Abstract Head and neck squamous cell carcinoma (HNSCC) is the seventh most prevalent type of cancer globally with 940,000 new cases and 480,000 fatalities yearly. Oral squamous cell carcinoma (OSCC) is characterized by molecular alterations that drive progression of some preneoplastic lesions to invasive cancer. Not all dysplastic premalignant lesions evolve into carcinoma in situ or full-blown carcinoma. Moreover, this transition may occur as these lesions evolve over years. Identifying precancerous lesions likely to progress to invasive cancer remains challenging. Comprehensive immunogenomic profiling may reveal whether immune tolerance is a hallmark of early dysplasia and/or an acquired prerequisite for malignant transformation. We thus conducted a longitudinal study with matched dysplastic and invasive lesions to uncover early molecular drivers, reveal clonal relationships, and to identify genomic and immune mechanisms underlying SCC progression. Samples were obtained from The Ohio State University (OSU) and The Johns Hopkins University (JH). RNA was extracted from a total of 111 FFPE samples from 34 patients with precursor lesions and/or HNSCC across different stages (OSU) and submitted for RNA sequencing. Differential gene expression analysis was performed in invasive versus dysplastic lesions. Immune-related transcripts were annotated by curated immune gene sets to characterize microenvironmental remodelling. To investigate the interactions between tumor and immune cells at the subcellular resolution, spatial transcriptomics of matched dysplasia and invasive SCC was performed in a subset of patients using 10X Genomics Visium HD platform followed by Seurat based downstream analysis. For tumor-immune interaction analysis, we integrated spatial data with a single-cell reference atlas containing annotated immune cell subtypes and tumor cell states. Bulk RNA-seq revealed 164 differentially expressed genes in invasive versus dysplastic lesion, with enrichment in extracellular matrix remodelling, epithelial-mesenchymal transition and cytokine signalling pathways. Moreover, 80% of immune genes (e.g. CXCL9/10, CCL5, STAT1) are upregulated in invasive SCC. Preliminary spatial transcriptomics data revealed distinct immune microenvironments between invasive and dysplastic regions, with enrichment of immune cells at the tumor-stroma interface. The current comparative analysis revealed distinct immune infiltration patterns between dysplastic and invasive lesions, suggesting the spatial evolution of immune evasion mechanisms during cancer progression. Our cohort of samples is unique with paired patient samples that progressed from a biopsy proven premalignant state to invasive cancer over time. Our preliminary data suggest that we can identify premalignant lesions likely to progress and targetable immunogenomic drivers to intercept cancer progression at an earliest stage. Citation Format: Foram Ujjvalkumar Vaidya, Alvaro Gutierrez, Abberly Lott Limbach, Bhavna Kumar, Martin P. Alphonse, James W. Rocco, Mariana Brait, Amanda Ewart Toland, David Sidransky. Immunogenomic alterations driving premalignant lesions to invasive lesions of the head and neck [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 3617.

Oral Cavity Squamous Cell Carcinoma (OCSCC) is an aggressive tumor that develops within the mouth of patients. Tumor-suppressor gene loss and genomic arrangements fuel tumorigenesis and transcriptional reprogramming. Understanding how these alterations contribute to OCSCC growth and cell survival may identify new therapeutic vulnerabilities or biomarkers. We profiled the role of long non-coding RNAs (lncRNAs) in the growth of three OCSCC cell lines using a CRISPRi-screen and identified 19 lncRNAs that contribute to OCSCC proliferation. By comparing these lncRNAs to other screens, we find that these lncRNAs are uniquely required in OCSCC and not other malignancies. We show that these lncRNAs are abundantly expressed in OCSCC cells and tumors. Independent testing of candidate lncRNAs confirms their role in supporting OCSCC growth. Our results show that a novel subset of lncRNAs are required for the growth of OCSCC cancer cells and that these lncRNAs are cell lineage specific.

