Abstract Purpose: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer globally, representing roughly 6% of all malignancies. Each year it accounts for an estimated 660,000 new diagnoses and over 325,000 deaths, with incidence projected to rise by nearly 30% by 2030. Despite advances in surgery, radiation, and immunotherapy, patients with HPV-negative HNSCC continue to face poor outcomes, with five-year overall survival rates often below 55%. Adoptive cell therapy (ACT) in solid tumors remains limited by the scarcity of tumor-reactive T cells and their functional exhaustion after expansion. The sentinel lymph node (SLN), the initial site of tumor antigen presentation and T-cell priming, may provide a rich and minimally exhausted source of tumor-specific lymphocytes. Here, we evaluated the immunologic and therapeutic advantages of SLN-derived leukocytes over those from tumor tissue in an orthotopic model of HPV- HNSCC. Methods: CD45+ immune cells isolated from donor SLNs, NSLNs, tumors, or SLNs preconditioned with low-dose tdRT (4 Gy) in the 4MOSC1 murine model were adoptively transferred into syngeneic tumor-bearing recipients to evaluate their therapeutic potential. Recipient groups included untreated controls, preconditioned mice with 4 Gy tdRT, or a single PD-1 blockade (PD1i) dose before ACT. Phenotypic and functional characterization was performed using flow cytometry, multiplex immunofluorescence, ELISA, and CITE-seq to evaluate cell populations, activation, exhaustion, clonal overlap, and tissue distribution. Results: SLN-CD45+ populations were enriched in activated CD4+/CD8+ T cells with elevated CD69, CD137, and IFN-γ expression compared with tumor-derived counterparts. mIHC confirmed higher densities of activated T cells in SLN and tumor regions after SLN-CD45+ transfer. TCR analysis revealed substantial overlap between SLN and tumor clonotypes, suggesting selective enrichment of tumor-reactive clones. Functionally, ACT with SLN-CD45+ cells induced greater tumor regression and survival benefits than ACT from tumor source, even without lymphodepletion or expansion in culture. Notably, cryopreserved SLN-derived cells retained comparable efficacy to freshly isolated ones. Preconditioning recipients with 4 Gy tdRT or PD1i further improved intratumoral infiltration, activation, and persistence of transferred cells without added toxicity. Conclusions: The sentinel lymph node provides a potent, readily accessible reservoir of tumor-reactive immune cells for ACT. Combining SLN-derived ACT with tdRT and PD1i significantly enhances therapeutic efficacy in HPV- HNSCC. These findings highlight the translational potential of leveraging tumor-draining lymphoid niches as tumor-primed immune reservoirs for next-generation ACT in solid tumors. Citation Format: Pardis Mohammadzadeh, Tomoya Kurokawa, Kelsey Decker, Peter Vo, Prakriti Sen, Riley N. Jones, Sayuri Miyauchi, Santiago Fassardi, Anais Zourelidis, Robert Saddawi-Konefka, Joseph A. Califano, . Sentinel lymph node-derived CD45+ cells combined with low-dose radiotherapy and PD-1 blockade as a next-generation adoptive cell therapy in 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 3703.

Use of quantitative polymerase chain reaction (qPCR) to measure viral DNA in plasma has promise for the detection and/or monitoring of high-risk human papillomavirus (hrHPV)-associated cancers. We examined Roche's cobas 6800 HPV test for detection of hrHPV in plasma or peripheral blood mononuclear cells (PBMC) of patients with HPV-associated dysplasia or cancer. In our pilot study, this assay detected hrHPV DNA in 1 mL pre-treatment plasma of patients with advanced (stage II-IV) cervical cancer, but not stage I or dysplasia cases, or in PBMC. Plasma of oropharyngeal cancer patients also tested positive in most cases. Seeking to enhance sensitivity, we re-tested these pilot samples with qPCR employing three published primer sets (E7, BR E1-5, HR E5L2-4) selected for improved detection of HPV16 in cancer. This triplex assay detected quantifiable levels of HPV16 in pre-treatment plasma of advanced (stage II-IV) cervical cancer cases, and lower levels in some stage I and dysplasia cases. In serial sampling of HPV16 + cervical cancer patients post-treatment with no evidence of disease over ~2 years, no reproducible signal was detected by cobas HPV testing, whereas the triplex qPCR sporadically detected HPV DNA in plasma and PBMC samples of a subset of these patients.

