Context.—:The International Collaboration on Cancer Reporting is a not-for-profit organization whose goal is to develop evidence-based, internationally agreed standardized data sets for each anatomic site to be used throughout the world. Objective.—:To update the changes in the 2nd edition of the data set suite, including carcinomas of the hypopharynx, larynx and trachea, major salivary glands, nasal cavity and paranasal sinuses, oropharynx and nasopharynx, and oral cavity, and ear and temporal bone tumors, malignant odontogenic tumors, mucosal melanomas of the head and neck, and nodal excisions and neck dissection specimens. Design.—:International consensus by expert data set authoring committees, especially authors of the World Health Organization head and neck tumor classification. Results.—:The unique features have been updated based on current research and developments in histologic classification and standardized reporting guidelines. Separation between core and noncore elements is based on data meaningful to prognosis and stratification. The changes are in conjunction with publication of the 5th edition of the World Health Organization head and neck tumor classification. Conclusions.—:Increased harmonization of reporting and benchmarking improves patient outcomes and international collaborative research.

Lacrimal sac squamous cell carcinoma (LSSCC) is the most common tumor of the rare disease subsite of the nasolacrimal system. Circulating tumor DNA (ctDNA) has emerged as a biomarker for non-invasive monitoring of tumor burden and treatment response across a variety of solid tumors. In this case study, we report on two patients with histologically confirmed HPV + (HPV16 and HPV33) LSSCC managed with definitive chemoradiotherapy who underwent serial ctDNA monitoring prior to, during, and following treatment using an ultrasensitive multi-feature HPV whole genome sequencing liquid biopsy. In this case study, we report on two patients with histologically confirmed HPV + (HPV16 and HPV33) LSSCC managed with definitive chemoradiotherapy who underwent serial ctDNA monitoring prior to, during, and following treatment using an ultrasensitive multi-feature HPV whole genome sequencing liquid biopsy. This report suggests that ctDNA monitoring is a feasible approach for detection and monitoring of HPV + LSSCC. We advocate for continued p16 immunohistochemistry in all squamous cell carcinomas arising in the lacrimal sac. Confirmation with HPV ISH or PCR should be performed given the potential impact on management and surveillance.

Thyroid tumors with questionable capsular invasion (CI) or angioinvasion (AI) pose diagnostic and management challenges. The World Health Organization (WHO) recognizes these borderline entities as follicular tumors of uncertain malignant potential (FT-UMP) or well-differentiated tumors of uncertain malignant potential (WDT-UMP). We have conducted a retrospective, single-center study of all thyroid tumors diagnosed as UMP (2005–2025). Of 534 tumors initially labeled “uncertain malignant potential,” application of exclusion criteria yielded 324 tumors from 318 patients with questionable CI and/or AI. Clinicopathological characteristics, molecular testing data, postoperative laboratory values, imaging, and outcomes were analyzed. UMP was diagnosed in 0.5

Tumor-associated neutrophils (TANs) are abundant across cancers, yet their phenotypic diversity and functional states remain poorly defined. Here, we introduce a cell-type probability classifier that recovers low-transcript neutrophils from scRNAseq datasets, enabling pan-cancer analyses of TAN heterogeneity. Across >190 human and murine tumors, we identify a conserved differentiation trajectory that culminates in a terminal CCL3hi state. This state exhibits pro-tumor transcriptional programs, including those involved in hypoxic adaptation and senescence. Consistently, CCL3hi TANs are enriched in hypoxic tumor niches in both humans and mice. Through mechanistic perturbations of neutrophil-derived CCL3 in mice, we show that it sustains TAN survival in hypoxic tumor regions via CCR1-dependent signaling. These findings establish CCL3 as a conserved marker and functional driver of pro-tumor neutrophils in growing tumors, and provide a scalable framework for dissecting neutrophil biology across cancer types.