B cells can account for a substantial proportion of tumor-infiltrating lymphocytes (TILs) and have been associated with improved outcomes in several malignancies. Yet, their antigen specificity, a key factor for ascribing a distinct function, is mostly unknown. Here, we demonstrate that in tumors of patients with human papillomavirus-positive head and neck cancer (HPV+ HNC) a substantial proportion of intratumoral antibody-secreting cells (ASCs) are specific for HPV E proteins. Using IgG ELISpots on TILs, we show that HPV E protein-specific ASCs are detectable in virtually all patients and can account for up to almost a third of all intratumoral IgG+ ASCs. ASC responses against the viral proteins E1 and E2 consistently outnumbered those against the classic oncoproteins E6 and E7. HPV-specific intratumoral ASCs correlated strongly between primary tumor and metastatic lymph nodes as well as with plasma IgG titers, indicating that intratumoral tumor-specific ASCs, at least partially, contribute to systemic anti-tumor responses. To further resolve the makeup of tumor-specific antibody responses, we developed a novel Multiplex Antigen Flow-based ImmunoAssay (MAFIA) enabling simultaneous and standardized quantification of HPV E-specific antibodies across all major Ig isotypes and IgG subclasses. Applying MAFIA to plasma samples of patients with HPV+ HNC revealed that HPV-specific plasma antibodies mainly consisted of IgG1, IgG3, and IgA, with the highest median responses directed against E1 and E2. Overall, our study delineates the antigenic hierarchy and makeup of HPV-specific humoral responses in HPV+ HNC, both intratumorally and systemically, thus providing a framework for future biomarker as well as next-generation therapeutics development.

Methionine aminopeptidase (MetAP2) is a multifunctional protein that plays a significant role in regulating protein synthesis and post-translational modifications. Although its involvement in the development of various cancers is well-documented, its precise function in head and neck squamous cell carcinoma (HNSCC) remains unclear. This study aimed to investigate the specific role of MetAP2 in HNSCC and its potential as a biomarker and therapeutic target. We utilized data from The Cancer Genome Atlas (TCGA)-HNSCC dataset to analyze MetAP2 expression levels in HNSCC tissues. To further validate these findings, MetAP2 mRNA expression was confirmed through RT-qPCR analysis on oral squamous cell carcinoma (OSCC) samples, the most common subtype of HNSCC. In addition, the relationship between MetAP2 expression and clinicopathological features, as well as patient prognosis, was examined using the TCGA-HNSCC dataset. To explore the functional role of MetAP2 in HNSCC pathogenesis, gene-gene interactions and associated pathways were analyzed using GeneMANIA and Metascape tools. The results showed that HNSCC tissues exhibit significantly higher levels of MetAP2 compared to non-tumor tissues. Abnormal MetAP2 expression was found to be strongly associated with key clinicopathological factors, such as tumor grade and stage. Furthermore, high MetAP2 expression was negatively correlated with immune characteristics, which had a detrimental impact on overall survival rates. In silico analysis revealed that MetAP2 interacts with several oncogenes, including MetAP1, GNAT1, and RFC5, and is involved in pathways related to ribosome biogenesis. MetAP2 appears to be a promising biomarker and therapeutic target in HNSCC. Further research is warranted to explore novel therapeutic strategies and to identify prognostic biomarkers for better HNSCC management.

Conditional reprogramming is a cell culture technique that effectively immortalizes epithelial cells with normal genotypes by renewing epidermal stem cells. Y-27632, a compound that promotes conditional reprogramming through an unknown mechanism, was developed to inhibit the two Rho-associated kinase (ROCK) isoforms. We used human foreskin keratinocytes (HFKs) to study the role of Y27632 in conditional reprogramming and learn how ROCKs control epidermal stem cell renewal. In conditional reprogramming, Y-27632 increased HFK adherence to culture dishes, progression through S, G2 and M phases of the cell cycle, and epidermal stem cell marker levels. Although this correlated with ROCK inhibition by Y-27632, we generated CRISPR-Cas9-mediated HFK ROCK knockouts to test the direct role of ROCK inhibition. Knockout of single ROCK isoforms was insufficient to disrupt ROCK activity or promote HFK propagation without Y-27632. Although ROCK activity was reduced, HFKs with double knockout of ROCK1 and ROCK2 still required Y-27632 to propagate. Y-27632 was the most effective among the ROCK inhibitors we tested at promoting HFK proliferation and epidermal stem cell marker expression. Thus, the ability of Y-27632 to promote an epidermal stem cell state in conditional reprogramming not only depends upon ROCK inhibition but also acts via as-yet-unidentified mechanisms. Epidermal stem cell renewal might in part be regulated by ROCKs, but also involves additional pathways.

PDF file, 2045KB, Patient tumor characteristics (Group 2).

PDF file, 1581KB, HNSCC cell line characteristics.

PDF file, 5124KB, YM155 and cisplatin combination treatment significantly inhibits tumor cell motility. Tumor cell motility was examined by scratch assay.

PDF file, 839KB, YM155 and cisplatin combination treatment significantly inhibits tumor cell proliferation. UM-SCC-74A cells were treated with YM155 or cisplatin (CDDP) alone or in combination. After 72 hrs, cell proliferation was assessed by MTT assay.

