Purpose/Objective(s) Cetuximab (CTX) is an approved anti-EGFR monoclonal antibody for the treatment of metastatic head and neck squamous cell carcinoma (HNSCC) and as a radiosensitizer during radiation therapy (RT) for locally advanced cases. However, resistance to CTX, either intrinsic or acquired, remains a major clinical challenge. Preclinical studies identified phosphorylation of tyrosine 821 (Y821) on AXL as a mediator of resistance to CTX and RT through activation of the tyrosine kinase c-Abl. Blocking of c-Abl with the tyrosine kinase inhibitor imatinib (IMT) restored sensitivity to CTX and RT, ultimately resulting in complete tumor regression without recurrence in models of head and neck cancer (HNC). This window-of-opportunity trial evaluated the biological effects of combining CTX with IMT in HNSCC patients. Materials/Methods Eligible patients (pts) with HNSCC scheduled for definitive surgery, radiation, or chemoradiation underwent baseline research biopsy prior to treatment. Pts received CTX (400 mg/m2 loading dose on Day 1; 250 mg/m2 on Day 8) plus IMT (400 mg orally daily for 8-14 days). The primary endpoint was the change in Ki67 expression between pre- and post-treatment biopsies/surgical specimens. Results Nineteen pts consented to enroll; 15 were eligible and treated (2 recurrent, 13 newly diagnosed). All pts completed CTX; 13 had complete IMT dosing. All underwent definitive surgical resection. One grade ³ 4 AE (tumor hemorrhage) occurred related to disease. Common treatment-related AEs included fatigue (27%), acneiform rash (47%), nausea (20%), headache (20%) and lymphopenia (13%). Ki67 decreased in 7 pts (median: -13%; range -32 to +7), increased in 1 pt, and 1 pt did not have sufficient carcinoma at the time of surgical resection. Ki67 results remain pending for 6 pts. Reduction in primary tumor size was observed in 6 pts by physical examination (median -0.4 cm; range -8 to -0.2 cm). Conclusion This window trial demonstrated that IMT plus CTX produced consistent decrease in Ki67 with preliminary evidence of tumor shrinkage. Physical measurements were limited by tumor location. A future trial is planned to incorporate cross-sectional imaging and targeted biopsies to better assess pathologic response. These findings support further investigation of IMT plus CTX in HNSCC.

PURPOSE:RTOG 1016 was a noninferiority phase III trial comparing the efficacy of radiation with either cisplatin (RT + Cis) or cetuximab (RT + Cetux) for patients with Humman Papillomavirus (HPV)+ oropharyngeal cancer (OPC). Perceived hearing handicap was included as a patient-reported outcome (PRO) secondary endpoint. The primary hypothesis was that perceived hearing handicap would be greater for patients receiving RT + Cis compared with RT + Cetux. METHODS AND MATERIALS:Perceived hearing handicap was measured at baseline, end of treatment, and 3, 6, and 12 months posttreatment using the Hearing Handicap Inventory for Adults Screening Version (HHIA-S), a 10-item self-assessment questionnaire designed to measure patients' reactions to their hearing loss. Mixed ordinal logistic models were used to determine the treatment effect on HHIA-S scores and handicap categories (2-sided α = 0.05). RESULTS:The PRO substudy included 375 eligible patients. No significant differences in patient/tumor characteristics were found between patients who participated in the HHIA-S study versus those excluded. For total HHIA-S scores and social and emotional subscales, RT + Cetux had significantly lower (ie, better) scores from end of treatment. Change score from baseline to end of treatment for RT + Cis (4.32; 95% confidence interval [CI], 2.57-6.07]) was greater than RT + Cetux (0.08; 95% CI, -1.15 to 1.31). For hearing handicap category, RT + Cis had a significantly higher percentage of mild/moderate and severe cases at the end of treatment (32%) compared with RT + Cetux (20%) (P < .0001). Adjusted conditional odds of higher self-perceived hearing handicap category for RT + Cis compared with RT + Cetux was 3.73 (95% CI, 2.10-6.62). CONCLUSION:Patients have significantly worse self-perceived hearing handicap after receiving RT + Cis treatment than with RT + Cetux. This was consistent across time through 1 year posttreatment. These findings inform hearing-related outcomes for patients with HPV-associated OPC and indicate the need for ototoxicity monitoring with RT + Cis treatment.

