TP53 Induced Glycolysis and Apoptosis Regulator and Monocarboxylate Transporter 4 drive metabolic reprogramming with c-MYC and NFkB activation in breast cancer.

Breast cancer is composed of metabolically coupled cellular compartments with upregulation of TP53 Induced Glycolysis and Apoptosis Regulator (TIGAR) in carcinoma cells and loss of caveolin 1 (CAV1) with upregulation of monocarboxylate transporter 4 (MCT4) in fibroblasts. The mechanisms that drive metabolic coupling are poorly characterized. The effects of TIGAR on fibroblast CAV1 and MCT4 expression and breast cancer aggressiveness was studied using coculture and conditioned media systems and in-vivo. Also, the role of cytokines in promoting tumor metabolic coupling via MCT4 on cancer aggressiveness was studied. TIGAR downregulation in breast carcinoma cells reduces tumor growth. TIGAR overexpression in carcinoma cells drives MCT4 expression and NFkB activation in fibroblasts. IL6 and TGFB drive TIGAR upregulation in carcinoma cells, reduce CAV1 and increase MCT4 expression in fibroblasts. Tumor growth is abrogated in the presence of MCT4 knockout fibroblasts and environment. We discovered coregulation of c-MYC and TIGAR in carcinoma cells driven by lactate. Metabolic coupling primes the tumor microenvironment allowing for production, uptake and utilization of lactate. In sum, aggressive breast cancer is dependent on metabolic coupling.

Porocarcinoma with YAP1-NUTM1 fusion presenting as a NUT immunohistochemistry-positive lymph node metastasis.

INTRODUCTION: Porocarcinoma is a rare, malignant adnexal tumor that recently has been shown to contain YAP1-NUTM1 and YAP1-MAML2 fusion transcripts, with nuclear protein in testis (NUT) immunohistochemistry (IHC) positivity in a subset of these tumors. Consequently, NUT IHC may either aid in the differential diagnosis, or represent a confounding factor depending on the clinical scenario. Here, we present a case of NUTM1-rearranged sarcomatoid porocarcinoma of the scalp presenting as a NUT IHC-positive lymph node metastasis. CASE REPORT: A mass was excised from the right neck level 2 region with a lymph node initially diagnosed as metastatic NUT carcinoma with unknown primary site. An enlarging scalp mass was identified 4 months later, excised and diagnosed as NUT-positive carcinoma. Additional molecular testing was performed to detect the fusion partner in the NUTM1 rearrangement, confirming a YAP1-NUTM1 fusion. Given this molecular data along with the histopathologic characteristics, the clinicopathologic picture was retrospectively determined to be most consistent with a primary sarcomatoid porocarcinoma of the scalp with metastasis to a right neck lymph node and the right parotid. DISCUSSION: Porocarcinoma is a rare entity, and typically only enters the differential diagnosis when the clinical consideration is a cutaneous neoplasm. In an alternative clinical scenario such as the approach to tumors of the head and neck, porocarcinoma is not typically a consideration. In the latter scenario, as seen in our case, positivity with NUT IHC led to the initial misdiagnosis of NUT carcinoma. This case represents an important presentation of porocarcinoma that will occur not infrequently, and pathologists must be aware of this presentation to avoid this pitfall.

Periocular Microcystic Adnexal Carcinoma: A Case Report and a Major Review.

PURPOSE: To describe a patient with periocular microcystic adnexal carcinoma (MAC) and to review the clinical presentation, systemic work-up, histopathologic features, and outcome of all previously reported periocular MAC. METHODS: A major literature review. PubMed/MEDLINE and Google Scholar databases were searched for all well-documented cases of periocular MAC. RESULTS: The final analysis yielded 93 patients with MAC, 48 (52%) females, 39 (42%) males, and 6 with sex not specified (6%) with an average age of 56 years (range 3 days-95 years). Most tumors were localized to the eyebrow (26/93, 28%) and lower eyelid (20/93, 22%). Of patients with known information, MAC most commonly presented as a nodule (37/68, 54%) or plaque (20/68, 29%) with poorly-defined margins (20/51, 39%) and distortion of eyelid margin (13/51, 25%). Orbital involvement at any point of the disease course was seen in 20 of 93 (22%) patients. An accurate histopathologic diagnosis on initial biopsy was made in 25 of 70 (36%) cases. Initial management included surgical excision (47/93, 51%), Mohs micrographic surgery (17/93, 18%), and excision with frozen section control of margins (8/93, 9%). Aggressive or recurrent MAC was managed with multimodal therapies, including adjuvant radiation (10/34, 29%). The average follow-up after the last treatment was 3 years (median 2, range 0.2-20 years). In total, 33 of 86 (38%) tumors recurred, and 6 of 87 (7%) metastasized. Disease-related mortality occurred in 3 of 79 (4%) of patients. CONCLUSIONS: Periocular MAC is frequently misdiagnosed on initial biopsy and has a tendency for recurrence and locally aggressive behavior, highlighting the importance of accurate timely diagnosis, and appropriate management.