Surgery is a standard treatment for early-stage human papillomavirus-associated head and neck cancer (HPV+HNC), yet adjuvant therapy decisions rely on clinicopathologic features with limited prognostic accuracy. Circulating tumor HPV DNA is a promising biomarker, but current assays lack the sensitivity needed for detecting minimal residual disease after surgery. This study evaluated the prognostic value of an HPV whole-genome sequencing assay, HPV-DeepSeek, and compared this with existing methods and clinical standards. One hundred three patients with stage I to IV HPV+HNC treated with surgery were prospectively enrolled (NCT06730412). Blood samples were collected before surgery, postoperatively, and during surveillance and were analyzed using HPV-DeepSeek and droplet digital polymerase chain reaction (PCR) assays. After a median follow-up of 27 months, patients with circulating tumor HPV DNA detected after surgery had worse 2-year disease-free and overall survival than those without detection. Detection after completion of all treatment was also associated with poorer outcomes. Minimal residual disease status was a stronger predictor of recurrence than standard clinicopathologic criteria. Molecular recurrence was identified up to 17.5 months earlier than clinical recurrence and nearly twice as early as with droplet digital PCR. These findings demonstrate that sensitive HPV whole-genome sequencing enables accurate detection of minimal residual disease, early identification of recurrence, and improved postoperative risk stratification, supporting its potential as a tool for guiding personalized adjuvant therapy in HPV+HNC.

Sinonasal squamous cell carcinoma (SNSCC) is an aggressive head and neck cancer of the sinonasal cavity which has not benefitted from therapeutic advances over decades 1 . Though historically attributed to inhaled carcinogens such as hardwood dust and tobacco smoking 2 , SNSCC is incidentally associated with human papillomavirus (HPV) 3,4 . Importantly, HPV is the primary oncogenic driver of >80% of anatomically adjacent oropharyngeal cancers 5 . While viral status drives clinical staging and treatment guidelines in these malignancies 6,7 , the potentially oncogenic consequences and prognostic value of host-virus interactions in SNSCC remain incompletely defined. Here, through paired host and viral whole-genome sequencing (WGS), we map the genomic footprint of HPV in SNSCC. Strikingly, lesser studied strains such as HPV45, 51, and 39 constitute driver infections in this rare but clinically credentialed cancer, where extrachromosomal DNA (ecDNA)-associated viral integration and APOBEC mutagenesis are shown to underpin somatic tumor evolution. Statement of Significance:Paired host viral and whole-genome sequencing of SNSCC nominates HPV as a primary oncogenic driver of SNSCC. HPV-human ecDNA amplicons harboring noncanonical strains such as HPV45, 51 mediate viral carcinogenesis. Routine clinical diagnostic HPV panels should be expanded to capture the activity of lesser studied strains.

BACKGROUND:Pleomorphic adenoma (PA) is the most common benign salivary gland neoplasm detected by fine-needle aspiration (FNA). However, metaplastic changes within PA can complicate its cytologic interpretation. This study evaluates the impact of metaplasia on the FNA interpretation in a large, multi-institutional cohort. METHODS:A two-armed study design was employed: one included 200 consecutive PA resections and the corresponding FNAs, and the second included 53 PA cases with known metaplastic features identified by cytologic or pathologic review. RESULTS:Histologic assessment revealed metaplasia in 16% of the 200 PA cases. Although the majority (83%) were cytologically classified as Neoplasm: Benign, indeterminate diagnoses were more common in cases with metaplasia (25%) than in those without (15.5%). Squamous metaplasia was frequently interpreted as indeterminate relative to other types of metaplasia, and a statistical trend was observed between the extent of metaplasia and indeterminate categorization. In the multicenter arm, FNA cases with metaplasia were more frequently interpreted as indeterminate compared to FNA cases without metaplasia (61.8% vs. 15.8%, p < .01). CONCLUSIONS:Metaplastic change in PA can pose diagnostic challenges. Although most FNA cases with limited metaplastic features are appropriately classified, extensive metaplasia is more likely to result in an indeterminate interpretation.