Background:Approximately 15-20% of head and neck cancer squamous cell carcinoma (HNSCC) patients respond favorably to immune checkpoint blockade (ICB). Previous single-cell RNA-Seq (scRNA-Seq) studies identified immune features, including macrophage subset ratios and T-cell subtypes, in HNSCC ICB response. However, the spatial features of HNSCC-infiltrated immune cells in response to ICB treatment need to be better characterized. Methods:Here, we perform a systematic evaluation of cell interactions between immune cell types within the tumor microenvironment using spatial omics data using complementary techniques from both 10X Visium spot-based spatial transcriptomics and Nanostring CosMx single-cell spatial omics with RNA gene panel including 435 ligands and receptors. In this study, we used integrated bioinformatics analyses to identify cellular neighborhoods of co-localizing cell types in single-cell spatial transcriptomics and proteomics data. In addition, we used both publicly available scRNA-Seq and in-house spatial RNA-Seq data to identify spatially constrained Ligand-Receptor interactions in Responder patients. Results:With 522,399 single cells profiled with both RNA and protein from 26 patients, in addition to spot-resolved spatial RNA-Seq from 8 patients treated with ICB together with bioinformatics analysis of publicly available single-cell and bulk RNA-Seq, we have identified a spatial and cell-type specific context-dependency of myeloid and T cell interaction difference between Responders and Non-Responders. We defined further cellular neighborhood and the sources of chemokine CXCL9/10-CXCR3 interactions in Responders, emerging targets in ICB, as well as CXCL16-CXCR6, CCL4/5-CCR5, and other underappreciated and potential markers and targets for ICB response in HNSCC. In addition, we have contributed a rich data resource of cell-cell Ligand Receptor interactions for the immunotherapy and HNSCC research community. Discussion:Our work provides a comprehensive single-cell and spatial atlas of immune cell interactions that correlate with response to ICB in HNSCC. We showcase how integrating multiple technologies and bioinformatics approaches can provide new insights into potential immune-based biomarkers of ICB response. Our results suggested refining future studies using preclinical animal models in a more context-specific manner to elucidate potential underlying mechanisms that lead to improved ICB responses.

BACKGROUND: SMARCB1-deficient and SMARCA4-deficient sinonasal carcinomas are rare, with only a few systematic studies available in the literature. Secondary EWSR1 gene abnormalities have been reported in SMARCB1-deficient tumors. This study aimed to systematically investigate SWI/SNF complex-deficient sinonasal carcinomas in a single-institution cohort, perform clinicopathologic characterization, and explore the underlying molecular mechanisms. METHOD: Immunohistochemistry (IHC) of INI1 and BRG1 was performed on tissue microarrays containing tumor tissue from 149 consecutive sinonasal carcinomas. Single nucleotide polymorphism (SNP) array and EWSR1 gene fluorescence in situ hybridization (FISH) analyses were conducted on SMARCB1-deficient sinonasal carcinomas. Clinicopathologic characterization was studied. RESULT: Of the 149 sinonasal carcinomas, 7 (4.7%) showed SMARCB1 loss, while none demonstrated SMARCA4 loss. All patients were male and presented with advanced-stage tumors. Four SMARCB1-deficient sinonasal carcinomas exhibited basaloid morphology, two displayed eosinophilic tumor morphology, and one had mixed morphology. Homozygous and heterozygous SMARCB1 deletions were identified in 4/6 and 2/6 cases respectively. Heterozygous loss involving genes neighboring SMARCB1 gene, including EWSR1, was observed in four cases. One tumor showed a heterozygous loss of the entire chromosome 22q. EWSR1 FISH assay revealed concordant heterozygous EWSR1 loss in these five cases. CONCLUSION: SMARCB1-deficient carcinomas account for 4.7% of sinonasal carcinomas in this single-institution cohort, while SMARCA4-deficient tumors are even rarer, with none identified. SMARCB1-deficient sinonasal carcinomas exhibit a broad spectrum of morphologic and immunohistochemical features. These carcinomas show complex genetic alterations, with homozygous SMARCB1 deletions present in the majority of cases.