Abstract Introduction: Extrachromosomal DNA (ecDNA) contributes to oncogene amplification in human cancers, yet the mechanisms underlying the formation and function of human-viral hybrid ecDNA (hybrid ecDNA), circular elements harboring both HPV and human sequences, remain unclear. We recently identified hybrid ecDNA in human papillomavirus-associated oropharyngeal cancer (HPVOPC), a rapidly increasing malignancy. Here, we show that de novo enhancer formation at viral-human junctions activates viral oncogenes and promotes hybrid ecDNA self-amplification, revealing a tractable therapeutic vulnerability. Methods: Hybrid ecDNA was identified in HPVOPC cell lines and patient-derived xenografts (PDX) using whole-genome sequencing and AmpliconArchitect. Validation was performed by multicolor FISH. Chromatin accessibility and enhancer activity were profiled by ATAC-seq and H3K27ac ChIP-seq. Hi-C sequencing was used to assess 3D chromatin interactions and potential contacts among distinct ecDNA species. Functional significance was evaluated by CRISPR interference (CRISPRi) targeting ecDNA-derived enhancers. Therapeutic relevance was assessed by BET inhibition in vitro and in vivo. Results: Hybrid ecDNA was detected in both cell lines and PDX tumors and validated by overlapping human and HPV FISH signals. Epigenomic profiling revealed de novo active enhancers flanking HPV sequences exclusively in hybrid ecDNA(+) tumors. Hi-C demonstrated enhancer-promoter loops linking host enhancers to HPV oncogenes. CRISPRi targeting these enhancers significantly inhibited proliferation in hybrid ecDNA(+) models only (P = 0.006). BET inhibition selectively suppressed hybrid ecDNA(+) tumor growth in vivo (P = 2×10-5). Importantly, Hi-C contacts also indicated enriched interaction patterns among distinct ecDNA species, suggesting potential extrachromosomal cooperation that enhances viral oncogene expression. Conclusions: Hybrid ecDNA is a functionally critical structure in HPVOPC by generating de novo enhancers and reorganizing chromatin interactions to activate viral oncogenes, creating a therapeutic vulnerability. Contact enrichment among distinct ecDNA species further suggests participation in a broader extrachromosomal regulatory network in HPV-driven malignancies. Citation Format: Takuya Nakagawa, Jens Luebeck, Kaiyuan Zhu, Sasik Roman, Brin Rosenthal, Kathleen Fisch, Toyoyuki Hanazawa, Atsushi Kaneda, Paul S. Mischel, Vineet Bafna, Joseph A. Califano. De novo enhancer formation at human-viral junctions drives hybrid extrachromosomal DNA amplification in HPV-associated 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 1925.

Abstract Emerging evidence highlights the potential of lymphatic-sparing strategies in head and neck squamous cell carcinoma (HNSCC), as well as the sentinel lymph node (SLN) as a critical immunological niche1-3. Within this context, CCR7+ dendritic cells (DCs) have been identified as an essential subpopulation for the response to immunoradiotherapy4. Here, we investigated the therapeutic effect of intranodal CCR7 agonists CCL19/21 into the SLN using a sustained-release fibrin gel-conjugate system in the syngeneic, orthotopic, partially programmed death-1 inhibitor (PD-1i) sensitive 4MOSC1 model of HNSCC5, aiming to enhance DC function along the tumor-SLN axis. C57Bl/6 mice were injected with 4MOSC1 cells in the buccal space and underwent SLN mapping and injection of CCL19/21 conjugated (con.) to fibrinogen intranodal (i.n.), CCL19/21 unconjugated (uncon.) to fibrinogen i.n., PD-1i intraperitoneal (i.p.), CCL19/21 con. to fibrinogen i.n. + PD-1i i.p., or sham surgery following 6 days after tumor implantation. Animals were monitored for tumor volume and survival. Primary tumors were assayed by flow cytometry. Intranodal con. CCL19/21 led to a significantly reduced tumor growth (p= <0,0001 vs. sham) and complete response (CR) rate of 60%, whereas uncon. CCL19/21 i.n. (p=0,3354 vs. sham) and sham groups showed no CR. Combination of con. CCL19/21 + PD-1i significantly improved overall survival with 80% survival by day 34, compared to 60% for con. CCL19/21 i.n., and 0% for uncon. CCL19/21 i.n. and controls. Flow cytometric analysis 48 h following treatment shows enhanced DCs and enhanced activated DCs in tumors in con. CCL19/21 treated mice. Intranodal delivery of CCR7 agonists CCL19/21 significantly enhances DCs and activated DCs in tumors, reduces tumor growth and prolongs survival in the HNSCC 4MOSC1 mouse model. 1. Delclaux I et al. 2024;10(1):28-37.2. Saddawi-Konefka R et al. 2022;13(1):4298.3. Cochran AJ et al. 2006;6(9):659-670.4. Saddawi-Konefka R et al. 2025;16(1):6578.5. Wang Z et al. 2019;10(1):5546. Citation Format: Anais Aurelia Zourelidis, Jonathan Gunn, Tomoya Kurokawa, Sayuri Miyauchi, Kelsey Decker, Peter Vo, Pardis Mohammadzadeh, Jason Wu, Ester J. Kwon, Joseph A. Califano. Intranodal chemokine therapy targeting the sentinel lymph node in head and neck squamous cell carcinoma [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 448.