BACKGROUND:Gene fusion testing, essential for the diagnosis and classification of many salivary gland and soft tissue neoplasms, is typically performed on formalin-fixed paraffin-embedded material, which may be limited or unavailable in cytology specimens. Although cytology smears have been validated for single-gene and DNA-based assays, evidence supporting routine diagnostic use of RNA-based next-generation sequencing fusion testing on smears remains limited. METHODS:The authors evaluated the clinical utility of a clinically validated anchored multiplex polymerase chain reaction (adenosine monophosphate)-based RNA fusion assay performed on digitized, scraped, and sacrificed cytology smears. Alcohol-fixed Papanicolaou-stained and air-dried modified Giemsa-stained smears meeting predefined cellularity thresholds were digitally archived, manually scraped, and processed for RNA extraction. Fusion testing targeted solid tumor-associated genes, and sequencing was performed on an Illumina platform. RESULTS:Of 8706 fusion assays performed during the study period, 807 (9.3%) were applied to cytology specimens, including 14 cases (1.7%) using sacrificed direct smears. Adequate nucleic acid was obtained in all cases. Gene fusions were detected in 64% (9/14), including MYB::NFIB, ACTB::PLAG1, NCALD::PLAG1, CRTC1::MAML2, COL1A1::PDGFB, COL1A2::USP6, and FGFR1::NOL4. Fusion results enabled definitive diagnosis for adenoid cystic carcinoma, mucoepidermoid carcinoma, pleomorphic adenoma, nodular fasciitis, and dermatofibrosarcoma protuberans. CONCLUSIONS:Adenosine monophosphate-based RNA fusion testing on sacrificed cytology smears is feasible, reliable, and diagnostically impactful, expanding the role of cytology specimens in definitive tumor classification.

A variety of factors, including the extent of invasion, determine the clinical outcome in patients with carcinoma ex pleomorphic adenoma (Ca ex PA). A Head and Neck Consensus Language for Ease of Reproducibility (HN CLEAR) Steering Committee organized a working group (WG) to harmonize diagnostic and research approaches for assessing invasion in Ca ex PA.WG of head and neck pathologists and a radiation oncologist conducted 6 iterative rounds of online voting (Modified Delphi), using Google Forms, over an 8-month period on invasion in the setting of Ca ex PA. Agreement was defined by the same opinion of at least 50% of the responders. The list of parameters with predetermined options was developed.Minimally invasive Ca ex PA was defined as a pT1-2 pN0 carcinoma, resected en bloc with negative margins, with or without perineural invasion, without vascular invasion and without distant metastasis at presentation. Consensus was not reached on whether high grade histology is compatible with the concept of "minimal invasion". Diagnosis of minimal invasion is possible when tumor excision is complete (with negative margins), en bloc (without fragmentation), and requires sampling of the entire tumor capsule or tumor-normal interface. The consensus guidelines characterizing Ca ex PA and the list of challenging aspects are provided. The WG proposed a checklist for future research, aiming to refine the diagnosis of in situ and minimally invasive Ca ex PA.