Background: Microphysiological systems (MPS), advanced 3D models that reproduce organ form and function, show promise for modeling patient-specific treatment outcomes in head and neck cancer (HNC) as they aim to recapitulate the architecture and function of the tumor microenvironment (TME). However, cell and extracellular matrix (ECM) composition within these environments is often decided arbitrarily and may struggle to mimic different microenvironments i.e. primary tumors or metastatic lymph nodes. To guide the cellular and physical composition of the TME in vitro we employed spatial transcriptomic profiling of a tissue microarray (TMA) from patients with HNC to aid in identification of ECM components and cell populations that can be translated into MPS to improve their fidelity to tumor tissue. Methods: Spatial transcriptomic profiling was performed using the GeoMx whole transcriptome atlas on a TMA containing matched primary and lymph node tissue from 44 patients with HNC. Each patient was analyzed in duplicate, with segmentation between tumor (pan-cytokeratin staining) and stroma (collagen staining) for a total of 366 areas of interest profiled. Data was analyzed using differential gene expression, gene set enrichment analysis and linear mixed effect models. MPS were constructed using cells isolated from HNC tumor tissue, comprising tumor epithelial cell spheroids surrounded by a hydrogel matrix containing fibroblasts and tumor infiltrating leukocytes. Molded lumens seeded with blood and lymphatic endothelial cells form vasculature. This MPS permits visualization of readouts such as tumor spheroid growth, migration and viability, angiogenesis, and treatment response modulated by different ECM and cell combinations. Results: 91% of the samples returned acceptable data with a gene detection rate of >5%. Differential gene expression analysis demonstrated that 68.9% of the expressed genes were shared between primary and lymph node tumors while 58.3% were shared between primary and lymph node stroma. Pathway analysis identified significant differences between primary and lymph node tumor in pathways associated with ECM organization and degradation and immune cell cytokine signaling. Identification of ECM proteins enriched in primary and lymph node stroma can guide modulation of biological hydrogels for MPS construction by including these additional proteins in the matrix. Deconvolution of cell type composition using CIBERSORTx also permits modulation of cell proportions in the MPS to more closely mimic tumor tissue. MPS representing primary or lymph node TME’s created with a standard collagen/fibronectin or enhanced guided matrix or standard vs guided cell ratio’s will be built and analyzed for metrics of cell growth, viability, angiogenesis and response to cisplatin. Conclusions: We performed spatial transcriptomic analysis of primary and lymph node tumor tissue from patients with HNC. These data are guiding the ECM and cellular composition of MPS to develop models that faithfully represent patient tumor tissue and response to treatment. Citation Format: Rene Welch Schwartz, Adeel Ahmed, Rong Hu, Randall J Kimple, Paul M Harari, Irene M Ong, Sheena C Kerr. Spatial transcriptomic profiling of the head and neck cancer tumor microenvironment to guide design of microphysiological systems for reporting treatment response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B027.

Background: Head and neck cancer (HNC) treatment outcomes are highly variable among patients, and lack of actionable data on individual treatment responses limits effective clinical decision-making. Microphysiological systems (MPS) are advanced in vitro models that replicate the biological, physical, and functional properties of human tissues, offering a promising approach to testing treatment responses in vitro. Despite their potential, MPS adoption for evaluating standard clinical treatments—such as radiation and chemotherapy—has been hindered by the challenge of recreating the complexity of the tumor microenvironment. To address this, we developed an HNC MPS that mimics patient-specific tumor microenvironments using primary cells from patient tumors embedded in a 3D matrix with vascular components. This model was used to evaluate individualized responses to radiation and chemotherapy through cell-scale readouts, including DNA damage, proliferation, and migration. Methods: The HNC MPS comprises a microscale chamber containing two removable rods. A collagen-fibronectin matrix incorporating patient-derived tumor spheroids, fibroblasts, and immune cells was cast in the chamber. The matrix polymerized around the removable rods, which were subsequently extracted to create molded luminal structures. These lumens were lined with vascular and lymphatic endothelial cells to recreate a vascularized tumor microenvironment. An optimized media formulation supported the growth of all cell types within the MPS. The model was treated with radiation, cisplatin, or both to simulate the current standard of care. Cellular responses, including viability, proliferation, migration, and DNA damage, were assessed using immunofluorescence and cytokine secretion. A custom image processing pipeline analyzed multiple orthogonal readouts to reveal treatment responses at the cellular level with metrics to compare to actual patient outcomes under development. The model was further validated by comparing it with tumor tissue using single-cell RNA sequencing (scRNA-seq). Experiments were conducted with samples from multiple patients. Results: We successfully developed a patient-specific model of the HNC microenvironment using primary cells isolated from patient tumor tissue. This model captured key patient-specific features, such as angiogenesis and cell migration, and provided insights into treatment responses across different modalities. Cellular responses, such as proliferation and migration, were quantified, and scRNA-seq demonstrated close concordance between the MPS and patient tumor tissue. Our model presents a valuable functional assay for predicting patient-specific responses to therapy and validating biomarkers for treatment stratification. Conclusion: The HNC MPS is a robust in vitro platform that recapitulates patient-specific tumor microenvironments and treatment responses. It offers a promising approach for personalized cancer therapy, enabling functional assays to guide clinical decision-making and improve treatment outcomes for patients with HNC. Citation Format: Adeel Ahmed, Nathan W Hendrikse, Marcos A Lares, Fauzan Ahmed, Adam R Burr, Paul M Harari, David J Beebe, Sheena C Kerr. Patient-specific, organotypic head and neck cancer model for personalized medicine [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B023.