6016 Background: Standard chemoradiation (CRT) for unfavorable risk p16+ head and neck squamous cell carcinoma (HNSCC) is associated with high rates of toxicity and treatment failure. We hypothesized that concurrent and adjuvant pembrolizumab (pembro) could achieve superior PFS with acceptable toxicity compared to CRT in this population. Methods: KEYCHAIN was an open-label randomized phase II trial conducted at 6 US academic centers. Eligible patients had newly diagnosed unresected p16+ HNSCC with unfavorable risk (AJCC 8 th edition stage II-III oropharynx or stage III-IVB non-oropharynx HNSCC). Patients were randomized 1:1 to RT (70 Gy in 35 fractions) plus pembro (200 mg, 2 weeks prior to RT then every 3 weeks starting day 1 of RT up to 20 total cycles) or RT plus concurrent cisplatin (cis) (100 mg/m 2 every 3 weeks). Age, ECOG status, and oropharynx site were stratification factors. The primary endpoint was PFS, defined as time from randomization to first progression or death from any cause. Primary analysis was in the modified intent-to-treat population, including all patients having ≥ 1 dose of study medication and ≥ 1 efficacy evaluation after baseline. The study design had 80% power with 1-sided α = 0.15 (log-rank test) and planned sample size of 50 analyzable subjects per arm. The study is registered with clinicaltrials.gov (NCT03383094). Results: Between Feb 2019 and Aug 2025, 108 patients were randomized (53 pembro, 55 cis), with 102 analyzable (50 pembro, 52 cis). Median follow-up was 26.5 months. 79% had minimum follow-up of 2 years. At 2 years, there were 7 PFS events and 1 death in the pembro arm, and 14 PFS events and 7 deaths in the cis arm. Two-year PFS was 84% (95% CI: 74-96%) vs. 70% (95% CI: 58-85%) in the pembro vs cis arm (HR 0.57, 95% CI: 0.25-1.31; 1-sided p = 0.09) (see Table). Two-year OS was 98% (95% CI: 94-100%) vs. 85% (95% CI: 76-96%) for pembro vs. cis (HR 0.33, 95% CI: 0.09-1.28; 1-sided p = 0.048). HRs adjusted for T3-4 and N2-3 category were 0.68 (PFS) and 0.31 (OS). As of the latest follow-up, there were 24 total PFS events and 12 deaths. Grade ≥ 3 adverse events definitely or probably related to treatment were 36% vs. 46% in the pembro vs. cis arm. The median number of cycles received in the pembro arm was 18. In the cis arm, 94% completed ≥ 2 cycles. Results by CPS status will be reported when available. Conclusions: Pembro plus RT met the pre-specified significance criterion for improvement in PFS compared to cisplatin plus RT in this phase II trial. A phase III trial of RT with pembrolizumab for unfavorable risk p16+ HNSCC is warranted. Clinical trial information: NCT03383094 . Pembro Cisplatin N 50 52 Mean age 63.5 62.5 Male 87% 89% White 85% 89% Age > 65 42% 35% ECOG 0 83% 70% Oropharynx 94% 96% T1 8% 10% T2 22% 12% T3 38% 38% T4 32% 40% N0 6% 8% N1 42% 29% N2 46% 54% N3 6% 10% 2-yr PFS 84% 70% 2-yr OS 98% 85% Completed RT w/in 56 days 96% 92% Grade ≥ 3 mucositis 18% 17% Grade ≥ 2 mucositis 72% 63% Grade ≥ 3 dysphagia 14% 6% Grade ≥ 2 dysphagia 48% 35%

Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HPV+ HNSCC) is one of the most rapidly increasing cancers in the United States. Although patients with HPV+ HNSCC exhibit better responses to chemoradiation compared with patients with HPV- HNSCC, a subset still develops recurrent/refractory disease. In this study, we investigated the role and mechanisms by which the HPV E5 oncogene promotes resistance to conventional chemotherapy and radiation. After treatment with chemotherapy and radiation, HNSCC cells expressing HPV E5 had decreased apoptosis, improved cell viability, enhanced DNA damage response, and increased tumor growth in murine xenograft models when compared with empty vector controls. Transcriptomic analysis showed an enrichment of signaling pathways associated with epithelial development and increased expression of stem cell markers, including SOX9 and SOX4, in HPV E5 cells compared with empty vector cells. Knockdown of SOX9 restored sensitivity to chemo- and/or radiotherapy in HPV E5-expressing cells. In patients with HNSCC, RNA sequencing analysis showed that high expression of HPV E5 was associated with enrichment of pathways involved in cellular senescence and stem cell pluripotency. Furthermore, an HPV E5-associated gene set was identified that correlates with poor patient prognosis and is significantly overexpressed in recurrent HPV+ HNSCC tumors. Taken together, these data identify HPV E5 as a viral mediator of resistance to standard-of-care therapies for HPV+ HNSCC by promoting a stem cell-like phenotype that enhances DNA damage responses and tumor cell survival. Thus, HPV E5 may contribute to chemotherapy resistance, tumor recurrence after radiation, and poor prognosis of patients with refractory or metastatic disease.Significance: HPV E5 upregulates SOX9 to induce stem cell-like characteristics in head and neck cancer that drive resistance to chemotherapy and radiation, providing rationale for developing therapeutic agents targeting HPV E5.

Surgical ablation or broad radiation of tumor-draining lymph nodes can eliminate the primary tumor response to immunotherapy, highlighting the crucial role of these nodes in mediating the primary tumor response. Here, we show that immunoradiotherapy efficacy is dependent on treatment sequence and migration of modulated dendritic cells from tumor to sentinel lymph nodes. Using a tamoxifen-inducible reporter paired with CITE-sequencing in a murine model of oral cancer, we comprehensively characterize tumor immune cellular migration through lymphatic channels to sentinel lymph nodes at single-cell resolution, revealing a unique immunologic niche defined by distinct cellular phenotypic and transcriptional profiles. Through a structured approach of sequential immunomodulatory radiotherapy and checkpoint inhibition, we show that sequenced, lymphatic-sparing, tumor-directed radiotherapy followed by PD-1 inhibition achieves complete and durable tumor responses. Mechanistically, this treatment approach enhances migration of activated CCR7+ dendritic cell surveillance across the tumor-sentinel lymph node axis, revealing a shift from their canonical role in promoting tolerance to driving antitumor immunity. Overall, this work supports rationally sequencing immune-sensitizing, lymphatic-preserving, tumor-directed radiotherapy followed by immune checkpoint inhibition to optimize tumor response to immunoradiotherapy by driving activated dendritic cells to draining sentinel lymph nodes.

