Abstract A02: High-depth sequencing reveals the presence of multi-clonality in colorectal premalignancy
Authors: Borras Ester , Chang Kyle , Lucas Anthony San , Masand Gita , Delgado Amador Axel , Fowler Jerry , Davies Gareth E. , Ehli Erik A. , You Y. Nancy , Lynch Patrick M. , Hawk Ernest T. , Capella Gabriel , Scheet Paul A. , Vilar Eduardo 
Published: March 2017

Clonality in carcinomas is thought to arise from the accumulation of mutations and changes in selective pressure during carcinogenesis and further stages of tumor development. However, the identification of clonality through different molecular and systems biology tools has challenged the current understanding of carcinogenesis and progression. Familial Adenomatous Polyposis (FAP) is a genetic condition secondary to germline mutations in APC and characterized by the development of hundreds of polyps in the colorectum. Therefore, FAP is an ideal model to study the presence of clonality during early stages of colorectal carcinogenesis in humans. Second hits in APC are a valuable and intuitive method to establish the presence of clonality in FAP, as additional somatic APC mutations will reflect the presence of additional clones. Here, we present a targeted high-depth sequencing analysis of APC and KRAS in conjunction with high density SNP array data in 37 colorectal adenomas and matched adjacent uninvolved mucosa samples. Ampliseq sequencing was performed using the next-generation sequencing platform IT Personal Genome Machine (PGM; Life Technologies) using the Ion PGM 200 Sequencing Kit on an Ion 318 Chip Kit (Life Technologies, average of 70% bases at 500x). Ion Torrent Variant Caller v4.2 was run in the somatic low stringency proton mode to detect variants in APC and KRAS against hg19. Then, normal variants were subtracted from matched adenoma variants to create a list of somatic candidates for each adenoma and normal pair. SNP array analysis was performed using the HumanOmniExpressExome-8 v1.2 BeadChip (Illumina, San Diego, CA). To validate the presence of double somatic hits in APC we performed a colony genotyping assay from one adenoma sample. First, we identified APC somatic mutations or loss of heterogeneity (LOH) in 5q (second hits) in 81% (30/37) and KRAS somatic mutations in 35% (16/37) of adenomas analyzed. Six adenomas (16%) presented double somatic APC mutations with different allelic frequencies with three of them showing a significant difference in allelic frequencies among the somatic APC hits (&gt;30%); in addition, one adenoma presented two KRAS mutations. The APC clonality was validated in one adenoma using colony analysis. The region that contains the two somatic mutations (c.4348C&gt;T and c.4267_4280del) and the germline mutation (c.2894delA) was amplified by Long-Range PCR and cloned into the pGEM-T plasmid. DNA from one hundred colonies was extracted and genotyped using Taqman assay probes. All samples were genotyped in triplicate and positive and negative controls for all mutations were included in every single plate. A total of 25% of the colonies harbored a wild-type allele, 36% the known germline APC mutation, 15% the APC somatic c.4348C&gt;T mutation and 24% the APC somatic c.4267_4280del mutation. In conclusion, our analysis showed that there are multiple mutated APC subclones driving initiation of colorectal carcinogenesis, thus supporting experimentally a finding of multi-clonality in premalignant colonic lesions. These findings support a model of colorectal carcinogenesis that is based on polycryptal polyclonal adenomas and calls for revisiting the “Big-Bang” model based on the presence of multiple initiator clones. Further analyses aimed to understand the functional impact and the hierarchical relations among the different identified subclones are warranted.

Citation Format: Ester Borras, Kyle Chang, Anthony San Lucas, Gita Masand, Axel Delgado Amador, Jerry Fowler, Gareth E. Davies, Erik A. Ehli, Y. Nancy You, Patrick M. Lynch, Ernest T. Hawk, Gabriel Capella, Paul A. Scheet, Eduardo Vilar. High-depth sequencing reveals the presence of multi-clonality in colorectal premalignancy. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A02.