Thyroid tumors of uncertain malignant potential (UMP) are recognized by the World Health Organization (WHO) as tumors of borderline malignancy, encompassing follicular tumor of uncertain malignant potential (FT-UMP) and well-differentiated tumor of uncertain malignant potential (WDT-UMP). Tumors classified as UMP have questionable capsular invasion (CI) and/or angioinvasion (AI); however, biological uncertainty extends beyond the possibility for invasiveness alone. We conducted a retrospective, single-center study of all thyroid tumors diagnosed as UMP (2005-2025). Upon review of our institutional UMP cohort, we identified 39 tumors that lack questionable CI and/or AI, and therefore, would not meet WHO-defined UMP criteria. This retrospective analytic group of borderline thyroid nodules (BTN) was used to interrogate the overlap between non-invasive follicular-patterned thyroid lesions and worrisome morphologic and/or molecular features. BTN show features of neoplasia (solid growth or microfollicular architecture, papillary thyroid carcinoma (PTC)-like nuclear atypia, oncocytic morphology, conspicuous mitoses, and/or abnormal molecular results) with or without procedural artifacts (mechanically disrupted capsule, squamous metaplasia or ischemic necrosis). BTN were diagnosed in 0.1% of thyroid surgeries; median age 55 years and 1.3:1 female-to-male ratio. Median tumor size was 4 cm. RAS or RAS-like variants were most frequent (25%), followed by gene fusions (10%). Negative molecular testing was identified in 10% of BTN and high-risk gene alterations, including TERT promoter and PIK3CA, were seen in 20%. Most patients underwent hemithyroidectomy (62%), with median follow-up of 2.5 years (range 1-12.5 years), and no recurrences or metastases were documented. Our findings suggest indolent behavior of BTN and support conservative management, such as hemithyroidectomy. Longer-term, multi-institutional studies will refine risk stratification and management strategies, with possible consideration for expanding UMP diagnostic criteria.

Anaplastic thyroid carcinomas (ATCs) harboring oncogenic kinase fusion drivers are rare and highly aggressive malignancies that may benefit from targeted therapy. While fine needle aspiration (FNA) provides an opportunity for early diagnosis, the cytomorphologic features of these kinase fusion ATCs have not been specifically detailed in the literature. We present a focused analysis of three kinase fusion-positive ATCs with detailed cytologic, histologic, and molecular correlations. Our cases demonstrate cytologic features typical of conventional ATC, including marked nuclear atypia, sarcomatoid morphology, increased/atypical mitotic activity, and tumor necrosis. Although no microscopic features were identified cytologically or histologically that specifically distinguish kinase fusion-positive ATC from conventional ATC, negative immunohistochemistry for BRAF V600E and RAS Q61R may serve as an indication for early molecular testing that includes fusion analysis. Rapid genetic analysis would facilitate triage to optimal primary or neoadjuvant systemic therapy. This potentially allows for early patient triage for initiation of systemic and/or targeted therapies. The modest success observed with fusion-targeted therapy as part of a multimodal treatment strategy in our cohort is encouraging, although outcomes may be constrained by co-occurring oncogenic drivers, limited case numbers, and acquired resistance mechanisms.

Abstract Adenoid cystic carcinoma (ACC) is a rare malignancy characterized by indolent growth but a high rate of metastasis, with approximately 40-60% of patients developing distant disease. Systemic therapies, including immune checkpoint inhibitors (ICIs), have shown minimal efficacy, and multiple clinical trials have failed to identify effective treatments for recurrent or metastatic ACC. In previous work, we profiled the ACC immune microenvironment using multiplex immunofluorescence (mIF) and found that ACCs are “cold” tumors, with scarce tumor-infiltrating lymphocytes (TILs) and uniformly low expression of B2M and HLA class I across nearly all analyzed samples in our cohort of 24 ACCs. Only metastatic lesions displayed focal HLA class I expression. Spatial transcriptomic analysis revealed that these focally positive regions were associated with an interferon-γ-driven transcriptional program. Short-term ex vivo treatment of ACC tissues with interferon-γ or a STING agonist strongly upregulated HLA class I, B2M, and PD-L1, suggesting that immune visibility of ACC can be pharmacologically restored. However, using surrogate cell lines (given the lack of established ACC cell lines), we observed that STING activation induced a negative feedback loop leading to suppression of STING signaling pathway with reduced STING, IRF3 and TBK1 expression after 24 hours. Co-treatment with a range of agents - including proteasome and lysosome inhibitors, kinase pathway inhibitors, epigenetic modifiers, and modulators of NF-κB or PI3K/AKT signaling - did not prevent this feedback inhibition. Clinically, one ACC patient treated with a combination of a STING agonist dazostinag and pembrolizumab over nine months showed a ∼70% reduction in tumor burden, while other patients experienced stable or progressive disease. One hypothesis is that differential activation of the STING feedback mechanism may underlie these varied clinical responses. These findings indicate that while ACC cells retain the machinery to upregulate antigen-presentation pathways, intrinsic mechanisms of STING pathway repression may limit the durability of immunostimulatory responses, underscoring the need for strategies to sustain interferon signaling in ACC. Ongoing work focuses on identifying strategies to overcome this feedback inhibition and on testing alternative agents capable of enhancing antigen presentation without triggering the same suppressive cascade, with the goal of developing more durable immunostimulatory therapies for ACC. Citation Format: Annie Li, Samantha E. Flynn, Prinjali Kalyan, Dawn R. Mitchell, Chengzhou Gao, Edwin Zhang, Diane Yang, Ross D. Merkin, William C. Faquin, Daniel L. Faden, Xin Gao, Jong Chul Park, Lori J. Wirth, A. John Iafrate. STING pathway activation reveals feedback inhibition potentially limiting immunostimulatory response in adenoid cystic 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 4299.

