IDENTIFICATION OF MUC1-C AS A TARGET FOR SUPPRESSING PROGRESSION OF HEAD AND NECK SQUAMOUS CELL CARCINOMAS.

The MUC1-C protein is aberrantly expressed in adenocarcinomas of epithelial barrier tissues and contributes to their progression. Less is known about involvement of MUC1-C in the pathogenesis of squamous cell carcinomas (SCCs). Here, we report that the MUC1 gene is upregulated in advanced head and neck SCCs (HNSCCs). Studies of HNSCC cell lines demonstrate that the MUC1-C subunit regulates expression of (i) RIG-I and MDA5 pattern recognition receptors, (ii) STAT1 and interferon (IFN) regulatory factors, and (iii) downstream IFN-stimulated genes (ISGs). MUC1-C integrates chronic activation of the STAT1 inflammatory pathway with induction of the ∆Np63 and SOX2 genes that are aberrantly expressed in HNSCCs. In extending those dependencies, we demonstrate that MUC1-C is necessary for NOTCH3 expression, self-renewal capacity and tumorigenicity. The findings that MUC1 associates with ∆Np63, SOX2 and NOTCH3 expression by scRNA-seq analysis further indicate that MUC1-C drives the HNSCC stem cell state and is a target for suppressing HNSCC progression.

Enhanced oral versus flank lymph node T cell response parallels anti-PD1 efficacy in head and neck cancer.

OBJECTIVES: Understanding head and neck tissue specific immune responses is important for elucidating immunotherapy resistance mechanisms to head and neck squamous cell carcinoma (HNSCC). In this study, we aimed to investigate HNSCC-specific immune response differences between oral and subcutaneous flank tumor transplantation in preclinical models. MATERIALS AND METHODS: The MOC1 syngeneic mouse oral carcinoma cell line or versions expressing either the H2Kb-restricted SIINFEKL peptide from ovalbumin (MOC1OVA) or ZsGreen (MOC1ZsGreen) were inoculated into mouse oral mucosa (buccal space) or subcutaneous flank and compared for immune cell kinetics in tumors and tumor-draining lymph nodes (TDLNs) and for anti-PD1 response. RESULTS: Compared to subcutaneous flank tumors, orthotopic oral MOC1OVA induced a higher number of OVA-specific T cells, PD1 + or CD69 + activated OVA-specific T cells in both primary tumors and TDLNs. Tumors were also larger in the flank site and CD8 depletion eliminated the difference in tumor weight between the two sites. Oral versus flank SIINFEKL peptide vaccination showed enhanced TDLN lymphocyte response in the former site. Notably, cDC1 from oral TDLN showed enhanced antigen uptake and co-stimulatory marker expression, resulting in elicitation of an increased antigen specific T cell response and increased activated T cells. Parental MOC1 in the oral site showed increased endogenous antigen-reactive T cells in TDLNs and anti-PD1 blockade rejected oral MOC1 tumors but not subcutaneous flank MOC1. CONCLUSION: Collectively, we find distinct immune responses between orthotopic oral and heterotopic subcutaneous models, including priming by cDC1 in TDLN, revealing important implications for head and neck cancer preclinical studies.

Targeting Dendritic Cell Dysfunction to Circumvent Anti-PD1 Resistance in Head and Neck Cancer.

PURPOSE: Neoadjuvant anti-PD1 (aPD1) therapies are being explored in surgically resectable head and neck squamous cell carcinoma (HNSCC). Encouraging responses have been observed, but further insights into the mechanisms underlying resistance and approaches to improve responses are needed. EXPERIMENTAL DESIGN: We integrated data from syngeneic mouse oral carcinoma (MOC) models and neoadjuvant pembrolizumab HNSCC patient tumor RNA-sequencing data to explore the mechanism of aPD1 resistance. Tumors and tumor-draining lymph nodes (DLN) from MOC models were analyzed for antigen-specific priming. CCL5 expression was enforced in an aPD1-resistant model. RESULTS: An aPD1-resistant mouse model showed poor priming in the tumor DLN due to type 1 conventional dendritic cell (cDC1) dysfunction, which correlated with exhausted and poorly responsive antigen-specific T cells. Tumor microenvironment analysis also showed decreased cDC1 in aPD1-resistant tumors compared with sensitive tumors. Following neoadjuvant aPD1 therapy, pathologic responses in patients also positively correlated with baseline transcriptomic cDC1 signatures. In an aPD1-resistant model, intratumoral cDC1 vaccine was sufficient to restore aPD1 response by enhancing T-cell infiltration and increasing antigen-specific responses with improved tumor control. Mechanistically, CCL5 expression significantly correlated with neoadjuvant aPD1 response and enforced expression of CCL5 in an aPD1-resistant model, enhanced cDC1 tumor infiltration, restored antigen-specific responses, and recovered sensitivity to aPD1 treatment. CONCLUSIONS: These data highlight the contribution of tumor-infiltrating cDC1 in HNSCC aPD1 response and approaches to enhance cDC1 infiltration and function that may circumvent aPD1 resistance in patients with HNSCC.