Supplementary Date from Multi-scale Pan-cancer Integrative Analyses Identify the STAT3-VSIR Axis as a Key Immunosuppressive Mechanism in Head and Neck Cancer

PDF file, 2741KB, YM155 treatment induces survivin migration to nucleus in a time-dependent manner. CAL27-CisR cells were treated with YM155 (10 nM) for different time intervals and survvin localization was examined by staining these cells with survivin (red), β-catenin (green, membrane staining), and DAPI (blue, nucleus).

PDF file, 995KB, YM155 inhibits tumor cell proliferation in a dose-dependent manner. Cell proliferation in tumor cells (UM-SCC-74A or CAL27-CisR) or human oral keratinocytes (HOK) was assessed by MTT assay after 72 hrs.

Supplementary Table S1. Characteristics of study population (n=84). Supplementary Figure S1. Validation of cIAP1 antibody by immunohistochemistry. Supplementary Figure S2. Sensitivity of HNSCC cells to radiation or LCL161 as a single agent. Supplementary Figure S3. LCL161 does not alter the phosphorylation of STAT3, AKT and ERK in HNSCC cells. Supplementary Figure S4. Altered treatment schedule of LCL161 and radiation does not affect the radiosensitivity of Cal27 cells. Supplementary Figure S5. LCL161 and radiation do not substantially increase tumor cell expression of TNFα(after 24-48 hrs) but does increase TNFα secretion, and TNFα potentiates radiation mediated-apoptosis in tumor cells. Supplementary Figure S6. Kaplan-Meier survival curves demonstrating percent of mice free from tumor doubling are shown for both Cal27 and FaDu xenografts.α

Abstract Purpose: VSIR is a novel immune checkpoint protein whose expression on tumor cells across cancers remains largely uncharacterized. Here we purposed to decode the pan-cancer biologic and clinical significance of VSIR overexpression in the tumor compartment. Experimental Design: We performed multi-omics integrative analyses of 9,735 tumor samples to identify cancers with non-leukocytic expression of VSIR (VSIR High), followed by association with overall survival and immune cell infiltration levels. Orthogonal assessments of VSIR protein expression and lymphocytic infiltration were performed using quantitative immunofluorescence (QIF). Results: Integrative modeling identified a subset of cancer types as being enriched for VSIR High tumors. VSIR High tumors were associated with significantly poorer overall survival in immunogenic ovarian serous adenocarcinoma (SA) and oral cavity squamous cell carcinoma (SCC). QIF assessments in an independent validation cohort confirmed overexpression of VSIR as being associated with poorer overall survival within immunogenic oral cavity SCC. VSIR overexpression was associated with lower CD4 helper T-cell infiltration in both ovarian SA and oral cavity SCC, but did not impact CD8 T-cell infiltration. VSIR overexpressing tumors in both cancer types exhibited significantly higher STAT3 signaling activity. Pharmacologic inhibition of STAT3 signaling resulted in dose-dependent reduction of VSIR expression in ovarian SA and oral cavity SCC cells. Conclusions: The STAT3-VSIR axis is a potentially significant immunomodulatory mechanism in oral cavity and ovarian cancers, whose activation is associated with poorer survival and an immune microenvironment marked by decreased CD4 helper T-cell activity. The role of VSIR as a tumor-intrinsic modulator of resistance to immunotherapy warrants further exploration.

Objective To identify predictors of overall survival (OS) and to stratify patients according to significant prognostic variables. Methods A retrospective study of 274 consecutive patients with primary Oral Cavity Squamous Cell Carcinoma. Kaplan-Meier, Cox proportional hazard models, and recursive partitioning analysis (RPA) were used for analysis of OS. These results were further validated using National Cancer Database cohort of 21 895 patients. Results Median OS was 3.65 years. T-classification and N-classification, alcoholic beverages/week, age, and adjuvant treatment were significant predictors of OS. RPA identified high-risk subpopulations: N0-1 patients with CCI >= 4.5 and N2-3 patients ordered by those not receiving adjuvant treatment, those with T3-4 disease despite adjuvant therapy, and those having T1-2 disease with adjuvant therapy. Conclusions This study utilized significant prognostic indicators and RPA to highlight the importance of age, N-classification, T-classification, comorbidity, and adjuvant therapy in conjunction with American Joint Committee on Cancer staging to improve preoperative counseling.

