Lewis, James S.

3118 Background: Improved risk stratification is critically needed across multiple cancers to guide treatment decisions. This includes reducing overtreatment in Ductal Carcinoma in Situ (DCIS), addressing late recurrence risk in estrogen receptor-positive/lymph node-negative (ER+/LN-) breast cancer (BC), and refining prognosis in head & neck squamous cell carcinomas (HNSCC), and high-grade serous ovarian carcinoma (HGSOC). Tumor-infiltrating lymphocytes (TILs) reflect anti-tumor immunity, whereas tumor-associated macrophages (TAMs) polarize toward anti-tumor M1-like (CD68+) or immunosuppressive M2-like (CD163+) phenotypes. Integrating TILs with TAM polarization markers may better capture inflammatory balance and improve outcome prediction. We developed a hematoxylin and eosin (H&E)-only AI method (VAIP) to quantify TILs and virtual CD68/CD163 TAMs to develop multi-cancer based potential prognostic models. Methods: TILs were detected on H&E with HoVer-UNet, and virtual CD68 and CD163 macrophages were inferred with VISTA. In cancer regions, we computed cell densities and immune-balance ratios and trained cancer-specific Cox models with compact feature sets (DCIS 6; HGSOC 7; ER+ 4; HNSCC 4). Prognostic stratification used disease-free survival (DFS) for DCIS (N=273), HGSOC (training N=165, independent test N=220), and ER+ BC (training N=144, test N=622), and overall survival (OS) for HNSCC (N=77), reflecting clinical endpoints. Evaluation used cross-validation for cohorts without an external test set (DCIS, HNSCC) and independent training and testing when independent cohort was available (HGSOC, ER+). Results: Risk stratification performance across cancer types is in Table 1. The DCIS model used 6 features, largely TIL-macrophage contrast ratios (TIL/M2, TIL/M1). HGSOC used 7 features with a similar ratio-heavy immune-balance signature, plus macrophage-normalized density and polarization metrics (e.g., M1/N, M/N). In contrast, ER+ BC and HNSCC achieved best performance with 4 features dominated by macrophage measures, particularly M2 density and the M1/M2 ratio. Across sites, CD163-related macrophage metrics and immune-balance ratios were consistently informative. Conclusions: Integrating H&E-visible TILs with virtual CD68/CD163 TAMs enables fully automated, H&E-only prognostic biomarkers that stratify OS and DFS across multiple cancers, demonstrating consistent prognostic value in DCIS, ER+ BC, HGSOC, and HNSCC. These findings support further study of this histomorphometric risk classifier through broader external and prospective validation. Performance across cancer types. Cancer Type Cohort C-index HR [95% CI] p-value DCIS All 0.616 1.76 [1.20-2.59] <0.001 DCIS RT/No-treatment 0.624 1.94 [1.15-3.26] 0.003 HGSOC Test 0.553 1.38 [1.02-1.87] 0.030 ER+ BC Test 0.577 1.51 [1.00-2.28] 0.004 HNSCC All 0.603 1.92 [1.03-3.36] 0.012

Current spatial T cell receptor (TCR) profiling approaches lack the resolution needed to link clonal identity, transcriptional state, and spatial positioning of individual T cells in the tumor microenvironment. Here, we introduce a spatial TCR profiling strategy that resolves individual T cell clones together with their transcriptional states at single-cell resolution and applied the method to human head and neck squamous cell carcinoma. Presumed tumor-specific T cells were broadly dispersed throughout the tumor microenvironment, and cells of the same clone occupied distinct transcriptional states in different locations: Immune-rich regions contained more plastic or progenitor cells, whereas tumor-dense regions were enriched for exhausted states. Patients exhibited notably different spatial architectures of antitumor T cell responses, revealing variation that was not captured by high-resolution, spatially agnostic methods such as spectral flow cytometry and single-cell RNA sequencing. These results provide a blueprint for dissecting antigen-specific T cell states in human tumors and reveal how T cell states are spatially coordinated with local cues across the tumor microenvironment.

