Somatic L1 retrotransposition occurs early during colorectal tumorigenesis. S. Solyom1, A. D. Ewing2, N. Baker2, A. Gacita2, L. D. Wood3, S. J. Meltzer4, B. Vogelstein5, K. W. Kinzler5, H. H. Kazazian1 1) McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD; 2) Center for Biomolecular Science and Engineering, University of California, Santa Cruz, CA; 3) Gastrointestinal & Liver Pathology, Johns Hopkins Hospital, Baltimore, MD; 4) Johns Hopkins University School of Medicine & Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD; 5) The Ludwig Center and The Howard Hughes Medical Institute at Johns Hopkins Kimmel Cancer Center, Baltimore, MD.

   Long INterspersed Element-1 (L1) retrotransposons are autonomous mobile elements that comprise 17% of the human genome. L1s retrotranspose by a copy and paste mechanism via an RNA intermediate. Although insertional mutagenesis by these elements is a known cause of various human Mendelian diseases, their somatic mobilization in the cancer genome has only recently been established as a new mutational phenomenon. Here we investigate the timing of insertional events and the extent of tumor heterogeneity conferred by retrotransposons, as well as their impact on malignancy. We studied DNA from 4 colon cancer patients who had been previously diagnosed with colonic polyps (3 adenomas and one hyperplastic), as well as from 5 additional patients with colorectal dysplasia arising in inflammatory bowel disease (IBD). In contrast to the paired polyp-cancer samples, the IBD cancers were immediately adjacent to, and likely originated from, their matched dysplasias. Two of the 4 patients with both colon adenomas and carcinomas also had metastases. After dissection of abnormal from normal tissue, next-generation L1-targeted resequencing (L1-seq) was carried out on DNA from these tissues. After PCR-validation and Sanger sequencing of putative somatic L1 insertions, we found for the first time that certain pre-cancerous lesions were mutagenized by somatic L1 insertions. We validated 56 somatic insertions in cancerous or pre-cancerous lesions, of which 31 occurred in pre-malignant lesions. We found no verifiable insertions in normal colon. Surprisingly, 2 adenomas contained more than 9 insertions each. Two IBD dysplasias contained insertions as well, and 6 L1 insertions were present not only in these lesions, but also in their paired cancers. Finally, we validated 2 metastasis-specific insertions and 6 insertions found in both primary colon cancers and their matched metastases. Since 6 of 7 insertions in colon cancers were present in their matched metastases, and 6 of 9 insertions in IBD dysplasias were also present in their paired carcinomas, many insertions may be present in every cell of the cancer or the dysplasia. These data suggest that L1s may serve as novel biomarkers of malignant progression. Numerous genes - some with unknown function - were targeted by L1s and may play a role in malignancy. To conclude, somatic retrotransposition is involved early in the pathogenesis of some colon cancers and may provide useful biomarkers of neoplastic progression.

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