Continuous chromosome missegregation over successive mitotic divisions, known as chromosomal instability (CIN), is common in cancer. Increasing CIN above a maximally tolerated threshold leads to cell death due to loss of essential chromosomes. Here, we show in two tissue contexts that otherwise isogenic cancer cells with higher levels of CIN are more sensitive to ionizing radiation, which itself induces CIN. CIN also sensitizes HPV-positive and HPV-negative head and neck cancer patient derived xenograft (PDX) tumors to radiation. Moreover, laryngeal cancers with higher CIN prior to treatment show improved response to radiation therapy. In addition, we reveal a novel mechanism of radiosensitization by docetaxel, a microtubule stabilizing drug commonly used in combination with radiation. Docetaxel causes cell death by inducing CIN due to abnormal multipolar spindles rather than causing mitotic arrest, as previously assumed. Docetaxel-induced CIN, rather than mitotic arrest, is responsible for the enhanced radiation sensitivity observed in vitro and in vivo, challenging the mechanistic dogma of the last 40 years. These results implicate CIN as a potential biomarker and inducer of radiation response, which could provide valuable cancer therapeutic opportunities. Statement of Significance:Cancer cells and laryngeal tumors with higher chromosome missegregation rates are more sensitive to radiation therapy, supporting chromosomal instability as a promising biomarker of radiation response.

BACKGROUND:The increased incidence of human papillomavirus (HPV)-related cancers has motivated efforts to optimise treatment for these patients with excellent prognosis. Validation of surrogates for overall survival could expedite the investigation of new therapies. We sought to evaluate candidate intermediate clinical endpoints in trials assessing definitive treatment of p16-positive oropharyngeal cancer with chemotherapy or radiotherapy. METHODS:We did a retrospective review of five multicentre, randomised trials (NRG/RTOG 9003, 0129, 0234, 0522, and 1016) that tested radiotherapy with or without chemotherapy in patients (aged ≥18 years) with p16-positive localised head or neck squamous-cell carcinomas. Eight intermediate clinical endpoints were considered as potential surrogates for overall survival: freedom from local progression, freedom from regional progression, freedom from distant metastasis, freedom from locoregional progression, freedom from any progression, locoregional progression-free survival, progression-free survival, and distant metastasis-free survival. We used a two-stage meta-analytical framework, which requires high correlation between the intermediate clinical endpoint and overall survival at the patient level (condition 1), and high correlation between the treatment effect on the intermediate clinical endpoint and the treatment effect on overall survival (condition 2). For both, an r2 greater than 0·7 was used as criteria for clinically relevant surrogacy. FINDINGS:We analysed 1373 patients with oropharyngeal cancer from May 9, 2020, to Nov 22, 2023. 1231 (90%) of patients were men, 142 (10%) were women, and 1207 (88%) were White, with a median age of 57 years (IQR 51-62). Median follow-up was 4·2 years (3·1-5·1). For the first condition, correlating the intermediate clinical endpoints with overall survival at the individual and trial level, the three composite endpoints of locoregional progression-free survival (Kendall's τ 0·91 and r2 0·72), distant metastasis-free survival (Kendall's τ 0·93 and r2 0·83), and progression-free survival (Kendall's τ 0·88 and r2 0·70) were highly correlated with overall survival at the patient level and at the trial-group level. For the second condition, correlating treatment effects of the intermediate clinical endpoints and overall survival, the composite endpoints of locoregional progression-free survival (r2 0·88), distant metastasis-free survival (r2 0·96), and progression-free survival (r2 0·92) remained strong surrogates. Treatment effects on the remaining intermediate clinical endpoints were less strongly correlated with overall survival. INTERPRETATION:We identified locoregional progression-free survival, distant metastasis-free survival, and progression-free survival as surrogates for overall survival in p16-positive oropharyngeal cancers treated with chemotherapy or radiotherapy, which could serve as clinical trial endpoints. FUNDING:NRG Oncology Operations, NRG Oncology SDMC, the National Cancer Institute, Eli Lilly, Aventis, and the University of Michigan.

