Abstract LB102: Triaging oral premalignant lesions: Plasma ultrashort cell-free DNA-based liquid biopsy approach
Authors: Swarup Neeti , Cheng Jordan C. , Choi Irene , Aziz Mohammad Arshad , Liu Kelly Y. , Wei Fang , Li Feng , Kim Yong , Poh Catherine , Wong David T. 
Published: April 2024

Abstract
Background: Rapid advancements in cell-free DNA (cfDNA) based liquid biopsy (LB), has opened avenues of critical clinical importance. Recent discovery of a novel class of cell-free DNA 40-70 bps in length, ultrashort cfDNA (uscfDNA) is demonstrating immense potential as a cancer early detection biomarker. Globally, oral cavity squamous cell carcinomas (OCSCC) make up a significant proportion of head and neck squamous cell cancers. OCSCC commonly arises after the occurrence of oral premalignant lesions (OPLs), making it an ideal target for plasma-based LB screening initiatives.
Methods: Current work focuses on preferential extraction of low molecular weight nucleic acids and single-stranded library preparation and sequencing, Broad Range cell-free DNA- Sequencing (BRcfDNA-Seq), to obtain uscf- & 167 bps mononucleosomal cfDNA (mncfDNA), for disease detection. Sequencing data was bioinformatically processed: paired-end reads merged (BBmerge), aligned with BWA-mem, deduplicated using SRSLY UMI, and blacklisted portions removed. This was followed by analysis for non-mutational features like fragmentomics, occurrence of G-Quad structures, 4-mer Motif sequences at the end of cfDNA sequences, functional genomic element peak formation by uscfDNA. These features have been recently described non-mutation metrics which have demonstrated evidence of clinical relevance. With non-mutational multi-modal analysis of uscfDNA, obtained using BRcfDNA-Seq, we were able to categorize oral premalignant disorders into two cohorts: one which progressed into OCSCC (PG-OPL) (n=19) & one which did not (nPG-OPL) (n=19).
Results: Bioinformatic processing for fragmentomics, of the relative proportion of 40-53bps fragments to 54-70bps fragments in the uscfDNA population demonstrated a significant difference across 1700 chromosomal bins between PG & nPG-OPL (multiple t test, FDR 10%, p value = 0.044, AUROC = 0.7258). The occurrence of G-quadruplex structures in uscfDNA was also significantly different between the two groups (student t test, p= 0.02, AUROC= 0.6953). Examination of the end-motifs profiles of uscfDNA between PG-OPL & nPG-OPL samples showed the randomness of the motifs as revealed by the Shannon entropy score is significant between the two groups (p = 0.046, AUROC = 0.6482). We further evaluated individual motifs that could discriminate between the two groups and identified CCCC, CCCT, TGGG, etc. are highly discriminatory. uscfDNA contained a substantially higher proportion of functional genomic element sequences in the PG-OPL group than the nPG-OPL group like exon (p = 0.00067, AUROC = 0.7147) & 5’ UTR (p= 0.000006, AUROC= 0.7673) are different between the groups.
Conclusion: Results demonstrated clinical relevance of BRcfDNA-Seq for plasma-derived cfDNA in assessment of malignancy progression of OPLs. These non-mutational multi-modal features of cfDNA maybe indicative of local and systemic factors associated with disease affecting cfDNA.
Citation Format: Neeti Swarup, Jordan C. Cheng, Irene Choi, Mohammad Arshad Aziz, Kelly Y. Liu, Fang Wei, Feng Li, Yong Kim, Catherine Poh, David T. Wong. Triaging oral premalignant lesions: Plasma ultrashort cell-free DNA-based liquid biopsy approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB102.