Abstract Background: Sinonasal squamous cell carcinoma (SNSCC) is a rare, aggressive malignancy with limited molecular characterization. Emerging evidence implicates human papillomavirus (HPV) in a subset of cases, but the genomic interplay between viral and host factors remains poorly defined. Methods: We performed paired host and viral whole-genome sequencing (WGS) on 21 SNSCC tumors with matched normal samples, including ultra-deep viral WGS (10,000×) and comprehensive HPV PCR genotyping. Somatic mutations, viral integration, structural variants, and mutational signatures were analyzed using established computational pipelines. Results: Eighteen of 21 (86%) tumors were HPV-positive by WGS or PCR, with 94% concordance across assays. Unlike oropharyngeal squamous cell carcinoma (OPSCC), where HPV16 predominates, SNSCC exhibited nine HPV genotypes, including HPV11, 18, 35, 39, 45, 51, 56, and 59. Viral integration events were detected in most HPV-positive tumors, including an HPV45-TP63 fusion amplified as extrachromosomal DNA (ecDNA), representing, to our knowledge, the first HPV-associated ecDNA-like amplicon in SNSCC. Multi-genotype infections showed a single dominant integrating type, indicating that one “driver” genotype typically initiates tumorigenesis. Host mutational analysis revealed recurrent clonal mutations in PIK3CA, CHEK2, and KDM6A, with depletion of TP53 and CDKN2A alterations in high-risk HPV+ cases. Mutational signature analysis identified predominant APOBEC3 activity, independent of smoking, which extended to viral genomes, providing direct evidence of concurrent host-virus APOBEC mutagenesis. Structural variants were common, including focal PTEN loss and complex rearrangements such as chromothripsis and chromoplexy, underscoring pervasive genomic instability. Conclusions: Paired human and viral WGS reveals that HPV drives tumorigenesis in SNSCC through diverse genotypes, APOBEC-mediated mutagenesis, and integration-linked ecDNA formation. These mechanisms expand the known spectrum of viral oncogenesis beyond the oropharynx, highlighting novel routes of genomic instability and supporting the inclusion of broad HPV genotyping and viral-genomic assays in diagnostic and translational frameworks for SNSCC. Citation Format: Harrison B. Chong, Michael E. Bryan, Maoxuan Lin, Magdy Gohar, William C. Faquin, Lisa J. Mirabello, James S. Lewis, Michael S. Lawrence, Daniel Faden. Human and viral whole genome sequencing identify HPV and APOBEC as oncogenic drivers in sinonasal 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 1988.