Circulating tumor-tissue modified HPV DNA testing in the clinical evaluation of patients at risk for HPV-positive oropharynx cancer: The IDEA-HPV study.

OBJECTIVES: While survival outcomes are favorable for Human Papillomavirus (HPV)-positive oropharyngeal squamous cell carcinomas (OPSCCs), early diagnosis may minimize treatment-related morbidity and mortality. This study evaluated circulating tumor tissue-modified viral (TTMV)-HPV DNA plasma testing to facilitate early diagnosis of HPV-positive OPSCCs. METHODS: In this prospective exploratory cohort study, patients presenting to an Otolaryngology-Head and Neck Surgery clinic with unexplained signs or symptoms considered high-risk for HPV-positive OPSCC were recruited between March 2021-October 2022. Circulating TTMV-HPV DNA testing was performed, and results were shared with subjects and treating clinicians. Clinicians were surveyed regarding the perceived clinical utility of the test. RESULTS: Thirty-nine subjects were included. Most subjects were women (N = 23, 59 %), white (N = 32, 82 %) and never-smokers (N = 20, 51 %) with median age 60 years. Circulating TTMV-HPV DNA was detected in 2/39 subjects, both subsequently diagnosed with HPV-positive OPSCC. Both were white men aged 70-80 years with a neck mass. One subject with undetectable TTMV-HPV DNA was also diagnosed with HPV-positive OPSCC through excisional neck mass biopsy. Other eventual diagnoses included 3 HPV-negative head and neck squamous cell carcinomas and 4 other malignancies. Testing was perceived as helpful in clinical decision-making for 26/38 (68 %) subjects, and useful for similar future patients for 32/37 (86 %) subjects. CONCLUSION: Circulating TTMV-HPV DNA testing is feasible and holds potential as a diagnostic aid for HPV-positive OPSCC alongside standard clinical workup. Clinicians should be cognizant of its limitations, as a negative test does not necessarily indicate the absence of disease. Further studies to evaluate its utility are warranted.

Preexisting tumor-resident T cells with cytotoxic potential associate with response to neoadjuvant anti-PD-1 in head and neck cancer.

About 50% of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) experience recurrences after definitive therapy. The presurgical administration of anti-programmed cell death protein 1 (PD-1) immunotherapy results in substantial pathologic tumor responses (pTR) within the tumor microenvironment (TME). However, the mechanisms underlying the dynamics of antitumor T cells upon neoadjuvant PD-1 blockade remain unresolved, and approaches to increase pathologic responses are lacking. In a phase 2 trial (NCT02296684), we observed that 45% of patients treated with two doses of neoadjuvant pembrolizumab experienced marked pTRs (≥50%). Single-cell analysis of 17,158 CD8+ T cells from 14 tumor biopsies, including 6 matched pre-post neoadjuvant treatment, revealed that responding tumors had clonally expanded putative tumor-specific exhausted CD8+ tumor-infiltrating lymphocytes (TILs) with a tissue-resident memory program, characterized by high cytotoxic potential (CTX+) and ZNF683 expression, within the baseline TME. Pathologic responses after 5 weeks of PD-1 blockade were consistent with activation of preexisting CTX+ZNF683+CD8+ TILs, paralleling loss of viable tumor and associated tumor antigens. Response was associated with high numbers of CD103+PD-1+CD8+ T cells infiltrating pretreatment lesions, whereas revival of nonexhausted persisting clones and clonal replacement were modest. By contrast, nonresponder baseline TME exhibited a relative absence of ZNF683+CTX+ TILs and subsequent accumulation of highly exhausted clones. In HNSCC, revival of preexisting ZNF683+CTX+ TILs is a major mechanism of response in the immediate postneoadjuvant setting.