BACKGROUND:Limited understanding of the biology predisposing certain human papillomavirus-related (HPV+) oropharyngeal squamous cell carcinomas (OPSCCs) to relapse impedes therapeutic personalization. We aimed to identify molecular traits that distinguish recurrence-prone tumors. METHODS:Fifty HPV+ OPSCCs that later recurred (cases) and 50 nonrecurrent controls matched for stage, therapy, and smoking history were RNA-sequenced. Groups were compared by gene set enrichment analysis, and select differences were validated by immunohistochemistry. Features discriminating groups were scored in each tumor using gene set variation analysis, and scores were evaluated for recurrence prediction ability. RESULTS:Cases downregulated pathways linked to antitumor immunity (FDR-adjusted P < .05) and contained fewer tumor-infiltrating lymphocytes (P < .001), including cytotoxic T-cells (P = .005). Cases also upregulated pathways related to cell division and other aspects of tumor progression. Upregulated and downregulated pathways were respectively used to define a tumor progression score (TPS) and immune suppression score (ISS) for each tumor. Correlation between TPS and ISS (r = .603, P < .001) was potentially explained by observed upregulation of DNA repair pathways in cases, which might enhance their progression directly and by limiting cytosolic DNA-induced inflammation. Accordingly, cases contained fewer double-strand breaks based on staining for phospho-RPA32 (P = .006) and γ-H2AX (P = .005) and downregulated the cytosolic DNA sensing pathway. A combined score derived from TPS and ISS optimized recurrence prediction and stratified survival in a manner generalizable to 3 external cohorts. CONCLUSIONS:We describe a potential link in HPV+ OPSCCs between reduced DNA damage and other tumor-intrinsic and immune-related contributors to recurrence risk, opening opportunities to detect and target this high-risk biology.

Immune checkpoint inhibition has demonstrated no benefit when combined with chemoradiotherapy for locally advanced HNSCC, raising the possibility that standard therapies compromise the response to immunotherapy. We previously demonstrated that ablating tumor draining lymphatics abolishes the response to immune-oncology therapy. Specifically, we defined that the response to immunotherapy is coordinated by the cDC1 population in tumor-draining lymph nodes. These findings support the premise that successful tumor responses to immuno-oncology therapies is predicated upon intact and functional, locoregional anatomy. Accordingly, we hypothesize that lymphatic-preserving immune oncology therapy will promote antitumor immunity by enhancing surveillance along the tumor-immune-lymphatic axis. To explore this, we employ a novel mouse reporter model that allows precise spatiotemporal labeling of immune effector cells. Combined with sentinel lymph node mapping, our reporter model enables selective labeling of immune effectors within the tumor microenvironment and subsequent tracking and characterization of these cells as they migrate to the sentinel lymph node. This approach led us to define the “cancer immune migratome” - the repertoire of immune cells actively migrating from tumors to lymph nodes during an antitumor response. For precise cellular identification, we employed CITE-sequencing, which integrates single-cell RNA expression with surface protein markers, allowing us to comprehensively define the characteristics of migrating immune cells. This work represents the first characterization of immune cell populations migrating from the tumor to the sentinel lymph node, revealing the diversity and dynamics of these populations in antitumor immunity. We then develop a tumor-directed, lymphatic-sparing radiotherapy model to modulate the tumor immune microenvironment without inducing tumor regression. Sequential administration of tumor-directed radiotherapy followed by PD-1 checkpoint inhibition achieves complete and durable responses. Analysis of the cancer immune migratome during immunoradiotherapy revealed a crucial role for an activated population of CCR7+ dendritic cells. These migratory dendritic cells are reprogrammed, via rational sequencing of radiation and immunotherapy, from their canonical role in immune tolerance to actively drive antitumor immunity. This reprogramming enhances T cell priming, clonotypic expansion, and, ultimately, the successful tumor response to immunoradiotherapy. Disruption of sentinel lymphatic channels or selective blockade of CCR7+ DC entry into the SLN abrogates the response to immunoradiotherapy. Overall, this work supports rationally sequencing lymphatic-preserving radiotherapy followed by immune checkpoint inhibition to optimize tumor response. Robert Saddawi-Konefka, Riyam Al Msari, Cynthia Tang, Chad Philips, Sayed Sadat, Lauren M. Clubb, Sarah Luna, Santiago Fassardi, Riley Jones, Ida Franiak Pietryga, Farhoud Faraji, Shiruyeh Schokrpur, Bryan S. Yung, Michael M. Allevato, Chanond A. Nasamran, Daisy Chilin- Fuentes, Sara Brin Rosenthal, Shawn M. Jensen, Bernard A. Fox, Richard Bell, J. Silvio Gutkind, Andrew Sharabi, Joseph A. Califano. Mapping the tumor-sentinel node immune migratome demonstrates a key role for CCR7+ dendritic cells in the successful response to immunoradiotherapy [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 3764.