PURPOSE:HPV-associated carcinomas(HPV+cancers) account for 5% of all cancers. Circulating tumor HPV DNA(ctHPVDNA) assays for HPV+ cancer surveillance have limited prognostic utility at the time of cancer diagnosis. While HPV integration into the host genome is a proven tissue-based biomarker predicting poor clinical outcomes, existing clinically utilized ctHPVDNA assays cannot classify the viral physical state. METHODS:We previously developed HPV-DeepSeek, a multi-feature HPV whole-genome sequencing liquid biopsy with 99% diagnostic accuracy at the time of HPV+ oropharynx cancer diagnosis. We test the diagnostic accuracy of HPV-DeepSeek in a cohort of 235 HPV+ cancers across nine anatomic sites and employ a novel blood-based computational classifier to infer HPV genome physical state from plasma, termed HPV-SIGNAL, to assess its prognostic potential. RESULTS:HPV-DeepSeek demonstrated a sensitivity and specificity of 99%. In 189 eligible samples, HPV-SIGNAL identified four viral physical states: episomal-only(N=71), episomal-rearranged(N=52), integrated-mixed(N=57), and integrated-clonal(N=9), which were confirmed and further elucidated via three orthogonal tissue and blood approaches. Patients harboring integrated viral states in the blood exhibited significantly worse age- and AJCC stage-adjusted progression-free survival(HR 2.78, 95% CI1.30-5.99, p=0.009) and overall survival(HR2.95, 95% CI 1.01-8.61, p=0.048) compared to patients with episomal states. CONCLUSION:HPV whole-genome sequencing liquid biopsy has high diagnostic accuracy across HPV+ cancer types and can be used to identify and classify HPV physical state from blood. Patients with integrated viral states detected in the blood demonstrated worse progression-free and overall survival, suggesting blood-based HPV physical state classification could be used as a prognostic tool at the time of cancer diagnosis.

BACKGROUND:Fine-needle aspiration (FNA) is critical in the initial diagnosis of many high-risk human papillomavirus (HR-HPV)-associated, metastatic oropharyngeal squamous cell carcinomas. Updated guidelines recommend HR-HPV-specific polymerase chain reaction (PCR) analysis over p16 immunohistochemistry on FNA specimens because p16 performs poorly on cytology material. PCR-based assays on liquid cytology material have demonstrated excellent analytic performance; however, the diagnostic utility of a positive HR-HPV PCR result in specimens with indeterminate cytomorphology remains uncharacterized. METHODS:The authors retrospectively identified 279 head and neck FNA specimens that had paired HR-HPV PCR testing on residual liquid cytology material over a 5-year period. The positive predictive value for histopathologically confirmed squamous cell carcinoma on surgical follow-up was calculated within each cytologic interpretive category. RESULTS:The HR-HPV PCR results were positive in 50.2% of specimens, negative in 40.9%, and indeterminate in 9.0%. The HR-HPV positivity rate ranged from 0% in specimens categorized as negative for malignancy to 57.3% in cytologically positive specimens, with 19.0%, 41.2%, and 50.0% positivity in the atypical, suspicious, and nondiagnostic categories, respectively. Among cytologically indeterminate specimens with positive HR-HPV PCR results (n = 14), the positive predictive value was 100% (95% confidence interval, 78.5%-100.0%). Blinded slide review additionally identified 15 cytologically positive specimens in which the definitive malignant interpretation depended substantially on HR-HPV positivity; all 15 were confirmed as squamous cell carcinoma. CONCLUSIONS:A positive HR-HPV PCR result on liquid cytology material carries a positive predictive value of 100% for malignancy in cytologically indeterminate head and neck FNA specimens. These findings support integrating HR-HPV PCR analysis into routine cytologic interpretation with the potential to upgrade some indeterminate specimens to malignant when HR-HPV is detected, expediting definitive treatment and sparing patients additional, invasive sampling.

