also reported the possibility of a 'genetic field effect' in patients with FAP based on finding that physically separated crypts share hotspot mutations in KRAS. 29 However, the inference of the evolutionary relationship based on several mutation sites limits the reliability of this conclusion. Our analysis based on WES further confirmed the theory of field cancerisation in patients with FAP.…”

Colorectal Cancer: Genetic Abnormalities, Tumor Progression, Tumor Heterogeneity, Clonal Evolution and Tumor-Initiating Cells

“…Interestingly, in this study, it was also observed that the at-risk colon mucosa in part displayed the genetic alterations observed in adenomas, thus indicating the evolution from normal tissue to premalignant adenoma and surrounding mucosa and then to carcinoma [ 218 ]. Gausachs et al, in a recent study, provided evidence that the mutational heterogeneity in APC and KRAS observed in adenomas arises at the crypt level [ 220 ]. This conclusion was based on the ultrasensitive genotyping of matched bulk biopsies and individual crypts of colorectal adenomas and carcinomas: this analysis observed the presence of novel APC mutations, abundant wild-type APC crypts coexisting with mutant crypts, and mutational heterogeneity in KRAS within crypts, events not detectable through the analysis of standard biopsies [ 220 ].…”

Intratumor heterogeneity: A new perspective on colorectal cancer research

“…22 Multiple trunk APC mutations and heterogeneity of KRAS mutations were also found in early colorectal adenomas, suggesting that ITH is already present in such lesions. 23 The early evolutionary progress of CRC is branched, while most mutations in advanced CRC are neutral. Time is a natural process that enables the expansion of the early selection effect.…”