Ultrasensitive Detection of Circulating LINE-1 ORF1p as a Specific Multicancer Biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. Although proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible expression in normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 mol/L) ORF1p concentrations in plasma across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multianalyte panel, provides early therapeutic response monitoring in gastroesophageal cancers, and is prognostic for overall survival in gastroesophageal and colorectal cancers. Together, these observations nominate ORF1p as a multicancer biomarker with potential utility for disease detection and monitoring. SIGNIFICANCE: The LINE-1 ORF1p transposon protein is pervasively expressed in many cancers and is a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 µL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring. See related commentary by Doucet and Cristofari, p. 2502. This article is featured in Selected Articles from This Issue, p. 2489.

7-UP: Generating in silico CODEX from a small set of immunofluorescence markers.

Multiplex immunofluorescence (mIF) assays multiple protein biomarkers on a single tissue section. Recently, high-plex CODEX (co-detection by indexing) systems enable simultaneous imaging of 40+ protein biomarkers, unlocking more detailed molecular phenotyping, leading to richer insights into cellular interactions and disease. However, high-plex data can be slower and more costly to collect, limiting its applications, especially in clinical settings. We propose a machine learning framework, 7-UP, that can computationally generate in silico 40-plex CODEX at single-cell resolution from a standard 7-plex mIF panel by leveraging cellular morphology. We demonstrate the usefulness of the imputed biomarkers in accurately classifying cell types and predicting patient survival outcomes. Furthermore, 7-UP's imputations generalize well across samples from different clinical sites and cancer types. 7-UP opens the possibility of in silico CODEX, making insights from high-plex mIF more widely available.

Identification of HPV16 E1 and E2-specific T cells in the oropharyngeal cancer tumor microenvironment.

BACKGROUND: High-risk human papillomavirus (HPV) is a primary cause of an increasing number of oropharyngeal squamous cell carcinomas (OPSCCs). The viral etiology of these cancers provides the opportunity for antigen-directed therapies that are restricted in scope compared with cancers without viral components. However, specific virally-encoded epitopes and their corresponding immune responses are not fully defined. METHODS: To understand the OPSCC immune landscape, we conducted a comprehensive single-cell analysis of HPV16+ and HPV33+ primary tumors and metastatic lymph nodes. We used single-cell analysis with encoded peptide-human leukocyte antigen (HLA) tetramers to analyze HPV16+ and HPV33+ OPSCC tumors, characterizing the ex vivo cellular responses to HPV-derived antigens presented in major Class I and Class II HLA alleles. RESULTS: We identified robust cytotoxic T-cell responses to HPV16 proteins E1 and E2 that were shared across multiple patients, particularly in HLA-A*01:01 and HLA-B*08:01. Responses to E2 were associated with loss of E2 expression in at least one tumor, indicating the functional capacity of these E2-recognizing T cells and many of these interactions validated in a functional assay. Conversely, cellular responses to E6 and E7 were limited in quantity and cytotoxic capacity, and tumor E6 and E7 expression persisted. CONCLUSIONS: These data highlight antigenicity beyond HPV16 E6 and E7 and nominate candidates for antigen-directed therapies.

Ultrasensitive detection of circulating LINE-1 ORF1p as a specific multi-cancer biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. While proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1, L1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible detectable expression in corresponding normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore the potential of ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 M) ORF1p concentrations in patient plasma samples across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multi-analyte panel, and provides early therapeutic response monitoring in gastric and esophageal cancers. Together, these observations nominate ORF1p as a multi-cancer biomarker with potential utility for disease detection and monitoring.

Graph deep learning for the characterization of tumour microenvironments from spatial protein profiles in tissue specimens.

Multiplexed immunofluorescence imaging allows the multidimensional molecular profiling of cellular environments at subcellular resolution. However, identifying and characterizing disease-relevant microenvironments from these rich datasets is challenging. Here we show that a graph neural network that leverages spatial protein profiles in tissue specimens to model tumour microenvironments as local subgraphs captures distinctive cellular interactions associated with differential clinical outcomes. We applied this spatial cellular-graph strategy to specimens of human head-and-neck and colorectal cancers assayed with 40-plex immunofluorescence imaging to identify spatial motifs associated with cancer recurrence and with patient survival after treatment. The graph deep learning model was substantially more accurate in predicting patient outcomes than deep learning approaches that model spatial data on the basis of the local composition of cell types, and it generated insights into the effect of the spatial compartmentalization of tumour cells and granulocytes on patient prognosis. Local graphs may also aid in the analysis of disease-relevant motifs in histology samples characterized via spatial transcriptomics and other -omics techniques.