Head and neck squamous cell carcinoma (HNSCC) shows variable response to anti-programmed cell death protein 1 (PD-1) therapy, which can be partially explained by a combined positive score (CPS) of tumor and immune cell expression of programmed death-ligand 1 (PD-L1) within the local tumor microenvironment (TME). To better define TME immune determinants associated with treatment efficacy, we conduct a study of n = 48 HNSCC tumors from patients prior to pembrolizumab therapy. Our investigation combines a rapid bioorthogonal multiplex staining method with computational analysis of whole-slide imaging to capture the single-cell spatial heterogeneity and complexity of the TME. Analyzing 6,316 fields of view (FOVs), we provide comprehensive PD-L1 phenotyping and cell proximity assays across the entirety of tissue sections. While none of the PD-L1 metrics adequately predict response, we find that the spatial organization of CCR7+ dendritic cells (DCs) in niches better predicts overall patient survival than CPS alone. This study highlights the importance of understanding the spatial context of immune networks for immunotherapy.

Background: Primary oncocytic salivary gland tumors and oncocytic subtypes of traditionally non-oncocytic salivary gland neoplasms are occasionally encountered in fine needle aspiration specimens, biopsies, and resections. Oncocytes are cells, either non-neoplastic or neoplastic, containing increased numbers of mitochondria resulting in cells with abundant eosinophilic cytoplasm and a low N/C ratio. Summary: A broad range of salivary gland tumors can be oncocytic including oncocytoma, Warthin tumor, mucoepidermoid carcinoma, salivary duct carcinoma, and others, especially those tumors where the oncocytic pattern represents a subtype of neoplasm; the oncocytic pattern can create a diagnostic challenge due to marked similarities in the oncocytic pattern of cells. Key Messages: While their microscopic cytologic and histologic features may be similar, these tumors differ intrinsically at the molecular level. Ancillary studies such as immunologic (e.g., androgen receptor for salivary duct carcinoma) and molecular analysis, e.g., FISH for detecting the MAML2 or PLAG1/HMGA2 gene alterations in mucoepidermoid carcinoma and pleomorphic adenoma, respectively, can be used to classify these oncocytic tumors in difficult cases. .

Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway (PI3K pathway) is a major intracellular regulatory pathway commonly involved in cancer survival, proliferation, migration, and metabolism. Activating mutations and amplifications of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha ( PIK3CA ) are frequent genomic features of head and neck squamous cell carcinoma (HNSCC). A growing body of evidence suggests that dysregulation of the PI3K pathway suppresses the anti-tumor immune response, thereby enabling tumor immune evasion. We retrospectively analyzed clinicopathologic and genomic data of patients with recurrent or metastatic (R/M) HNSCC treated with immune checkpoint inhibitors (ICI). PI3K pathway alterations were found in 44% and were associated with poor clinical outcomes in the human papillomavirus-negative (HPV-ve) HNSCC patients. PIK3CA expression was found to be inversely correlated with immune gene expression, decreased T-cell infiltration, and reduced CD8 T-cell cytotoxic activity. The pharmacologic inhibition of the PI3K pathway induced an increase in key immune-responsive gene expression in PIK3CA- mutant HPV-ve HNSCC cells. These findings suggest that the PI3K pathway activation promotes immune suppressive phenotype, and playing a role in conferring resistance to immune checkpoint inhibitors in HPV-ve subset of HNSCC patients. Highlights ### Competing Interest Statement LW receives honoraria for advisory roles from Bayer HealthCare Pharmaceuticals, Blueprint Pharmaceuticals, Coherus, Curie Therapeutics, Eli Lilly, Eisai, EMD Serono, Exelixis, Genentech USA, Merck, Morphic Therapeutics, Tome Biosciences and data safety monitoring board for PDS Biotechnology Corporation. JCP serves on the Scientific Advisory Boards, Consultant, or Expert Witness for GC Cell, Selecxine, Hanmi Pharmaceutical, and on the clinical Research Support/Data Safety Monitoring Board for MitoImmune. We thank all the members of Saladi lab for helpful comments and scientific discussions. NPB and SS are employees of Farcast Biosciences. This work is funded by Mike Toth Head and Neck cancer center funds to SVS. SVS is cofounder of a stealth mode startup not related to the work in this manuscript.