e18062 Background: Head and neck squamous cell carcinoma (HNSCC) is a significant global health concern, with over 60,000 new cases and approximately 14,000 deaths annually in the United States. Major risk factors include tobacco and alcohol use, as well as human papillomavirus (HPV) infection, which accounts for a growing subset of cases. Concurrently, the rising prevalence of cannabis use—reported in over 18% of U.S. adults—has raised concerns about its role in cancer pathogenesis. THC (Δ9-tetrahydrocannabinol), the primary psychoactive component of cannabis, interacts with CB1 and CB2 receptors, often overexpressed in cancers like HNSCC. While background studies have shown anti-tumor effects of THC, such as apoptosis and reduced metastasis in some preclinical models, its immunosuppressive properties and interactions with tumor biology remain concerning. Prior research highlights THC’s potential to promote tumor progression and metastasis in HPV-negative HNSCC through activation of oncogenic pathways, including p38 MAPK signaling. Epidemiological data further suggest a 25% increased risk of HNSCC among chronic cannabis users, underscoring the need for detailed investigation into its role in HPV-negative tumor progression. Methods: The pro-tumorigenic effects of THC at a recreational dosage (1 µg/mL) were assessed using HPV-negative head and neck squamous cell carcinoma (HNSCC) cell line JHU011. Cell proliferation was evaluated through Aqua Bluer assay, while the impact on cell migration and invasion was determined using wound healing and trans-well migration/invasion assays. THC-mediated activation of the ERK and p38 MAPK signaling pathways was analyzed through ELISA and Western blot. Additionally, the in vivo effects of THC on tumor growth were investigated using an immunocompetent lateralized oral cavity tumor model. In this model, 4MOSC1 cells were orthotopically injected into the buccal mucosa of C57BL/6 mice, serving as an HPV-negative HNSCC model. Results: Our findings indicate that treatment with 1μM THC promotes migration and invasion of HPV-negative HNSCC cell JHU011, without affecting cell proliferation. Additionally, THC treatment activated the p38 MAPK pathway as well as ERK MAPK pathway in these cells. In vivo studies involving daily administration of 1μM THC demonstrated no significant effect on tumor growth compared to controls in 4MOSC1 HPV-negative HNSCC model using immunocompetent animals. Conclusions: THC at recreationally relevant doses promotes cell migration and invasion in HPV-negative HNSCC via p38 and ERK MAPK activation without affecting proliferation or in vivo tumor growth. These findings underscore the complex role of THC in cancer biology and warrant further investigation into its implications for HNSCC progression.

Differential gene expression and pathway analysis

Prognosis of HNSCC patients based on the status of E5 signature and expression distribution of E5 in single cell RNA-sequencing dataset

Tumor initiation represents the first step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Capturing this process as it occurs in vivo, however, remains elusive. Here we employ cell tracing approaches with spatiotemporally controlled oncogene activation and tumor suppressor inhibition to unveil the processes underlying oral epithelial progenitor cell reprogramming into cancer stem cells (CSCs) at single cell resolution. This revealed the rapid emergence of a distinct stem-like cell state, defined by aberrant proliferative, hypoxic, squamous differentiation, and partial epithelial to mesenchymal (pEMT) invasive gene programs. Interestingly, CSCs harbor limited cell autonomous invasive capacity, but instead recruit myeloid cells to remodel the basement membrane and ultimately initiate tumor invasion. CSC transcriptional programs are conserved in human carcinomas and associated with poor patient survival. These findings illuminate the process of cancer initiation at single cell resolution, thus identifying candidate targets for early cancer detection and prevention.

The discovery of tumor-derived neoantigens which elicit an immune response through major histocompatibility complex (MHC-I/II) binding has led to significant advancements in immunotherapy. While many neoantigens have been discovered through the identification of non-synonymous mutations, the rate of these is low in some cancers, including head and neck squamous cell carcinoma. Therefore, the identification of neoantigens through additional means, such as aberrant splicing, is necessary. To achieve this, we developed the splice isoform neoantigen evaluator (SINE) pipeline. Our tool documents peptides present on spliced or inserted genomic regions of interest using Patient Harmonic-mean Best Rank scores, calculating the MHC-I/II binding affinity across the complete human leukocyte antigen landscape. Here, we found 125 potentially immunogenic events and 9 principal binders in a cohort of head and neck cancer patients where the corresponding wild-type peptides display no MHC-I/II affinity. Further, in a melanoma cohort of patients treated with anti-PD1 therapy, the expression of immunogenic splicing events identified by SINE predicted response, potentially indicating the existence of immune editing in these tumors. Overall, we demonstrate SINE’s ability to identify clinically relevant immunogenic neojunctions, thus acting as a useful tool for researchers seeking to understand the neoantigen landscape from aberrant splicing in cancer.

