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

Abstract

<b>Purpose:</b> The majority of genomic alterations causing intratumoral heterogeneity (ITH) in colorectal cancer are thought to arise during early stages of carcinogenesis as a burst but only after truncal mutations in <i>APC</i> 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 <i>APC</i> and driver <i>KRAS</i> mutations, thus challenging the prevailing monoclonal monocryptal model.<b>Experimental Design:</b> High-depth next-generation sequencing and SNP arrays were performed in whole-lesion extracts of 37 familial adenomatous polyposis colorectal adenomas. Also, ultrasensitive genotyping of hotspot mutations of <i>APC</i> and <i>KRAS</i> was performed using nanofluidic PCRs in matched bulk biopsies (<i>n</i> = 59) and crypts (<i>n</i> = 591) from 18 adenomas and seven carcinomas and adjacent normal tissues.<b>Results:</b> Multiple co-occurring truncal <i>APC</i> and driver <i>KRAS</i> alterations were uncovered in whole-lesion extracts from adenomas and subsequently confirmed to belong to multiple clones. Ultrasensitive genotyping of bulk biopsies and crypts revealed novel undetected <i>APC</i> mutations that were prominent among carcinomas, whereas abundant wild-type <i>APC</i> crypts were detected in adenomas. <i>KRAS</i> mutational heterogeneity within crypts was evident in both adenomas and carcinomas with a higher degree of concordance between biopsy and crypt genotyping in carcinomas. Nonrandom heterogeneity among crypts was also observed.<b>Conclusions:</b> The striking degree of nonrandom 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. <i>Clin Cancer Res; 23(19); 5936-47. ©2017 AACR</i>.