ABSTRACT Human papillomavirus (HPV) is comprised of >200 genotypes and has an ~8 kb, circular, double-stranded DNA genome. Transmission of HPV occurs through skin-to-skin contact and infection of squamous epithelial cells of cutaneous and mucosal surfaces. HPV genotypes are categorized as low- or high-risk (hrHPV) based on oncogenic potential. There are approximately 14 types of hrHPV that can cause several types of cancer, including HPV-associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). Detection of HPV(+)OPSCC is traditionally accomplished using p16 immunohistochemistry (IHC) and HPV-specific testing, either DNA or RNA in situ hybridization (ISH) staining or DNA-based PCR of suspected tumor biopsy tissue. More recently, platelet-poor plasma (PPP) samples from patients with HPV(+)OPSCC have proven useful for detection and quantitation of fragments of HPV circulating tumor DNA (ctDNA). ctDNA has been shown to be useful in determining treatment response and monitoring for disease recurrence. In this study, a novel droplet digital PCR assay (ddPCR) was developed and validated for the detection and quantitation of ctDNA from 5 hrHPV genotypes in PPP. Analytical sensitivity ranged from 7.71 to 19.45 fragments of HPV ctDNA per milliliter of PPP across five hrHPV genotypes. In patients with confirmed primary or recurrent HPV(+)OPSCC or HPV(-)OPSCC, testing of corresponding PPP samples ( n = 32) by ddPCR demonstrated 90.63% (29/32) overall agreement with p16/HPV-ISH biopsy results. Compared with reference ddPCR assays performed at outside laboratories, our ddPCR assay yielded 90% (9/10) overall agreement. This assay may provide clinicians with a tool for monitoring HPV ctDNA prior to, during, and after treatment of an HPV-associated cancer. IMPORTANCE At least 14 genotypes of human papillomavirus (HPV) have been identified to have high oncogenic potential. While molecular diagnostic testing for HPV is widely available for liquid cytologic cervical samples, testing is limited for other sample types, including liquid biopsy samples, such as platelet-poor plasma (PPP). With the rising incidence of HPV-associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC), laboratory testing is an essential part of patient diagnosis, management, and surveillance. Here, we summarize the development and analytical performance validation of a multiplexed, droplet digital PCR (ddPCR) assay for the detection and quantitation of HPV circulating tumor DNA (ctDNA) in PPP. This assay may provide clinicians with a tool to address minimal residual disease for patients with an HPV-associated cancer.

The head and neck region is a marvel of anatomic complexity and it encompasses a broad array of subsites, each with its own architectural nuances. This histologic richness begets a commensurate diversity of disease. Advances in our understanding of the genetic and epigenetic alterations that underlie many of these tumors have not only refined their classification, but also reshaped our approaches to diagnosis, prognosis, and therapy. This Fascicle reflects these scientific and diagnostic evolutions. Departing from prior editions this volume is organized by anatomic subsite. Each chapter delves into the neoplastic conditions and key differential diagnoses germane to a specific location. This anatomic framework is intended to provide readers with a more practical and intuitive means of navigating the field. This Fascicle serves as a useful companion—equal parts atlas, reference, and diagnostic confidant—for pathologists encountering the intricate, fascinating, and occasionally exasperating world of head and neck pathology.

Inverted papilloma (IP) is a benign neoplasm of the nasal cavity and paranasal sinuses, known for its variable risk of recurrence and potential for developing carcinoma. Emerging evidence has shown high rates of activating EGFR mutation, and a smaller subset is associated with low-risk human papillomavirus (lrHPV). While certain morphologic features, including an inverted growth pattern, are well-established, the presence of condylomatous features, such as large fungating lesions with thick undulating surface epithelium, hyperkeratosis, cytoplasmic clearing, raisinoid nuclei, and binucleation (koilocytic changes) in low-risk HPV-associated IP suggests that these tumors may be a distinct subtype of sinonasal papilloma (SP) with features similar to low-risk HPV-associated anogenital condylomas. This study presents a series of SP with condylomatous morphology and explores the association with lrHPV, the clinicopathologic features, and the rates of carcinoma development. In total, 17 cases of SP exhibiting condylomatous morphology were retrospectively identified. We performed lrHPV and high-risk HPV (hrHPV) RNA in situ hybridization and p16 immunohistochemistry and gathered detailed clinical and pathologic data along with treatment, disease follow-up, and outcomes. These condylomatous papillomas almost all developed in active smokers, were large, were primarily located in the nasal cavity (47%), and showed frequent transformation to invasive squamous cell carcinoma (29%). This malignant transformation rate is much higher than what has been reported for inverted, exophytic, and oncocytic papillomas. The tumors were almost uniformly associated with transcriptionally-active lrHPV (94%) and were consistently negative for p16 and hrHPV. This study shows that a subgroup of IPs with condylomatous morphology have a predilection for the nasal cavity, strong association with lrHPV, and high rates of carcinoma. These findings support the concept that these tumors are a distinct (fourth) type of SP with a higher risk of malignant transformation.