Cultured cell lines are the workhorse of cancer research, but it is unclear to what extent they recapitulate the cellular heterogeneity observed among malignant cells in tumors, given the absence of a native tumor microenvironment. Here, we used multiplexed single cell RNA-seq to profile ~200 cancer cell lines. We uncovered expression programs that are recurrently heterogeneous within many cancer cell lines and are largely independent of observed genetic diversity. These programs of heterogeneity are associated with diverse biological processes, including cell cycle, senescence, stress and interferon responses, epithelial-to-mesenchymal transition, and protein maturation and degradation. Notably, some of these recurrent programs recapitulate those seen in human tumors, suggesting a prominent role of intrinsic plasticity in generating intra-tumoral heterogeneity. Moreover, the data allowed us to prioritize specific cell lines as model systems of cellular plasticity. We used two such models to demonstrate the dynamics, regulation and drug sensitivities associated with a cancer senescence program also observed in human tumors. Our work describes the landscape of cellular heterogeneity in diverse cancer cell lines, and identifies recurrent patterns of expression heterogeneity that are shared between tumors and specific cell lines and can thus be further explored in follow up studies.

•Metformin protects against recurrence in adenoid cystic carcinoma.•Metformin users experience prolonged disease-free survival.•Perineural invasion and sinonasal location increase risk of recurrence.•Age, lymph node disease, and sinonasal location increase risk of death.

Abstract Targeting inhibitor of apoptosis proteins (IAP) with second mitochondria-derived activator of caspase (SMAC) mimetics may promote cancer cell death. We tested whether cIAP1 predicts poor prognosis in head and neck squamous cell carcinoma (HNSCC) and whether a novel Smac-mimetic, LCL161, could radiosensitize human papillomavirus–positive (HPV+) and -negative (HPV−) HNSCC. The association of BIRC2 (encoding cIAP1) mRNA level with HPV status in HNSCC was analyzed using The Cancer Genome Atlas (TCGA) database. cIAP1 was assessed by IHC on an HNSCC tissue microarray (TMA, n = 84) followed by correlation analysis with HPV status and patient outcomes. Human cell culture and animal models of HNSCC were used to analyze the outcome and molecular characteristics following radiotherapy in combination with LCL161. cIAP1 expression is increased in HPV− compared with HPV+HNSCC tumors in the TCGA database. In our TMA, cIAP1 was overexpressed in HNSCC compared with normal tissues (P = 0.0003) and associated with a poor overall survival (P = 0.0402). cIAP1 levels were higher in HPV− than that in HPV+HNSCC tumors (P = 0.004) and patients with cIAP1+/HPV− HNSCC had the worst survival. LCL161 effectively radiosensitized HPV− HNSCC cells, which was accompanied with enhanced apoptosis, but not HPV+ HNSCC cells. Importantly, LCL161 in combination with radiotherapy led to dramatic tumor regression of HPV− HNSCC tumor xenografts, accompanied by cIAP1 degradation and apoptosis activation. These results reveal that cIAP1 is a prognostic and a potential therapeutic biomarker for HNSCC, and targeting cIAP1 with LCL161 preferentially radiosensitizes HPV− HNSCC, providing justification for clinical testing of LCL161 in combination with radiation for patients with HPV− HNSCC.

ObjectivesTo elucidate predictive factors in the perioperative period resulting in gastrostomy tube (G‐tube) dependence for patients undergoing primary surgical treatment of oropharyngeal squamous cell carcinoma (OPSCC) in the modern era.MethodsTwo hundred and thirty patients with known OPSCC treated with primary surgery were screened and selected from a retrospective database spanning from 2002 to 2012 at The Ohio State University Wexner Medical Center (Columbus, Ohio), with univariable and multivariable logistic regression modeling used to determine independent predictive factors resulting in G‐tube dependence (defined as tube persistence/presence 1 year after surgery).ResultsSurgical approach, baseline characteristics, tumor (T)‐nodal‐metastasis stage, human papillomavirus status, extent of tissue resected, surgical complications, reconstructive technique, preoperative G‐tube presence, and adjuvant treatment were recorded. Patients undergoing open surgery for OPSCC without adjuvant treatment had 42.9% G‐tube dependence (44.6% with adjuvant chemoradiation [CRT]) compared to 0% for those undergoing transoral nonrobotic surgery (8.1% with adjuvant CRT) and 0% for those undergoing transoral robotic surgery (10.3% with adjuvant CRT). In multivariable analysis, greater than 25% of the oral tongue resected (odds ratio [OR] 12.29; P = 0.03), an open surgical approach (OR 5.72; P < 0.01) and T3/T4 tumor stage (OR 2.84; P = 0.02) were independent and significant predictors of G‐tube dependence.ConclusionSurgical approach, advanced tumor stage, and oral tongue resection may influence the development of nutritional dependence for surgically treated patients with OPSCC.Level of Evidence4 Laryngoscope, 129:415–421, 2019