Purpose HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) is a distinct disease from other head and neck tumors. This guideline provides evidence-based recommendations on the critical decisions in its curative treatment, including both definitive and postoperative radiation therapy (RT) management. Methods The American Society for Radiation Oncology convened a task force to address 5 key questions on the use of RT for management of HPV-associated OPSCC. These questions included indications for definitive and postoperative RT and chemoradiation; dose-fractionation regimens and treatment volumes; preferred RT techniques and normal tissue considerations; and post-treatment management decisions. The task force did not address indications for primary surgery versus RT. Recommendations were based on a systematic literature review and created using a predefined consensus-building methodology and system for grading evidence quality and recommendation strength. Results Concurrent cisplatin is recommended for patients receiving definitive RT with T3-4 disease and/or one node >3 cm, or multiple nodes. For similar patients who are ineligible for cisplatin, concurrent cetuximab, carboplatin/5-fluorouracil, or taxane-based systemic therapy are conditionally recommended. In the postoperative setting, RT with concurrent cisplatin (either schedule) is recommended for positive surgical margins (PSM) or extranodal extension (ENE). Postoperative RT alone is recommended for pT3-4 disease, >2 nodes, or a single node >3 cm. Observation is conditionally recommended for pT1-2 disease and a single node ≤3 cm without other risk factors. For patients treated with definitive RT with concurrent systemic therapy, 7000 cGy in 33 to 35 fractions is recommended, and for patients receiving postoperative RT without PSM and ENE, 5600-6000 cGy is recommended. For all patients receiving RT, intensity modulated RT over 3-D techniques with reduction in dose to critical organs-at-risk (including salivary and swallowing structures) is recommended. Reassessment with positron emission tomography-computed tomography (PET-CT) is recommended approximately 3 months following definitive RT/chemoradiation, and neck dissection is recommended for convincing evidence of residual disease; for equivocal PET-CT findings, either neck dissection or repeat imaging is recommended. Conclusions The role and practice of RT continues to evolve for HPV-associated OPSCC, and these guidelines inform best clinical practice based on the available evidence.

Head and neck cancers (HNCs) arise from the mucosal lining of the aerodigestive tract and are often associated with alcohol use, tobacco use, and/or human papillomavirus (HPV) infection. Over 600,000 new cases of HNC are diagnosed each year, making it the sixth most common cancer worldwide. Historically, treatments have included surgery, radiation, and chemotherapy, and while these treatments are still the backbone of current therapy, several immunotherapies have recently been approved by the Food and Drug Administration (FDA) for use in HNC. The role of the immune system in tumorigenesis and cancer progression has been explored since the early 20th century, eventually coalescing into the current three-phase model of cancer immunoediting. During each of the three phases-elimination, equilibrium, and escape-cancer cells develop and utilize multiple strategies to either reach or remain in the final phase, escape, at which point the tumor is able to grow and metastasize with little to no detrimental interference from the immune system. In this review, we summarize the many strategies used by HNC to escape the immune system, which include ways to evade immune detection, resist immune cell attacks, inhibit immune cell functions, and recruit pro-tumor immune cells.