6073 Background: Laryngeal Squamous Cell Carcinoma (LaSCC) has varying outcomes based on the stage of cancer patients present with. Currently, a high proportion of patients are diagnosed with advanced-stage LaSCC complicating the treatment landscape and over-treating low risk patients. Risk stratification of LaSCC can help tailor treatment plans. Numerous studies have identified spatial architecture of tumor-infiltrating lymphocytes (TILs) as a prognostic biomarker in oral cavity and oropharyngeal SCC. In this work, we evaluate the prognostic value of an artificial intelligence (AI)-leveraged approach that characterizes the spatial architecture of TILs on digitized hematoxylin and eosin (H&E)-stained slides from patients with LaSCC. Methods: H&E slides from 192 patients with LaSCC were collected from Baylor Medical Center. This dataset was randomly divided into two equal cohorts, A and B. The slides were digitized as whole slide images at 40x magnification. The nuclei of all cells were automatically segmented using a deep-learning model (Hover-Net). Each nucleus was then classified as TIL or non-TIL based on morphological features. TILs and non-TILs were clustered based on proximity, and features related to the density and spatial distribution were extracted. The top features, determined by the least absolute shrinkage and selection operator, were used to train a Cox Proportional Hazards regression model that assigned a risk score for recurrence of cancer to each patient in cohort A. For validation, the model was applied to patients in cohort B. The 25 th percentile training risk score was used as a cutoff for classifying patients as high or low risk. The performance of the model in prognosticating loco-regional recurrence (LRR) was evaluated using survival analysis. Results: Patients in Cohort B identified as “high risk” by the model based on spatial organization of TILs had a significantly shorter survival time. Univariate survival analysis showed this model was prognostic for DFS with a hazard ratio of 2.57 (95% Confidence Interval: 1.12-5.89, p-value=0.048), meaning that patients classified as “high risk” are approximately 2.5 times more likely to develop LLR. Conclusions: We used computational pathology to characterize the architecture of TILs and develop a model to predict risk of LLR in LaSCC. With additional validation, this approach can be used to assist clinicians with making clinical decisions. Multivariate analysis. Variable Reference vs Comparison Pr(>|z|) HR (95% CI) Race Black vs Caucasian 0.93 0.92 (0.40 - 2.31) N N0 vs N+ 0.67 1.80 (0.12 - 26.06) T 1-2 vs 3-4 0.83 1.28 (0.13 - 12.38) Chemo Yes vs No 0.38 3.49 (0.21 - 56.90) Tobacco Yes vs No 0.26 2.08 (0.58 - 7.47) Alcohol Yes vs No 0.43 1.53 (0.54 - 4.35) TIL Architecture Risk High vs Low 0.03 * 0.26 (0.08 - 0.89)

Context.— In 2018, an evidence-based guideline was published by the College of American Pathologists to develop recommendations for the testing, application, interpretation, and reporting of high-risk human papillomavirus and surrogate marker tests in head and neck carcinomas. Substantial new evidence has prompted a review, including data on human papillomavirus (HPV) in nonoropharyngeal anatomic sites, HPV global rates, p16 immunohistochemistry, and HPV testing performance in cytology specimens, and performance of p16 immunohistochemistry as a surrogate marker. Objective.— To assess research published since the release of the original 2018 guideline and to update evidence-based recommendations for HPV testing in head and neck carcinomas. Design.— The College of American Pathologists convened a panel of experts to update the guideline following the standards established by the National Academy of Medicine for developing trustworthy clinical practice guidelines. The expert panel defined the key questions and performed a systematic review of the literature. Using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, recommendations were updated on the basis of available evidence, certainty of that evidence, and key judgments. Results.— Seven strong recommendations, 4 conditional recommendations, and 5 good practice statements are offered in the guideline update. Conclusions.— The updated guideline statements provide direction on the nature of HPV testing in various head and neck specimens (including key updates based on new research on sinonasal squamous cell carcinoma) and expanded guidance on specific scenarios and practice settings. The goal is to improve and standardize, where possible, HPV testing across diverse pathology practice settings and different countries.