The mouse oral carcinoma (MOC) model: A 10-year retrospective on model development and head and neck cancer investigations.

Preclinical models of cancer have long been paramount to understanding tumor development and advancing the treatment of cancer. Creating preclinical models that mimic the complexity and heterogeneity of human tumors is a key challenge in the advancement of cancer therapy. About ten years ago, we created the mouse oral carcinoma (MOC) cell line models that were derived from 7, 12-dimethylbenz(a) anthracene (DMBA)-induced mouse oral squamous cell cancers. This model has been used in numerous investigations, including studies on tumor biology and therapeutics. We have seen remarkable progress in cancer immunology in recent years, and these cell lines, which are syngeneic to C57BL/6 background, have also been used to study the anti-tumor immune response. Herein, we aim to review the MOC model from its development and characterization to its use in non-immunological and immunological preclinical head and neck squamous cell carcinoma (HNSCC) studies. Integrating and refining these MOC model studies and extending findings to other systems will provide crucial insights for translational approaches aimed at improving head and neck cancer treatment.

YAP1 maintains active chromatin state in head and neck squamous cell carcinomas that promotes tumorigenesis through cooperation with BRD4.

Analysis of The Cancer Genome Atlas and other published data of head and neck squamous cell carcinoma (HNSCC) reveals somatic alterations of the Hippo-YAP pathway in approximately 50% of HNSCC. Better strategies to target the YAP1 transcriptional complex are sought. Here, we show that FAT1, an upstream inhibitor of YAP1, is mutated either by missense or by truncating mutation in 29% of HNSCC. Comprehensive proteomic and drug-screening studies across pan-cancer models confirm that FAT1-mutant HNSCC exhibits selective and higher sensitivity to BRD4 inhibition by JQ1. Epigenomic analysis reveals an active chromatin state in FAT1-mutant HNSCC cells that is driven by the YAP/TAZ transcriptional complex through recruitment of BRD4 to deposit active histone marks, thereby maintaining an oncogenic transcriptional state. This study reveals a detailed cooperative mechanism between YAP1 and BRD4 in HNSCC and suggests a specific therapeutic opportunity for the treatment of this subset of head and neck cancer patients.

Preoperative immunotherapy for head and neck cancers: state of art.

PURPOSE OF REVIEW: Programmed cell death 1 (PD-l)-targeting agents have been FDA-approved for treatment of recurrent/ metastatic head and neck squamous cell carcinoma (HNSCC). Clinical studies employing these agents preoperatively for HNSCC in the definitive setting are emerging and have important implications. RECENT FINDINGS: Preclinical studies demonstrate enhanced effectiveness of preoperative PD-1 targeting compared with postoperative treatment. Nine HNSCC clinical studies evaluating preoperative treatment with PD-1-targeted pembrolizumab/nivolumab alone or in combination therapy were recently reported. These studies differed by preoperative treatment type and duration and reported no surgical delays, no unexpected surgical complications and grade 3-4 immune-related adverse events consistent with the employed immunotherapeutic agent(s). Rates of major pathologic response (MPR), reduced residual viable tumour to 10% or less, ranged from 2.9-31% across eight trials without neoadjuvant radiation therapy. Higher PD-1 ligand (PD-L1) expression, increased inflammatory gene expression and enhanced immune cell tumour infiltration in baseline biopsies were associated with pathologic tumour response (pTR) in some studies. Any degree of pTR was associated with improved survival/relapse outcomes in two studies. SUMMARY: Emerging preoperative anti-PD-1 HNSCC clinical studies indicate that preoperative treatment does not impact surgical management. Defining predictive biomarkersand tumour pathologic response implications for patient survival are areas for further investigation.

Checkpoint blockade-induced CD8+ T cell differentiation in head and neck cancer responders.