PurposeTargeted radiopharmaceutical therapy (RPT) in combination with external beam radiotherapy (EBRT) shows promise as a method to increase tumor control and mitigate potential high-grade toxicities associated with re-treatment for patients with recurrent head and neck cancer. This work establishes a patient-specific dosimetry framework that combines Monte Carlo based dosimetry from the two radiation modalities at the voxel level using deformable image registration (DIR) and radiobiological constructs for patients enrolled in a phase I clinical trial combining EBRT and RPT.MethodsSerial SPECT/CT patient scans performed at approximately 24, 48, 72, and 168 hours post-injection of 577.2 MBq/m2 (15.6 mCi/m2) iodine-131 containing RPT agent called XXX 131. Clinical EBRT treatment plans were created on a treatment planning CT (TPCT) using RayStation; SPECT/CT images were deformably registered to the TPCT using the Elastix DIR module in 3D Slicer and assessed by measuring mean activity concentrations and absorbed doses. Monte Carlo EBRT dosimetry was computed using EGSnrc. RPT dosimetry was conducted using a GEANT4 based RPT dosimetry platform named XXX. Radiobiological metrics (BED, EQD2) were utilized to combine the two radiation modalities.ResultsThe DIR method provided good agreement for the activity concentrations and calculated absorbed dose in the tumor volumes for the SPECT/CT and TPCT; the maximum mean absorbed dose difference was -11.2%. Based on the RPT absorbed dose calculations, two to four EBRT fractions were removed from patients’ EBRT treatments. From the combined treatment, the absorbed dose to target volumes ranged from 57.14 – 75.02 Gy. When including partial volume corrections (PVC), the mean EQD2 to the PTV from EBRT+RPT was -3.11% to 1.40% different compared to EBRT alone.ConclusionThis work demonstrated the clinical feasibility of performing combined EBRT+RPT dosimetry on TPCTs. Dosimetry guides treatment decisions for EBRT and this work provides a bridge for the same paradigm to be implemented within the rapidly emerging clinical RPT space.

Head and neck squamous cell carcinoma (HNSCC) is diagnosed in more than 71,000 patients each year in the United States, with nearly 16,000 associated deaths. One significant hurdle in the treatment of HNSCC is acquired and intrinsic resistance to existing therapeutic agents. Over the past several decades, the University of Wisconsin has formed a multidisciplinary team to move basic scientific discovery along the translational spectrum to impact the lives of HNSCC patients. In this review, we outline key discoveries made throughout the years at the University of Wisconsin to deepen our understanding of therapeutic resistance in HNSCC and how a strong, interdisciplinary team can make significant advances toward improving the lives of these patients by combatting resistance to established therapeutic modalities. We are profoundly grateful to the many scientific teams worldwide whose groundbreaking discoveries, alongside evolving clinical paradigms in head and neck oncology, have been instrumental in making our work possible.

Radiation therapy (RT) activates multiple immunologic effects in the tumor microenvironment (TME), with diverse dose-response relationships observed. We hypothesized that, in contrast with homogeneous RT, a heterogeneous RT dose would simultaneously optimize activation of multiple immunogenic effects in a single TME, resulting in a more effective antitumor immune response. Using high-dose-rate brachytherapy, we treated mice bearing syngeneic tumors with a single fraction of heterogeneous RT at a dose ranging from 2 to 30 gray. When combined with dual immune checkpoint inhibition in murine models, heterogeneous RT generated more potent antitumor responses in distant, nonirradiated tumors compared with any homogeneous dose. The antitumor effect after heterogeneous RT required CD4 and CD8 T cells and low-dose RT to a portion of the tumor. At the 3-day post-RT time point, dose heterogeneity imprinted the targeted TME with spatial differences in immune-related gene expression, antigen presentation, and susceptibility of tumor cells to immune-mediated destruction. At a later 10-day post-RT time point, high-, moderate-, or low-RT-dose regions demonstrated distinct infiltrating immune cell populations. This was associated with an increase in the expression of effector-associated cytokines in circulating CD8 T cells. Consistent with enhanced adaptive immune priming, heterogeneous RT promoted clonal expansion of effector CD8 T cells. These findings illuminate the breadth of dose-dependent effects of RT on the TME and the capacity of heterogeneous RT to promote antitumor immunity when combined with immune checkpoint inhibitors.