Objective: Cutaneous Squamous Cell Carcinoma (cSCC) incidence has increased almost 200% in recent years. Blockade of immune checkpoint axes is an effective way to reactivate the host anti-tumor immune response across a variety of cancer types. We aimed to assess the presence of the Programmed cell Death-1 (PD-1):PD-L1/PD-L2 axis and associated CD8+ infiltration in head and neck cSCC, establish the co-localization criteria. Methods: This retrospective, cross-sectional study included 46samples of patients with high-risk cSCC. Multiplex staining for PD-1, PD-L1, PD-L2, and CD8 was performed, and images were captured by an epifluorescence microscope. 1%, 20% and 50% expression cutoff values were used, as well as the COX regression method to calculate the Hazard Ratio (HR) and 95% Confidence Interval (95% CI). Results: PD-1/PD-L1, PD-1/PD-L2 and PD-L1/PD-L2 co-localized in 80.4%, 45.7% and 47.8% of the patients, respectively. All patients exhibited CD8+ infiltration to some extent, with PD-1/PD-L1, PD-1/PD-L2 and PD-1/PD-L1/PD-L2 co-localized in 97.8%, 82.6% and 82.6% of the patients, respectively. There was no statistically significant relationship regarding the co-localization of immune checkpoint and the outcomes evaluated. Conclusion: This study has evidenced significant expression and co-localization of immune checkpoint axes in whole tissues and tumor infiltrate, emphasizing the potential application of co-localization as a valuable tool in the clinical management of cSCC. These findings provide important insights for understanding and optimizing therapeutic strategies in head and neck cSCC. Level of evidence: 4.

Poor tumor antigenicity is an important cause of non-response to immune checkpoint blockade (ICB)-therapy in many cancer patients and mandates new treatment strategies. Here we explore the use of proteolytically activated antibody-peptide epitope conjugates (APECs) to redirect the immunological effector activities of tumor-infiltrating antiviral bystander CTLs against head and neck squamous cell carcinoma (HNSCC) by loading cancer cell surface MHC I proteins with viral peptides. We find that inflationary T cell memory responses against common viral pathogens such as CMV and EBV unfold superior anti-tumor activity compared to conventional memory responses under APEC therapy in an ICB-resistant preclinical model of HNSCC. Mechanistically, APEC activation required cancer cell-intrinsic protease activity, even for proteases expressed by cells of the tumor stroma. Data mining and functional screening identified the protease activity of plasminogen activator urokinase (PLAU) as widely shared between human HNSCC cancers and as a highly efficient proteolytic activator of APECs. Furthermore, peripheral blood analysis in HNSCC patients reliably predicts the specificity, magnitude, and quality of intratumoral bystander CTL, thus allowing for the screening of HNSCC patients who may benefit most from APEC therapy. ### Competing Interest Statement MC and DGM are inventors on a patent (US9402916B2) related to the design and use of APECs for cancer therapy. SIP has received consultancy payments from Abbvie, AstraZeneca/MedImmune, CUE Biopharma, Fusion Pharmaceuticals, G1 Therapeutics, Incendia, Inovio, MSD/Merck, Newlink Genetics, Oncolys Biopharma, Parthenon Therapeutics, Recurrent Respiratory Papillomatosis Foundation, Replimune, Scopus Biopharma, Sensei Biotherapeutics, and Umoja; and research grants from Abbvie, AstraZeneca/MedImmune, CUE Biopharma, Eisai, Merck, Recurrent Respiratory Papillomatosis Foundation, Sensei Biotherapeutics, and Tesaro, outside of the submitted work. National Cancer Institute, CA240239, CA278212, CA232103 National Institute of Allergy and Infectious Diseases, https://ror.org/043z4tv69, AI123349