Abstract 1087: Genomic analysis reveals evidence of clonality in premalignant colonic polyps
Authors: Chang Kyle , Borras Ester , San Lucas Anthony , Fowler Jerry , Hawk Ernest T. , Capella Gabriel , Scheet Paul , Vilar Eduardo 
Published: August 2015

Abstract
Clonality of tumors arises from accumulation of mutations and changes in selective pressure during carcinogenesis and further stages of tumor development. The identification of clonality through different molecular and systems biology tools has challenged the current understanding of the carcinogenesis and progression process, as well as drug resistance, and clinical outcomes. With the advent of next-generation sequencing, numerous bioinformatics methods are being developed to study clonality of tumors at a genomic scale. However, these studies have mostly been focusing on late stage carcinomas and little is known about the clonality in pre-malignant lesions. Here we present an analysis of clonality on colonic polyps from patients diagnosed with Familial Adenomatous Polyposis (FAP). FAP is an autosomal dominant disease secondary to germline mutations in APC and characterized by the development of hundreds to thousands of polyps in the gastrointestinal tract at a young age. We performed whole-exome sequencing on 25 polyps and matched blood samples using an Illumina HiSeq2000 instrument. We obtained somatic mutations calls using MuTect and copy number variations from VarScan2. Then, we used two systems biology tools (ABSOLUTE and EXPANDS) to assess the levels of tumor purity and ploidy, and to determine the presence of clonal and subclonal populations. We found that EXPANDS predicted an average tumor purity of polyps (54%) lower than that of 20 TCGA stage I colorectal carcinomas (77%); and an average number of clones in polyps (2.72) lower than that of TCGA stage I colorectal carcinomas (7.04). ABSOLUTE predicted an average tumor purity in polyps (40%) lower than that in TCGA stage I colorectal carcinomas (68%) as well, although there were differences between the purity estimates between both tools. Second hits in APC are a valuable and intuitive method to establish the presence of clonality in FAP polyps as any additional APC mutation has to be acquired in the context of progression of carcinogenesis as a second hit. We identified 12 APC somatic mutations from 12 polyps and 2 KRAS somatic mutations from 2 polyps that were predicted to be in clonal populations by EXPANDS. Furthermore, we performed targeted high-depth IonTorrent Ampliseq in a second set of 37 polyps (average of 70% bases at 500x) and matched normal mucosa from 14 FAP patients. We identified evidence of multiple somatic APC mutations at different allelic frequencies in 7 polyp samples with 3 of them showing significant differences in allelic frequencies between different somatic APC hits (&gt;30%); in addition, 2 KRAS mutations were observed in a polyp. In conclusion, our analysis show that there are multiple mutated APC subclones driving progression and evidence of clonality in pre-malignant colonic lesions. Further analysis will be conducted to understand the functional impact and the relation among the different identified subclones.
Citation Format: Kyle Chang, Ester Borras, Anthony San Lucas, Jerry Fowler, Ernest T. Hawk, Gabriel Capella, Paul Scheet, Eduardo Vilar. Genomic analysis reveals evidence of clonality in premalignant colonic polyps. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1087. doi:10.1158/1538-7445.AM2015-1087