Whitfield, Damion

Abstract Purpose: Relapse is a major cause of failure in human papillomavirus–independent head and neck squamous cell carcinoma. Clinicopathologic criteria for adjuvant treatment remain imprecise and have not changed for decades. We investigated whether ctDNA in lymphatic exudate collected via surgical drains (“lymph”) 24 hours after surgery identified molecular residual disease (MRD) and compared its performance with time-matched plasma. Experimental Design: Using an ultra-sensitive tumor-informed sequencing approach, tumor variants were called in lymph and plasma to classify patients as ctDNA positive or ctDNA negative, trained in an initial cohort of 36 patients, and replicated in an independent cohort of 37 patients. Progression-free survival was compared in ctDNA(+) versus ctDNA(–) patients. Results: Lymph identified MRD in two independent multi-site cohorts (initial cohort sensitivity = 76% and specificity = 63%; log-rank P = 0.01; replication cohort sensitivity = 65% and specificity = 70%; log-rank P = 0.04). Lymph performance was enhanced in locoregional relapse (sensitivity = 78% and specificity = 67%; log-rank P = 0.0004) and generalized to early-stage patients. Analysis of matched plasma collected at this early timepoint was not predictive of recurrence (sensitivity = 35% and specificity = 72%; log-rank P = 0.7). In patients with intermediate-risk pathology, lymph ctDNA was associated with recurrence (sensitivity = 88% and specificity = 67%; log-rank P = 0.0008), suggesting an opportunity for improved stratification of patients who may benefit from additional adjuvant treatment. Conclusions: Postoperative lymph is a novel, proximal, and early source of MRD with the potential to introduce more precision into adjuvant therapy decision-making and improve outcomes, especially for patients with intermediate-risk human papillomavirus–independent head and neck squamous cell carcinoma.

Abstract Recurrence after surgery remains a major cause of failure in head and neck squamous cell carcinoma (HNSCC), particularly for HPV-negative patients whose 2-year failure rate is > 50%. There is unmet need for an accurate diagnostic test that predicts risk of recurrence prior to adjuvant therapy selection. We evaluated ctDNA in lymphatic exudate (“lymph”) collected via surgical drains at 24 hours to detect molecular residual disease (MRD) and compared its performance to plasma and adverse pathology features. Lymph, plasma and blood were collected from 76 HPV- HNSCC patients 24 hours after surgery. Tumor tissue was collected at surgery. 73 patients passed QC. Samples were sequenced using a 699-gene custom panel to a median of 6322x (lymph and plasma cfDNA) or 533x (tumor and blood genomic DNA). Tumor variants were directly genotyped in lymph and plasma using a tumor-informed pipeline. MRD status was determined using a calibrated cutoff based on mean variant allele fraction. Wilcoxon signed-rank test was used for comparisons. Kaplan-Meier estimator with log-rank test and Cox proportional hazards model were used for survival analysis. We previously demonstrated that 24 hour postoperative lymph ctDNA detects recurrence in HPV(-) HNSCC across two independent multi-site cohorts and by orthogonal sequencing approaches (sensitivity (SN) = 72%, specificity (SP) = 65%, p = 0.003, hazard ratio (HR) = 3.1, n = 73). When compared to other data available at this early time point (pathology and 24 hour postoperative plasma), lymph ctDNA outperformed each for recurrence detection (extranodal extension (ENE, p = 0.30), lymphovascular invasion (p = 0.02), perineural invasion (p = 0.38), positive lymph nodes (p = 0.03), plasma (p = 0.8)). We next evaluated lymph ctDNA in a critical subpopulation: patients considered intermediate risk by pathology (negative for ENE or margins but with one or more other adverse pathologic feature). Current NCCN guidelines recommend radiotherapy and to “consider systemic therapy” for these patients. Lymph ctDNA accurately stratified recurrence in this group with high sensitivity (SN = 88%, SP = 69%; P = 0.013, HR = 9.1, n = 24). Within this cohort, 77% of patients who received RT alone and later recurred were positive for lymph ctDNA at 24 hours, suggesting an opportunity to use molecular data in concert with pathology to identify patients who may benefit from adjuvant treatment intensification. Postoperative lymph represents a novel proximal analyte for MRD detection in HPV- HNSCC designed specifically for use in the immediate post-surgical window when adjuvant therapy decisions must be made. Lymph ctDNA is significantly associated with recurrence and outperforms both time-matched plasma and pathology. Accurate MRD identification at this early timepoint has the potential to augment traditional pathology and personalize adjuvant treatment paradigms in HPV-negative HNSCC. Citation Format: Seka Lazare, Zhuosheng Gu, Noah Earland, Adam Harmon, Maciej Pacula, Megan Long, Ashley Tellis, Damion Whitfield, Adam Benson, Lucien Khalil, Robert L. Ferris, Marra Francis, Aadel A. Chaudhuri, Jose P. Zevallos, Wendy Winckler. Postoperative lymphatic exudate in HPV-negative head and neck cancer detects recurrence prior to adjuvant treatment decisions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1946.