Earland, Noah

Abstract Background: Spatial cellular ecosystems in the tumor microenvironment (TME) form dynamic signaling hubs that critically influence cancer disease progression and response to therapy, including response to immune checkpoint inhibitors (ICIs). However, tumor sampling bias and the impracticality of acquiring serial tumor biopsies have made it challenging to profile the TME clinically. To address this challenge, we developed Liquid EcoTyper, a multi-analyte AI framework to quantify TME spatial cellular ecosystems—termed spatial ecotypes (SEs)—noninvasively from cell-free DNA (cfDNA) methylation data. Methods: In previous work, we discovered nine spatial ecotypes, including SEs associated with ICI response (e.g., SE7 with benefit, SE4 with resistance), and quantified them from 1,249 bulk tumor RNA-seq profiles of melanoma, non-small cell lung cancer (NSCLC), and bladder cancer (BC) patients (Cancer Res (2025) 85 (8_Supplement_1): 153). Here we trained Liquid EcoTyper, an interpretable deep learning model, to transfer SE profiling to cell-free DNA using CpG methylation profiles. Performance was quantified using simulated and real data, including methylation profiles (EM-seq) of plasma cfDNA paired with tumor biopsy-derived SE levels determined by EM-seq (n=20 pairs) or 10x Visium (n=15 pairs). Clinical outcome prediction was evaluated using pretreatment plasma from patients treated with ICI alone (n=78 melanoma pts), chemotherapy and ICI (n=25 NSCLC pts), and enfortumab vedotin and ICI (n=10 localized muscle-invasive BC (MIBC) pts). Tumor mutational burden (TMB), tumor PD-L1, or circulating tumor DNA (ctDNA) were profiled as a comparator where possible. Results: We observed striking concordance between plasma-derived SE levels and tumor biopsy-confirmed SE levels from the same patients, demonstrating that liquid biopsy analysis of cfDNA methylomes can recapitulate TME spatial biology. Underscoring specificity, no reliable SE signal was detectable from peripheral blood mononuclear cells. In pre-ICI plasma, liquid SE levels were significantly associated with ICI-based response (SE7) versus resistance (SE4) in patients with melanoma, NSCLC, and MIBC, with a mean cross-cancer area under the curve (AUC) of 0.87 for SE7 (range 0.8-0.97) and 0.81 for SE4 (range 0.76-0.85)—far surpassing TMB, tumor PD-L1, and ctDNA in evaluable patients, both in binary analyses of response and multivariable models of survival. Our results also showed potential to predict pathologic complete response in the neoadjuvant setting. Conclusion: Liquid EcoTyper is a scalable noninvasive AI-based framework for spatiotemporal assessment of the TME. By enabling TME-informed risk stratification without surgical biopsies, Liquid EcoTyper has potential to enable more precise and actionable decision making in patients with solid tumors. Citation Format: Erin L. Brown, Wubing Zhang, Abul Usmani, Noah Earland, Ayesha Hashmi, Chibuzor Olelewe, Anushka Viswanathan, Pradeep S. Chauhan, Minji Kang, Chloé B. Steen, Hyun Soo Jeon, Susanna Avagyan, Irfan Alahi, Nicholas P. Semenkovich, Matteo Bergsagel, Janella C. Schwab, Chloe M. Sachs, Faridi Qaium, Peter K. Harris, Antonella Bacchiocchi, Qingyuan Cai, Andrew J. Gentles, Rondell P. Graham, Peter C. Lucas, Ryan C. Fields, Jacob J. Orme, Aaron S. Mansfield, Mario Sznol, Ruth Halaban, David Y. Chen, Aaron M. Newman, Aadel A. Chaudhuri. Liquid biopsy profiling of the tumor microenvironment to determine response to immunotherapy regimens across solid tumors [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 94.

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.

Background: Multicellular programs in the tumor microenvironment (TME) drive cancer pathogenesis and response to therapy but remain challenging to identify and profile clinically. Here, we present Spatial EcoTyper, a multimodal machine learning framework for large-scale profiling of spatially dependent cell states and multicellular ecosystems, termed spatial ecotypes (SEs). Methods and Findings: By integrating five million single-cell and spot-level spatial transcriptomes from 10 diverse human neoplasms, including carcinomas and melanomas, we identified a rich tapestry of conserved regional plasticity across nine TME cell types. We then applied Spatial EcoTyper to deeply dissect these data, revealing nine novel SEs with broad conservation - each with unique biology, TME cell states, geospatial features, and clinical outcome associations. SEs were linked to specific biogeographic domains, including the tumor core, margin, and adjacent stroma, and were remarkably well-validated in 235 held-out tumor samples profiled by either single-cell RNA sequencing, sequencing-based spatial transcriptomics (10x Visium), or high-resolution imaging-based spatial transcriptomics (Vizgen MERSCOPE and NanoString CosMx). By deconvolving SEs from over 1,200 bulk tumor RNA-seq profiles, we discovered striking associations between SE levels and response to immune checkpoint inhibitors (ICIs), outperforming nearly 40 potential correlates of ICI response, including previously described signatures of tertiary lymphoid structures and tumor tissue PD-L1 expression. Notably, baseline levels of a proinflammatory SE - localized to the tumor core and defined by a hub of unique T cell, macrophage, fibroblast, and endothelial cell states - strongly forecasted ICI benefit, while another SE - localized to the tumor margin and defined by myofibroblast and hypoxia-associated endothelial cell states - portended ICI resistance. Conclusion: Our data reveal fundamental units of TME organization and demonstrate a platform for large-scale profiling of spatial cellular ecosystems in any tissue, with implications for improved forecasting of immunotherapy response and the development of novel therapeutic strategies. Citation Format: Wubing Zhang,Erin L. Brown,Abul Usmani,Noah Earland,Hyun Soo Jeon,Irfan Alahi,Nicholas P. Semenkovich,Janella C. Schwab,Chloé B. Steen,Chloe M. Sachs,Bilge Gungoren,Faridi Qaium,Peter K. Harris,Jacqueline Mudd,Qingyuan Cai,Antonella Bacchiocchi,Ryan C. Fields,Mario Sznol,Ruth Halaban,David Y. Chen,Aadel A. Chaudhuri,Aaron M. Newman. High-resolution profiling of the tumor microenvironment with spatial ecotypes [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 153.

