Winckler, Wendy

Abstract Our team has demonstrated that lymphatic exudate collected via surgical drains (“lymph”) is a proximal biofluid rich in ctDNA and prognostic of recurrence in HPV-independent Head and Neck Cancer (HPV- HNSCC). Other analytes such as proteins, metabolites, and cells are also abundant in lymph. To understand the liquid tumor microenvironment (TME) immediately after tumor resection, we characterized lymph at 24 hrs in HNSCC. We performed a multi-omic survey, demonstrating that lymph is distinct from plasma and represents a largely unexplored source of biomarkers relevant to immune modulation and metastasis.Metabolomics and proteomics were run on lymph supernatant and plasma. Proteomic screening was performed via Olink and normalized to internal standards for quantitative analysis. Metabolomic samples were assayed via LC-MS and normalized. 16s libraries were sequenced utilizing Illumina and analyzed in QIIME. 16s sequencing on 6 patients indicated an array of bacterial genera present in post-surgical lymph with consistency across patients. The distribution consisted primarily of microbes native to the oral cavity, suggesting post-surgical lymph may reflect the native microbiome prior to surgery. Pathogenic blooms were observed in a subset of patients. Ongoing analysis will correlate microbiome data with both cancer and surgical outcomes.Metabolite profiling was performed on post-surgical lymph and matched plasma from 4 patients, including 3 who recurred within 1 year of surgery. The metabolite profile in lymph of all 4 patients was more diverse than plasma with higher metabolite abundance per mL. 13% of identified metabolites were >2-fold enriched in lymph. Lymph from recurred patients showed high levels of saturated and monounsaturated fatty acids, reported to be associated with metastasis. Post-surgical lymph has a unique and largely uncharted metabolome to mine for biomarkers associated with recurrence.Proteomes were compared between lymph and plasma from 44 HPV-HNSCC patients. This cohort consists of 25 recurred (REC) and 19 patients with no evidence of disease (NED) with >1 year of follow up. Lymph and plasma displayed highly non-overlapping protein repertoires. The mean absolute volcano score was 25.2, indicating a strong global difference between lymph and plasma. Lymph was significantly enriched in 91 proteins (p range 4.1e-47 to 0.037), many associated with inflammation. REC lymph was also enriched in inflammatory markers compared to NED, including IL-1a, TNF, and TSLP (p = 0.002, 0.015, 0.003, respectively), which have been implicated in cancer metastasis, invasion, and angiogenesis. Lymph hits were largely non-overlapping with plasma, indicating the opportunity for novel biomarker discovery in this fluid.Analysis of the microbiome, metabolome, and proteome of post-surgical lymph reveals a rich and differentiated tumor-adjacent biofluid to study biomarkers relevant to metastasis and the TME. Citation Format: Zachary Costliow, Seka Lazare, Zhuosheng Gu, Adam Harmon, Maciej Pacula, Megan Rivera, Aadel A. Chaudhuri, Jose P. Zevallos, Jessalyn Marie Ubellacker, Wendy Winckler. Multi-omicsinpostoperative lymphatic exudate in HPV-negative head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6137.

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.

Abstract Introduction: Recurrence after tumor resection is a major cause of failure in HPV-negative head and neck squamous cell carcinoma (HNSCC), with up to 50% of patients recurring within 2 years. There is unmet need for an accurate diagnostic test that can predict risk of recurrence prior to adjuvant therapy selection. We previously demonstrated that ctDNA in lymphatic exudate collected via surgical drains (“lymph”) accurately identifies molecular residual disease (MRD) and outperforms plasma in HNSCC patients using a targeted sequencing (TS) approach. While TS assays are robust and sensitive, they have key limitations, namely limited ctDNA features, higher cost, longer turnaround times, and lower throughput. Here we evaluate whole genome sequencing (WGS) for detection of MRD in both lymph and plasma sampled 24 hours after surgery. Methods: Lymph, plasma, and whole blood were collected 24 hours postoperatively from 30 HPV-negative HNSCC patients (stages I – IV) along with resected tumor. To detect ctDNA, we utilized WGS analyses of patient matched tumor, germline (blood), and cfDNA from lymph or plasma samples at approximately 150x, 30x, and 30x depth, respectively. Tumor- specific single nucleotide variants were identified from tumor and germline datasets, from which a candidate variant set was used to determine the presence of ctDNA within lymph or plasma DNA through a random forest machine learning model. ctDNA status was determined based on the level of signal compared to a reference population of noncancerous donor plasma samples (n=80). 5 tumors and 1 lymph sample failed QC; in total, 24 patients remained, yielding 10 patients with disease recurrence and 14 with no evidence of disease with >1 year of follow-up. The Kaplan-Meier (KM) estimator with log-rank test and Cox proportional-hazards model were used for survival analyses. Results: KM survival analyses indicated that WGS of lymph ctDNA accurately predicts recurrence (sensitivity (SN) = 50%, specificity (SP) = 93%; p = 0.003, Hazard ratio (HR) = 6.0), consistent with previously published results using a TS MRD approach in this cohort (SN = 80%, SP = 64%, p = 0.02, HR = 5.3). WGS of lymph ctDNA outperformed plasma at this timepoint (SN = 30%, SP = 93%, p = 0.03, HR = 4.2). WGS of lymph ctDNA also showed enhanced performance in locoregional relapse (n=21, SN = 57%, SP = 93%; p = 0.006, HR = 6.7). Finally, the ctDNA fraction of proximal lymph was higher than paired plasma in all locoregional recurrences (n=7), while peripheral plasma had a higher ctDNA fraction in 2 out of 3 distant recurrences. Conclusions: Tumor-informed WGS analysis of lymph ctDNA represents a robust MRD approach in HPV-negative HNSCC. Lymph outperforms time-matched plasma for prediction of recurrence, particularly in patients with locoregional relapse. Accurate MRD identification in the immediate post-surgical timeframe suggests that lymph analysis has potential to augment pathology and longitudinal plasma testing to provide more personalized adjuvant treatment decision-making in patients with HPV-negative HNSCC. Citation Format: Zhuosheng Gu, Seka Lazare, Noah Earland, Marra S Francis, Adam Harmon, Megan Long, Amy Greer, Ellen Verner, Andrew Georgiadis, Mark Sausen, Shakti Ramkissoon, Jose P Zevallos, Aadel A Chaudhuri, Taylor J Jensen, Wendy Winckler. Tumor-informed whole genome sequencing of ctDNA from lymph and plasma detects molecular residual disease in HPV-negative head and neck cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A043.