Cell-free DNA fragmentomics and methylomics for thymic epithelial tumors.

Abstract

8118 Background: Development of circulating cell-free (cf) DNA assays for cancer detection and monitoring is an area of active research. Limited data are available on the utility of cfDNA for thymic epithelial tumors (TETs). Evaluation of cfDNA fragmentation patterns (fragmentomics) offers advantages over conventional cfDNA analysis. Studies have shown that concurrent generation of fragmentomics and methylomics features via enzymatic methyl-sequencing (EM-Seq) are highly predictive at classifying cancer vs. non-cancer and capture key cancer-related biological features. We conducted a study to determine the feasibility of fragmentomics and methylomics for evaluation of TETs. Methods: cfDNA was extracted from 1-2 mL of plasma EDTA derived from patients with advanced TETs using the QIAamp Circulating Nucleic Acid Kit, underwent enzymatic conversion via the NEBNext Enzymatic Methyl-Seq v2 Kit, and sequenced on a NovaSeq X 10B flowcell. Estimated tumor fractions were generated using ichorCNA with low tumor settings and via fragle . Recurrent copy number alterations (CNAs) were plotted and compared against external TET copy number datasets. Short fragment percentage (90-150 bps) was calculated and average DELFI scores (DNA evaluation of fragments for early interception) were generated via FinaleToolkit . Results: Plasma samples from 47 of 50 participants (thymic carcinoma: 26, thymoma: 24; median age: 55 years (range 23-77); females 22; stage distributions: I/II/III/IV = 1/0/1/48) yielded high-quality cfDNA which was sufficient for sequencing. Individuals with thymic carcinoma displayed higher estimated tumor fractions via ichorCNA (P<0.05) and Fragle (P=0.05). Moreover, although not statistically significant, individuals with thymic carcinoma exhibited a general trend of higher short fragment percentage and higher average DELFI scores. When evaluating recurrent CNAs, individuals with thymic carcinoma displayed greater enrichments in CNAs in general with noted amplifications/gains at chr 1, 5, 12, and 17 and deletions at chr 16, whereas individuals with thymoma showed deletions at chr 3. When grouped and compared to external thymoma tissue CNAs, recurrent CNAs detected in plasma showed partial concordance. Large alterations in fragmentation profiles were also identified across both groups, which showed increased variability near recurrent CNA regions. Conclusions: This proof-of-concept study demonstrates that plasma cfDNA is detectable and fragmentomics is feasible among individuals with TETs, thus providing a non-invasive means of capturing cancer-related aberrant signals. Validation of this result in larger studies would establish cfDNA fragmentomics as a novel non-invasive means for disease monitoring and biomarker identification in patients with TETs.