How Increased Nodal Metastasis and Recurrence in Cribriform Adenocarcinoma Relate to Polymorphous Adenocarcinoma and Survival: A Systematic Review.

BACKGROUND: Recently, histologic grade was removed from salivary tumor nomenclature by the WHO to include disease of higher grade. One such entity, cribriform adenocarcinoma (CAC), is an aggressive group of polymorphous adenocarcinoma (PAC), with frequent nodal metastasis and locoregional recurrence. We aim to examine the biologic behavior of this disease as compared with the PAC general cohort inclusive of all subtypes. METHODS: A systematic review of the literature on polymorphous adenocarcinoma and cribriform adenocarcinoma was completed. A descriptive analysis was performed for the following predictor variables: nodal and distant metastasis, in addition to recurrence. The outcome variables, disease free recurrence, and disease specific survival, where plotted using Kaplan-Meier curves. RESULTS: PAC and CAC both show median age of diagnosis in the sixth decade of life and a female predominance. CAC occurs most frequently in the tongue and PAC in the palate. The 2 groups show a similar biologic behavior in regards to incidence of distant metastasis (4.1 vs 5.5%), recurrence (12.5 vs 17.8%), and death from disease (3 vs 2.7%). However, there was an increased incidence of nodal metastasis in CAC (53%) as compared with that in PAC of all subtypes (14%). CONCLUSIONS: CAC exhibits more aggressive biologic behavior as compared with the PAC cohort. Although CAC is not an officially recognized entity, these tumors likely comprise a significant portion of the cases of PAC with poor outcomes and are deserving of attention and consideration for escalation in oncologic treatment.

Pathologist Opinions about EPIC Beaker AP: a Multi-Institutional Survey of Early Adopters.

EPIC Systems Corporation provides a widely used electronic medical record. Beaker Anatomic Pathology is a newly developed laboratory information system (LIS) that has been implemented at a small number of academic pathology departments. Pathologist opinions of EPIC Beaker AP have not been well described in the literature. A 37-question survey was administered to pathologists and pathology trainees to assess overall satisfaction and efficiency of report generation using Beaker AP. Data about experience in pathology, signout responsibilities, Beaker AP usage, and the legacy LIS was also collected. Seventy-four pathologists (51 faculty, 23 residents) responded to the survey (overall response rate 29.7%). Overall pathologist satisfaction with Beaker AP showed high inter-institutional variability; institutions with legacy LISs with a graphical interface had a generally neutral to negative assessment of Beaker AP. The majority of respondents disagreed with the statement "Beaker AP is easy to use and designed for my needs". Pathologists felt that Beaker AP was useful for reviewing clinical information and billing; areas of weakness included searching for prior cases and grossing efficiency. Overall, pathologists had a neutral opinion of whether generating and signing out a complete report was faster in Beaker AP, with marked inter-institutional variation. This variability was likely due to a combination of the efficacy of the legacy LIS, familiarity with Beaker AP at the time of the survey, and institution-specific optimization efforts.

Early squamous cell carcinoma of the oral tongue with histologically benign lymph nodes: A model predicting local control and vetting of the eighth edition of the American Joint Committee on Cancer pathologic T stage.

