Abstract 3353: Cell-free DNA (cfDNA) fragmentation profiles linked to copy number alteration analysis allows for early colorectal cancer detection
Authors: Noguer Pol Canal , Cernosa Iva , Alonso Fernando Trincado , Kruusmaa Kristi 
Published: April 2023

Abstract
Background: Colorectal cancer (CRC) is the 3rd most diagnosed cancer worldwide. Early detection and correct patient stratification are important components for cancer prevention, diagnosis, and successful treatment. Cell-free DNA (cfDNA) fragment sizes have been previously shown to differ between cancer and non-cancer patients, while loss of heterozygosity is one of the major types of genetic inactivation, with long arm of chromosome 18 being most frequently reported to be deleted in CRC patients. We are here looking at the cfDNA fragmentation signal in early-stage CRC in the context of 18q21 region reported to exhibit copy number variations (CNV).
Methods: This was a prospective, international multicenter observational cohort study. Plasma samples were collected either prior to a scheduled colonoscopy as part of standard colorectal cancer screening or prior to colonic surgery for primary CRC in several hospitals in Spain, Germany and Ukraine. Samples were prepared and analyzed with whole genome enzymatic sequencing approach in 2 separate sets (Training Set and Testing Set). Training set consisted of 24 CRC patients (3 stage 0, 10 stage I, 5 stage IIA, 3 stage III, 2 stage IIIA and 1 stage IIIB) and 24 matching controls. The proportion of aligned short fragments over the total number was computed for Chr18q21 based on pre-defined bins. From the total number of binned regions, those presenting an average correlation with the others &gt;0.5 were discarded, getting a final set of regions, which were used as the input data of the Linear Discriminant Analysis (LDA) model. LDA model was then applied to independent Testing Set consisted of 12 CRC patients (4 stage I, 4 stage IIA and 4 stage III) and 12 matching controls.
Results: Training Set analysis exhibited significant differences over whole Chr18q21 region, when comparing CRC and matching control patients. The 2959 region bins selected in the Training Set were used as the input data of the LDA model to test the performance. Model performance on an independent Testing set reached 83% sensitivity (10/12) at 92% specificity (11/12), with stage I sensitivity being as high as 75% (3/4) and stage IIA 100% (4/4). Sensitivity per cancer location was comparable with 83% sensitivity for proximal cancers (5/6) and 83% for distal cancers (5/6). Specificity was not affected by presence of benign findings such as hyperplastic polyps, diverticulosis/diverticulitis or hemorrhoids.
Conclusions: The study results indicate that combining CNV information with the cfDNA fragment size information could serve as a promising new avenue for early detection of colorectal cancer, with early-stage (I-II) CRC sensitivity of 87.5% at 92% specificity. Larger cohort validation of these results is in progress.
Citation Format: Pol Canal Noguer, Iva Cernosa, Fernando Trincado Alonso, Kristi Kruusmaa. Cell-free DNA (cfDNA) fragmentation profiles linked to copy number alteration analysis allows for early colorectal cancer detection [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 3353.