Preanalytical Validation of Circulating Tumor DNA from Surgical Drain Fluid as a marker of Postoperative Minimal Residual Disease in Head and Neck Squamous Cell Carcinoma - PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) remains a significant global health burden with one million new cases annually. HPV-negative (HPV-) HNSCC, which is the most common and deadliest form, is particularly problematic given its high rate of recurrence after curative-intent surgical resection. Unfortunately, current criteria for adjuvant therapy are imprecise and subject to variability, and there are no reliable biomarkers to detect minimal residual disease (MRD) post-surgery. Traditional plasma-based liquid biopsies are limited in sensitivity for locoregional recurrence and low tumor volume settings. To address these issues, our team has developed a novel approach using surgical drain fluid (SDF) collected postoperatively as a proximal bioanalyte to measure MRD in HNSCC patients. Our published preliminary data indicate that circulating tumor DNA (ctDNA) in SDF predicts locoregional relapse and progression-free survival with higher sensitivity and specificity than postoperative plasma ctDNA. Tumor-derived exosomes (TEX) in SDF also show potential as prognostic biomarkers. This proposal aims to systematically investigate the pre-analytical variables related to this new liquid biopsy analyte, with the goal of establishing standard operating procedures (SOPs) for SDF-based ctDNA and TEX assays to accurately measure MRD after HNSCC surgery. We propose three specific aims: (1) optimize postoperative collection and processing methods for SDF to enhance ctDNA recovery and stability by evaluating preanalytical variables such as collection timing, preservation solutions, centrifugation spins, temperature, and processing time; (2) optimize conditions for isolating TEX and TEX-associated DNA from SDF in HPV-positive and HPV-negative HNSCC patients using immune capture techniques and TEX luminal DNA quantification; and (3) validate SDF ctDNA for prognostication of relapse in a prospective HPV-negative HNSCC cohort, correlating SDF ctDNA detection after surgery with recurrence outcomes and comparing SDF ctDNA with SDF TEX. This research has the potential to revolutionize postoperative management and adjuvant therapy personalization for head and neck cancer patients based on optimized SDF SOPs, thus improving survival outcomes and reducing treatment-related morbidity.