BACKGROUND: Immune checkpoint blockade (ICB) response in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is limited to 15%-20% of patients and underpinnings of resistance remain undefined. METHODS: Starting with an anti-PD1 sensitive murine HNSCC cell line, we generated an isogenic anti-PD1 resistant model. Mass cytometry was used to delineate tumor microenvironments of both sensitive parental murine oral carcinoma (MOC1) and resistant MOC1esc1 tumors. To examine heterogeneity and clonal dynamics of tumor infiltrating lymphocytes (TILs), we applied paired single-cell RNA and TCR sequencing in three HNSCC models. RESULTS: Anti-PD1 resistant MOC1esc1 line displayed a conserved cell intrinsic immune evasion signature. Immunoprofiling showed distinct baseline tumor microenvironments of MOC1 and MOC1esc1, as well as the remodeling of immune compartments on ICB in MOC1esc1 tumors. Single cell sequencing analysis identified several CD8 +TIL subsets including Tcf7 +Pd1- (naïve/memory-like), Tcf7 +Pd1+ (progenitor), and Tcf7-Pd1+ (differentiated effector). Mapping TCR shared fractions identified that successful anti-PD1 or anti-CTLA4 therapy-induced higher post-treatment T cell lineage transitions. CONCLUSIONS: These data highlight critical aspects of CD8 +TIL heterogeneity and differentiation and suggest facilitation of CD8 +TIL differentiation as a strategy to improve HNSCC ICB response.

Neoadjuvant and Adjuvant Nivolumab and Lirilumab in Patients with Recurrent, Resectable Squamous Cell Carcinoma of the Head and Neck.

PURPOSE: Surgery often represents the best chance for disease control in locoregionally recurrent squamous cell carcinoma of the head and neck (SCCHN). We investigated dual immune-checkpoint inhibition [anti-PD-1, nivolumab (N), and anti-KIR, lirilumab (L)] before and after salvage surgery to improve disease-free survival (DFS). PATIENTS AND METHODS: In this phase II study, patients received N (240 mg) + L (240 mg) 7 to 21 days before surgery, followed by six cycles of adjuvant N + L. Primary endpoint was 1-year DFS; secondary endpoints were safety, pre-op radiologic response, and overall survival (OS). Correlatives included tumor sequencing, PD-L1 scoring, and immunoprofiling. RESULTS: Among 28 patients, the median age was 66, 86% were smokers; primary site: 9 oral cavity, 9 oropharynx, and 10 larynx/hypopharynx; 96% had prior radiation. There were no delays to surgery. Grade 3+ adverse events: 11%. At the time of surgery, 96% had stable disease radiologically, one had progression. Pathologic response to N + L was observed in 43% (12/28): 4/28 (14%) major (tumor viability, TV ≤ 10%) and 8/28 (29%) partial (TV ≤ 50%). PD-L1 combined positive score (CPS) at surgery was similar regardless of pathologic response (P = 0.71). Thirteen (46%) recurred (loco-regional = 10, distant = 3). Five of 28 (18%) had positive margins, 4 later recurred. At median follow-up of 22.8 months, 1-year DFS was 55.2% (95% CI, 34.8-71.7) and 1-year OS was 85.7% (95% CI, 66.3-94.4). Two-year DFS and OS were 64% and 80% among pathologic responders. CONCLUSIONS: (Neo)adjuvant N + L was well tolerated, with a 43% pathologic response rate. We observed favorable DFS and excellent 2-year OS among high-risk, previously treated patients exhibiting a pathologic response. Further evaluation of this strategy is warranted.See related commentary by Sacco and Cohen, p. 435.

Correction: Comprehensive Immunoprofiling of High-risk Oral Proliferative and Localized Leukoplakia.

[This corrects the article DOI: 10.1158/2767-9764.CRC-21-0060.][This corrects the article DOI: 10.1158/2767-9764.CRC-21-0060.].

Integrating CD4+ T cell help for therapeutic cancer vaccination in a preclinical head and neck cancer model.

Head and neck squamous cell carcinomas (HNSCC) are well suited for cancer vaccination strategies. In addition to tumor-associated antigens (TAAs) and endogenous retrovirus (ERV) encoded proteins, HNSCCs have a relatively high tumor mutational burden encoding potential neoepitopes. Peptide vaccine candidates are prioritized by predicted high-affinity to major histocompatibility complex (MHC) class I with MHC-II affinity largely not being considered. Herein, we extend previous studies to evaluate therapeutic vaccination in the mouse oral cancer (MOC) 22 model. Two distinct MOC22 derived SLPs were tested - a TSA-oriented mutant intercellular adhesion molecule 1 (mICAM1) and p15E, an ERV encoded antigen. In silico prediction revealed mICAM1 SLP bore strong MHC-I and MHC-II epitopes sharing a mutant residue with vaccination significantly increasing both antigen-specific IFN-γ producing CD4+ and CD8+ T cells. By contrast, p15E SLP had a predicted high-affinity MHC-I epitope but lacked an MHC-II epitope and vaccination induced antigen-specific CD8+ but not CD4+ T cell responses. Therapeutic mICAM1 vaccination attenuated tumor growth effectively with mICAM1-specific T cells displaying durable IFN-γ production compared with p15E SLP. Furthermore, mICAM1 SLPs carrying weakened MHC-II binding epitopes were unable to control tumor growth. These data underscore the potential value of therapeutic targeting of HNSCC epitopes and highlight the importance of studying distinct antigen classes in this setting. Moreover, they raise the possibility that, at least in part, CD4+ T cell help is critical for cancer vaccination in this preclinical HNSCC model and suggest in silico prediction approaches prioritize overlapping MHC-I and MHC-II epitopes to generate potent cancer vaccines.