DEK::AFF2 fusion nonkeratinizing squamous cell carcinoma (NKSCC) is an emerging entity in the sinonasal tract, temporal bone, and skull base. However, the clinical behavior of these tumors has not been well studied. Here, we report the largest cohort of DEK::AFF2 carcinomas to determine if morphology, mitotic rate, and/or Ki-67 IHC are associated with patient outcomes, including a comparison with high-risk human papillomavirus (HPV)-associated and independent patients. We solicited cases of molecularly or AFF2 immunohistochemistry (IHC) proven DEK::AFF2 SCC from surgical pathologists to collect patient demographic, clinical, and outcome data. Using representative H&E slides, we characterized the morphology and counted mitoses. Ki-67 immunohistochemistry was performed. We also compared the DEK::AFF2 survival rates to those in a cohort of AFF2 IHC-negative HPV-associated and HPV-independent SCC. DEK::AFF2 carcinomas most commonly arose in the nasal cavity (13/30, 43%), and the average number of recurrences was 1.8 (range: 0 to 10). At the last follow-up, most patients were disease free (19/30, 63%) or were alive with disease (9/30, 30%). There was an average mitotic rate of 2 per 2 mm2 (range: 0 to 9) and Ki-67 proliferation rate of 26% (range: 3% to 60%). Local recurrence was common, but morphology, mitotic activity, and Ki-67 index were not associated with recurrence or survival. On Kaplan-Meier survival analysis, DEK::AFF2 patients had lower disease-free survival but otherwise had similar outcomes to conventional SCC patients. Our multi-institutional study shows that local recurrence is common in DEK::AFF2 fusion nonkeratinizing SCC patients, but patients have survival rates similar to conventional SCC. Despite showing a range of different features and proliferation rates, traditional grading by morphology, mitotic rate, and/or Ki-67 activity does not seem to be predictive of outcome.

AbstractPurposeMultidisciplinary tumor boards (TB) are the standard for discussing complex head and neck cancer cases. During TB, imaging and microscopic pathology is reviewed, but there is typically no visualization of the resected cancer.MethodsA pilot study was conducted to investigate the utility of visual pathology reports at weekly TB for 10 consecutive weeks. Faculty‐level participants completed a pre‐survey and post‐survey to assess understanding of resected cancer specimens.ResultsProviders (n = 25) across seven medical specialties completed pre‐survey and post‐survey. Following intervention, providers reported significant improvement in understanding of anatomic orientation of the specimen and sites of margin sampling (mean 47.4–96.1, p < 0.001), ability to locate the site of a positive margin (mean 69.5–91.1, p < 0.001), and confidence in treatment plans created (mean 69.5–89.2, p < 0.001) with the addition of visual pathology reports.ConclusionsVisual pathology reports improve provider understanding of resected cancer specimens at multidisciplinary TB.

AbstractDue to the anatomic complexity of the head and neck and variable proximity between laboratory and operating room (OR), effective communication during frozen section analysis (FSA) between surgeons and pathologists is challenging. This proof‐of‐concept study investigates an augmented reality (AR) protocol that allows pathologists to virtually join the OR from the laboratory. Head and neck cancer specimens were scanned ex vivo using a 3‐dimensional scanner and uploaded into an AR platform. Eight head and neck specimens were discussed by surgeons and pathologists in an AR environment. AR‐guided intraoperative consultation was used for specimen orientation and discussion of FSA margin sampling sites. One patient had positive initial margins on FSA and was re‐resected to negative final margins. AR‐guided FSA is possible and allows pathologists to join the operating from any location for intraoperative discussion.