Abstract 2293: Comparisons of saliva versus plasma cfDNA and exosomal DNA as potential biomarkers for head and neck cancer
Authors: Barbirou Mouadh , Miller Amanda , Baek David , Hameier Allison , Wetzel Ashley , Linnenbach Alban , Nunes Kathryn , Llerena Pablo , Bar-Ad Voichita , Luginbuhl Adam , Outschoorn Ubaldo Martinez , Myers Ronald , Curry Joseph , Wang Chun , Yang Hushan 
Published: March 2024

Abstract
Purpose: Liquid biopsy approaches are revolutionizing cancer monitoring and have been considered potential mediators for therapeutic efficacy in multiple cancers. The investigation of different liquid biopsy biomarkers may lead to a better understanding of the cancers they are designed to detect and facilitate optimal management. This study aimed to identify and compare the epigenomic and genomic profiles of tumor biomarkers in circulation using cell-free DNA (cfDNA) and exosomal DNA (exoDNA) in plasma and saliva samples collected from patients with head and neck squamous cell carcinoma (HNSCC).
Methods: In a prospective trial, we performed whole-genome bisulfite sequencing on paired cfDNA and exoDNA from saliva versus plasma of 16 HNSCC patients. cfDNA was directly extracted from plasma and saliva, while exoDNA was extracted from purified exosomes after characterization. DNAs were then converted by Zymo EZ DNA Methylation-Direct kit and libraries were constructed with xGen™ Methyl-Seq Kit and sequenced on the Illumina NovaSeq platform. Sequencing data analyses were performed using Bismark, GATK, and methylKit for methylation pattern and cfTools for Tumor Fraction Estimation (TFE).
Results: Methylation data demonstrated heterogeneities across sample types. Comparisons of ctDNA as a reference for exoDNA showed 7147 hypermethylated and 9984 hypomethylated regions for plasma samples but showed 131 hypermethylated and 123 hypomethylated regions for saliva samples. Comparisons of plasma as a reference for saliva, 37 hypermethylated and 95 hypomethylated regions were noted in exoDNA, while 117 hypermethylated and 262 hypomethylated regions in ctDNA. The mean level of tumor-derived DNA was above 3%, the lower limit of sensitivity to indicate the presence of tumor cutoff as suggested by the cfTools. There were 3.24%, 3.14%, and 3.14% tumor-derived DNA in plasma cfDNA, plasma exoDNA, and saliva exoDNA, respectively. However, the tumor fraction in saliva cfDNA was below this cutoff (2.88%). A statistically significant (P &lt; 0.001) difference in TFE was observed between combined cfDNA and exoDNA data from plasma and saliva, but the difference between combined sample types cfDNA and exoDNA did not reach statistical significance (P = 0.306). The linear mixed-effects model for TFE influence between the different groups revealed a significant interaction between sample sources (plasma and saliva) and sample types (cfDNA and exoDNA) (P = 0.005), as well as a significant effect of sample source on TFE (P &lt; 0.001).
Conclusions: This pilot investigation provides evidence that exoDNA and cfDNA have different molecular profiles in plasma and saliva samples from the same HNSCC patient, and indicates that saliva carries additional tumor markers for HNSCC. These results help to advance our understanding of the potential impact of different liquid biopsy sample types in cancer management.
Citation Format: Mouadh Barbirou, Amanda Miller, David Baek, Allison Hameier, Ashley Wetzel, Alban Linnenbach, Kathryn Nunes, Pablo Llerena, Voichita Bar-Ad, Adam Luginbuhl, Ubaldo Martinez Outschoorn, Ronald Myers, Joseph Curry, Chun Wang, Hushan Yang. Comparisons of saliva versus plasma cfDNA and exosomal DNA as potential biomarkers for head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2293.