Abstract 222: High frequency of structural variants in FFPE colorectal cancer tissue detected by targeting selected common fragile site genes and LINE transposable elements
Authors: Rubio-Alarcon Carmen , Stelloo Ellen , Vessies Daan C. , van ‘t Erve Iris , Mekkes Nienke , Swennenhuis Joost , Lakbir Soufyan , van Bree Elise , Tijssen Marianne , Delis-van Diemen Pien , Lanfermeijer Mirthe , Linders Theodora C. , van den Broek Daan , Punt Cornelis J. , Heringa Jaap , Meijer Gerrit A. , Abeln Sanne , Feitsma Harma , Fijneman Remond J. 
Published: April 2023

Introduction: Structural variants (SVs) caused by chromosomal rearrangements or LINE retrotranspositions are a highly prevalent type of somatic DNA alterations in colorectal cancer (CRC). Studies about the function of SVs in tumor biology and their putative application as biomarkers are currently hampered by the need for fresh frozen tumor material to detect SVs by long-read or deep whole genome sequencing. This impedes the identification of SVs in retrospective cohorts and implementation in routine diagnostics (where only formalin-fixed paraffin-embedded (FFPE) tissue is available). In this study, we explored the applicability of FFPE-Targeted Locus Capture (FFPE-TLC) as a novel technology for targeted detection of SVs in CRC.

Materials and methods: We defined the regions most frequently presenting SVs in CRC and included these in a customized capture panel. FFPE-TLC followed by targeted sequencing was performed on primary tumors of 29 metastatic CRC patients and eight patient-matched normal colon tissues. SV-specific droplet digital PCR (ddPCR) assays were designed for orthogonal validation of SVs in tumor tissue and detection of SVs in cell-free plasma circulating tumor DNA (ctDNA).

Results: The regions associated with most SVs in metastatic CRC were four common fragile site (CFS) genes; MACROD2, PARK2, FHIT, WWOX, and three LINE source elements, and were included in a 2.7 Mb targeted panel. After filtering for germline variation, we identified 168 somatic SVs in 26/29 patients (90%), median SVs per patient: 3, range: 1-22. 110 SVs were found in CFS genes in 19/29 patients (66%), 12 of them presenting SVs in MACROD2. 58 SVs were caused by LINE integrations in 19/29 patients (66%), with different LINE behaviors observed: one source LINE generated 19 SVs in 13 patients, whilst another LINE caused 33 SVs in 5 patients. For selected cases, the tumor-specific origin of SVs was verified and its applicability as a plasma ctDNA biomarker demonstrated.

Discussion: This study demonstrates, for the first time, the feasibility of targeted detection of SVs in CFS genes and SVs caused by LINE retrotransposition in routinely obtained FFPE tissue without prior knowledge of the breakpoint location. The high prevalence of SVs offers opportunities to detect many somatic alterations by targeting few genes and LINEs, e.g. for translation into ctDNA biomarker assays. We conclude that FFPE-TLC enables the investigation of SV mutations in retrospective cohorts and opens new avenues for mutation analysis in routine diagnostics.

Citation Format: Carmen Rubio-Alarcon, Ellen Stelloo, Daan C. Vessies, Iris van ‘t Erve, Nienke Mekkes, Joost Swennenhuis, Soufyan Lakbir, Elise van Bree, Marianne Tijssen, Pien Delis-van Diemen, Mirthe Lanfermeijer, Theodora C. Linders, Daan van den Broek, Cornelis J. Punt, Jaap Heringa, Gerrit A. Meijer, Sanne Abeln, Harma Feitsma, Remond J. Fijneman. High frequency of structural variants in FFPE colorectal cancer tissue detected by targeting selected common fragile site genes and LINE transposable elements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 222.