Tumor-associated neutrophils (TANs) in human carcinoma-draining lymph nodes: a novel TAN compartment.

OBJECTIVES: The role of tumor-associated neutrophils (TANs) in the nodal spread of cancer cells remains unexplored. The present study evaluates the occurrence and clinical significance of human nodal TANs. METHODS: The relevance, derivation, phenotype and interactions of nodal TANs were explored via a large immunohistochemical analysis of carcinoma-draining lymph nodes, and their clinical significance was evaluated on a retrospective cohort of oral squamous cell carcinomas (OSCC). The tumor-promoting function of nodal TAN was probed in the OSCC TCGA dataset combining TAN and epithelial-to-mesenchymal transition (EMT) signatures. RESULTS: The pan-carcinoma screening identified a consistent infiltration (59%) of CD66b+  TANs in tumor-draining lymph nodes (TDLNs). Microscopic findings, including the occurrence of intra-lymphatic conjugates of TANs and cancer cells, indicate that TANs migrate through lymphatic vessels. In vitro experiments revealed that OSCC cell lines sustain neutrophil viability and activation via release of GM-CSF. Moreover, by retrospective analysis, a high CD66b+ TAN density in M-TDLNs of OSCC (n = 182 patients) predicted a worse prognosis. The analysis of the OSCC-TCGA dataset unveiled that the expression of a set of neutrophil-specific genes in the primary tumor (PT) is highly associated with an EMT signature, which predicts nodal spread. Accordingly, in the PT of OSCC cases, CD66b+TANs co-localised with PDPN+S100A9- EMT-switched tumor cells in areas of lymphangiogenesis. The pro-EMT signature is lacking in peripheral blood neutrophils from OSCC patients, suggesting tissue skewing of TANs. CONCLUSION: Our findings are consistent with a novel pro-tumoral TAN compartment that may promote nodal spread via EMT, through the lymphatics.

Yap1 Mediates Trametinib Resistance in Head and Neck Squamous Cell Carcinomas.

PURPOSE: In a head and neck squamous cell carcinoma (HNSCC) "window of opportunity" clinical trial, we reported that trametinib reduced MEK-Erk1/2 activation and resulted in tumor responses in a subset of patients. Here, we investigated resistance to trametinib and molecular correlates in HNSCC cell lines and patient samples. EXPERIMENTAL DESIGN: HNSCC cell lines were treated with trametinib to generate resistant lines. Candidate bypass pathways were assessed using immunoblotting, CRISPR knockout, and survival assays. Effectiveness of combined trametinib and verteporfin targeting was evaluated. Patient-derived xenografts (PDXs) from responder patients were treated with trametinib and resistant tumors were analyzed. Window trial clinical samples were subjected to whole-exome and RNA sequencing. RESULTS: HNSCC cell lines developed resistance (CAL27-TR and HSC3-TR) after prolonged trametinib exposure. Downstream effectors of the Hippo pathway were activated in CAL27-TR and HSC3-TR, and combined trametinib and verteporfin treatment resulted in synergistic treatment response. We defined the Hippo pathway effector Yap1 as an induced survival pathway promoting resistance to trametinib in HSC3-TR. Yap1 was necessary for HSC3-TR trametinib resistance, and constitutively active Yap1 was sufficient to confer resistance in parental HSC3. Analysis of trametinib neoadjuvant trial patient tumors indicated canonical MEK-Erk1/2 pathway activating mutations were infrequent, and Yap1 activity increased following trametinib treatment. Trametinib treatment of a PDX from a responder patient resulted in evolution of resistance with increased Yap1 expression and activity. CONCLUSIONS: These studies identify a Yap1-dependent resistance to trametinib therapy in HNSCCs. Combined Yap1 and MEK targeting may represent a strategy to enhance HNSCC response.