ABSTRACT Background Head and neck squamous cell carcinoma remains challenging due to high recurrence rates and poor survival outcomes. Developing precise technologies for disease burden assessment, treatment response, and minimal residual disease (MRD) surveillance is crucial for improving prognosis. Methods This review explores the potential of liquid biopsy for MRD and recurrence detection. A novel liquid TNM (LiTNM) staging system is introduced, integrating biomarkers from saliva, surgical drain lymphatic fluid (SLF), and peripheral blood. Results Proximal liquid biopsies, particularly saliva and SLF, offer advantages due to their proximity to the tumor microenvironment. Saliva demonstrates high sensitivity in HPV‐associated oropharyngeal cancers, while SLF holds potential in identifying early postoperative recurrence. Despite these advancements, standardization and validation remain challenges. Conclusions Liquid biopsy approaches show promise for postoperative disease monitoring, yet their clinical implementation remains in the early stages. The proposed LiTNM staging system could complement TNM staging by providing a molecular framework for risk stratification. However, rigorous prospective studies are necessary to validate its clinical utility and facilitate adoption.

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy globally, and the vast majority of cases are treated with primary surgical resection and adjuvant therapy. Our group has pioneered the use of HNSCC circulating tumor DNA (ctDNA) in surgical effluent collected postoperatively from surgical drains (surgical drain fluid, SDF) as a novel proximal bioanalyte for measuring minimal residual disease after surgery. We now profile the immune cell composition of SDF compared to paired peripheral blood mononuclear cells (PBMCs), and correlate SDF immune cell profiles with clinical variables and oncologic outcomes in HNSCC. Method: SDF and peripheral venous blood were collected 24 hours postoperatively from 58 HNSCC patients, including 11 HPV (+) and 47 HPV (-) patients undergoing standard of care surgical resection, lymph node dissection, and adjuvant therapy. Freshly collected SDF cells and PBMCs were cryopreserved until profiling by multicolor flow cytometry for respective leukocyte compositions. Analyses were used to define the following viable CD45+ leukocyte subsets: total T cells, CD4 T-cells CD8 T-cells, Treg cells, B-cells, non- classical monocytes, classical monocytes, natural killer (NK) cells, classical dendritic cells (cDC), plasmacytoid DC (pDC), macrophages, and monocytic myeloid-derived suppressive cells (MDSCs). Immune cell percentages were correlated with clinical characteristics and cancer recurrence. Results: CD45+ leukocytes comprised >99.5% of SDF cells, with very few EPCAM+ tumor cells (< 10^-4). HPV status did not significantly affect SDF immune cell composition, and no significant differences were noted by tumor site (larynx, oral cavity, oropharynx), or extracapsular lymph node extension. Among 13 HPV (-) HNSCC patients with paired peripheral blood drawn concurrently, SDF had a higher proportion of B-cells(16.8% vs. 9%, p=0.044), NK cells(56% vs. 12%, P<0.001), monocytes(14% vs. 1%, P<0.001), and macrophages(3.1% vs. 0.46%, P<0.001) compared to PBMCs, and a lower proportion of CD4+ T cells(1% vs. 24%, P<0.001), CD8+ T cells(0.6% vs. 18%, P<0.001), MDSCs(2.1% vs. 3.4%, P=0.029), and pDCs(0.1% vs. 0.2%, P=0.014). HPV (-) HNSCC patients that went on to recur (n=9) had a significantly higher proportions of SDF monocytes (total, classical, and non- classical) and CD56dimCD16+ NK cells (all P<0.001) compared to non-recurrent patients (n=38). Patients who went on to recur also had a significantly lower proportion of SDF B-cells (P<0.001), CD56bright NK cells(P=0.01), and macrophages (P=0.018) compared to non- recurrent cases. No significant differences were noted with respect to proportions of CD3+, CD4+, CD8+ T-cells, DC, and MDSC cells. Conclusion: SDF from HNSCC patients contains unique immune cell populations and may provide new insights into postoperative tumor immunity. Differences in SDF immune cell composition by oncologic outcome in patients that go on to recur, highlighting potential prognostic and predictive applications of SDF immune cell profiling for adjuvant therapy decision-making. Citation Format: Zhongping Xu, Noah Earland, Lucien Khalil, Lazar Vujanovic, Danny Azmi Elias, Riyue Bao, Jason J. Luke, Aadel A. Chaudhuri, Jose P. Zevallos. Immune cell composition in surgical drain fluid as a novel prognostic marker in head and neck squamous cell carcinoma [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 B031.

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.