e18061 Background: Prior smaller-scale studies have demonstrated elevated salivary CD44 and total protein (TP) in patients with oral cavity and oropharyngeal squamous cell carcinoma (OOPSCC), highlighting biomarker potential as an early detection tool. However, the effects of treatment remain poorly understood. This post-hoc analysis of a multi-institutional study aims to characterize the effects of primary surgery versus radiotherapy on these biomarkers. Methods: Oral rinses were collected from biopsy-proven, newly diagnosed OOPSCC patients at five institutions (UCSD, San Diego VA, Johns Hopkins, Greater Baltimore Medical Center, and NYU) from 2017-2019. Rinses were collected at enrollment and 3-, 6-, 12-, and 18-months post-treatment. CD44 ELISA assays and modified Lowry protein were performed to calculate biomarker levels. Patients were followed for 3 years, and we excluded those with recurrent or persistent disease. Paired samples T-tests were used to assess biomarker changes over time within groups, while independent samples T-tests compared levels and changes between treatment groups. Due to the non-normality of biomarker data, LN-transformed values were used for analysis. Results: In total,135 OOPSCC patients were analyzed. Our cohort had a mean age of 62.3 years (SD=10.82) and had 93 (68.9%) male participants. 38 (28.1%) of patients had oral cavity cancer, 94 (69.6%) had oropharyngeal cancer, and 3 (2.2%) had oral cavity and oropharyngeal involvement. At 3 months post-treatment, CD44(log) levels were significantly lower in the primary surgery group (mean = 1.361, SD = 0.691) compared to the radiation group (mean=1.688, SD=0.833, p = 0.032). Comparatively, CD44(log) increased in the radiation group (enrollment mean=1.520, SD=0.595) and decreased in the surgery group (enrollment mean=1.586, SD=0.654) from enrollment to 3 months post-treatment (p=0.035). Intragroup analysis of the radiation group showed decreased CD44 (log) levels at 12 months (mean=1.579, SD=0.644) vs the elevated 3-month values (mean=1.688, SD=0.833, p=0.048). TP levels in the radiation group decreased from enrollment (mean=0.386, SD=0.168) to 18 months (mean=0.324, SD=0.136; p=0.045). Intragroup analysis for surgical treatment revealed an initial decrease in TP levels from enrollment (mean=0.388, SD=0.168) to 3 months (0.318, SD=0.165; p=0.021). Conclusions: Our study demonstrates differing effects of primary surgery vs radiotherapy on salivary CD44 and TP in OOPSCC patients. CD44(log) levels were lower among patients receiving primary surgery at 3 months post-treatment, while radiated patients saw elevated levels. TP followed a similar short-term decrease in primary surgery patients but did not change among radiation patients. Although radiation patients had a sustained decrease in TP at 18-months vs. enrollment values, neither TP nor CD44 differed between radiation and surgery at 18 months.

ABSTRACT Background The role of alternative splicing events (ASEs) in immune evasion and prognosis in head and neck squamous cell carcinoma (HNSC) is not well characterized. Methods Using The Cancer Genome Atlas data, we identified ASEs (using our novel algorithm OutSplice) and characterized associations between splice burden, immune infiltration (quantified by xCell) and prognosis with multivariable logistic regression and survival models. Results HSNC tumors with low splice burden and high immune infiltration had significantly better prognosis than tumors with high splice burden and low immune infiltration when controlling for age, pathologic stage, HPV status, and tumor mutational burden (hazard ratio = 0.61). High splice burden predicted decreased immune infiltration in HNSC, which was validated in five other cancer types and supported by murine models of oral squamous cell carcinoma. Conclusions High splicing burden, as defined by OutSplice, is a novel biomarker to predict poor both immune infiltration and prognosis in HNSC.

Plasma human papillomavirus (HPV) DNA is a biomarker closely associated with HPV-mediated oropharynx cancer recurrence. This CCR Translations discusses the potential utility of plasma HPV DNA in predicting HPV oropharynx cancer recurrence and the challenges of application to clinical practice. See related article by Rettig et al., p. 1605.