Purpose/Objective(s) Chromosomal instability (CIN) is an ongoing rate of chromosome missegregation events over the course of multiple cell divisions and is common in cancer. There appears to be a range of CIN that allows for optimal tumor cell fitness, and when increased beyond a maximally tolerated threshold, can lead to cell death due to loss of both copies of one or more essential chromosomes. Radiation induces CIN and we have shown that head and neck cancer cells engineered to have higher CIN at baseline are more sensitive to radiation than their isogenic, wild-type counterparts. We hypothesized that laryngeal cancers from patients enrolled on RTOG 9512 with higher levels of CIN at baseline would be more sensitive to radiation therapy (RT), as reflected by decreased local failure (LF) and locoregional failure (LRF). Materials/Methods RTOG 9512 randomized patients with T2 squamous cell carcinoma of the glottic larynx to hyperfractionated (twice daily) versus standard fractionation (once daily) RT. CIN was quantified by 6-chromosome fluorescence in situ hybridization (FISH). Samples were stained with centromeric probes for chromosomes 3, 4, 7, 9, 10 and 17 and CIN was calculated as the average percentage of cells that deviate from the modal number for each centromeric probe. LF/LRF rates were estimated by the cumulative incidence method. Cox proportional hazards models (stratified by treatment) were used to associate (two-sided 0.10) CIN with LF, LRF, and overall survival (OS). Hazard ratios (HRs) for CIN are presented per 1-standard deviation (SD) increment. Results Of 239 eligible patients from RTOG 9512, 129 had samples available and 107 (44.8%) were of sufficient quality to quantify CIN. There were no notable differences in treatment assignment, patient characteristics, or outcomes between patients included in or excluded from analysis. Mean CIN was 0.48 (SD 0.09) and there was no difference between treatment arms. 5-year LF/LRF rates in this cohort were 23.5% [95% confidence interval (CI) 15.9, 32.0) and 26.3% (95% CI = 18.4, 35.0). CIN was significantly associated with LF (P = 0.04), HR 0.67 (90% CI = 0.48, 0.92), and LRF (P = 0.04), HR 0.68 (90% CI = 0.50, 0.92). After adjustment for covariates, CIN remained significantly associated with LF (P = 0.01), HR 0.58 (90% CI = 0.40, 0.82) and LRF (P = 0.009), HR 0.59 (90% CI = 0.42, 0.82). Before or after adjusting for covariates CIN was not significantly associated with OS (P = 0.61, P = 0.36) with HR 1.07 (90% CI = 0.85, 1.36) and 0.87 (90% CI = 0.68, 1.12). Conclusion Higher CIN in laryngeal tumors from RTOG 9512 was associated with decreased LF/LRF, potentially indicating enhanced radiation sensitivity. This suggests that CIN may serve as a prognostic biomarker for LF/LRF in laryngeal cancers treated with definitive radiation, though further validation is required.

BackgroundHigh-risk human papillomavirus (HR-HPV) infection has been increasingly recognized as a risk factor for sinonasal tract carcinomas. However the prevalence and prognostic significance of HPV-associated sinonasal carcinomas is not well known due to limited studies and inconsistency in HPV testing modalities in literatures. Morphologically, HPV-associated sinonasal carcinomas encompass a diverse group of tumors. HPV-associated sinonasal adenocarcinoma has not been reported. The purpose of this study was to determine the prevalence, morphologic spectrum and prognostic implication of HPV-associated sinonasal carcinomas.MethodsThis cohort included 153 sinonasal carcinomas. Tissue microarrays were constructed. P16 immunohistochemistry and HR-HPV E6/7 in-situ Hybridization (ISH) were performed. Carcinomas were deemed HPV-associated based on a positive ISH testing. Clinicopathologic data was collected.Results28/153 (18%) sinonasal carcinomas were HPV-associated. HPV-associated carcinomas consisted of 26 (93%) squamous cell carcinomas and variants, 1 (3.5%) HPV-related multiphenotypic sinonasal carcinoma and 1 (3.5%) adenocarcinoma. The HPV-associated adenocarcinoma closely resembled HPV-associated endocervical adenocarcinoma morphologically. HPV-associated carcinomas occurred in 8 (29%) women and 20 (71%) men with a median age of 66 years old. HPV-associated carcinomas were predominantly located at nasal cavity. A trend toward improved overall survival and progression free survival in HPV-associated carcinomas patients was observed, yet without statistical significance.ConclusionOur study identifies a novel HPV-associated sinonasal adenocarcinoma subtype, highlights the broad morphologic spectrum of HPV-associated sinonasal carcinomas, and supports routine p16 testing during pathology practice regardless of tumor subtype followed by a confirmatory HR-HPV testing. This practice is critical for studying the clinical behavior of HPV-associated sinonasal carcinomas.