BACKGROUND: The eighth edition of the American Joint Committee on Cancer staging manual (AJCC8) added depth of invasion to the definition of pathologic T stage (pT). In the current study, the authors assess pT stage migration and the prognostic performance of the updated pT stage and compare it with other clinicopathologic variables in patients with early squamous cell carcinoma of the oral tongue (OTSCC; tumors measuring ≤4 cm) with histologically benign lymph nodes (pN0). METHODS: A multi-institutional cohort of patients with early OTSCC was restaged as per AJCC8. Primary endpoints were local recurrence (LR) and locoregional recurrence (LRR). Influential variables were identified and an LR/LRR prediction model was developed. RESULTS: There were a total of 494 patients, with 49 LR and 73 LRR. AJCC8 pT criteria resulted in upstaging of 37.9% of patients (187 of 494 patients), including 34.5% (64 of 185 patients) from pT2 to pT3, without improving the prognostication for LR or LRR. Both LR and LRR were found to be similar for patients with AJCC8 pT2 and pT3 disease. On multivariate analysis, LR was only found to be associated with distance to the closest margin (hazard ratio, 0.36; 95% CI, 0.20-0.64 [P = .0007]) and perineural invasion (hazard ratio, 1.92; 95% CI, 1.10-0.64 [P = .046]). Based on these 2 predictors, a final proportional hazards regression model (which may be used similar to a nomogram) was developed. The proposed model appeared to be superior to AJCC pT stage for estimating the probability of LR and LRR for individual patients with early OTSCC. CONCLUSIONS: AJCC8 pT criteria resulted in pT upstaging of patients with pN0 disease without improved LR or LRR prognostication. The proposed model based on distance to the closest margin and perineural invasion, status outperformed pT as a predictor of LR and LRR in patients with early OTSCC.

INI1/SMARCB1-Deficient Carcinoma (Rhabdoid Tumor) of the Lacrimal Gland.

Integrase interactor 1 (INI1) is a tumor suppressor gene that is ubiquitously expressed in all nucleated cells. The loss of INI1 protein activity was first demonstrated in aggressive pediatric tumors, including atypical teratoid/rhabdoid (AT/RT) tumor of the central nervous system and malignant rhabdoid tumor of the kidney. Subsequently, INI1 deficiency was discovered in other pediatric and some adult neoplasms. The spectrum of INI1-negative tumors includes a wide variety of neoplasms that occur over a wide age range, are variably aggressive, and have a variable rhabdoid component on histopathologic evaluation. In this report, the authors describe a 27-year-old gravid woman with INI1-deficient carcinoma of the lacrimal gland, previously not described in this location.

Desmoglein 2 modulates extracellular vesicle release from squamous cell carcinoma keratinocytes.

Extracellular vesicles (EVs) are nanoscale membrane-derived vesicles that serve as intercellular messengers carrying lipids, proteins, and genetic material. Substantial evidence has shown that cancer-derived EVs, secreted by tumor cells into the blood and other bodily fluids, play a critical role in modulating the tumor microenvironment and affecting the pathogenesis of cancer. Here we demonstrate for the first time that squamous cell carcinoma (SCC) EVs were enriched with the C-terminal fragment of desmoglein 2 (Dsg2), a desmosomal cadherin often overexpressed in malignancies. Overexpression of Dsg2 increased EV release and mitogenic content including epidermal growth factor receptor and c-Src. Inhibiting ectodomain shedding of Dsg2 with the matrix metalloproteinase inhibitor GM6001 resulted in accumulation of full-length Dsg2 in EVs and reduced EV release. When cocultured with Dsg2/green fluorescence protein-expressing SCC cells, green fluorescence protein signal was detected by fluorescence-activated cell sorting analysis in the CD90+ fibroblasts. Furthermore, SCC EVs activated Erk1/2 and Akt signaling and enhanced fibroblast cell proliferation. In vivo, Dsg2 was highly up-regulated in the head and neck SCCs, and EVs isolated from sera of patients with SCC were enriched in Dsg2 C-terminal fragment and epidermal growth factor receptor. This study defines a mechanism by which Dsg2 expression in cancer cells can modulate the tumor microenvironment, a step critical for tumor progression.-Overmiller, A. M., Pierluissi, J. A., Wermuth, P. J., Sauma, S., Martinez-Outschoorn, U., Tuluc, M., Luginbuhl, A., Curry, J., Harshyne, L. A., Wahl, J. K. III, South, A. P., Mahoney, M. G. Desmoglein 2 modulates extracellular vesicle release from squamous cell carcinoma keratinocytes.

TP53-inducible Glycolysis and Apoptosis Regulator (TIGAR) Metabolically Reprograms Carcinoma and Stromal Cells in Breast Cancer.

A subgroup of breast cancers has several metabolic compartments. The mechanisms by which metabolic compartmentalization develop in tumors are poorly characterized. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is a bisphosphatase that reduces glycolysis and is highly expressed in carcinoma cells in the majority of human breast cancers. Hence we set out to determine the effects of TIGAR expression on breast carcinoma and fibroblast glycolytic phenotype and tumor growth. The overexpression of this bisphosphatase in carcinoma cells induces expression of enzymes and transporters involved in the catabolism of lactate and glutamine. Carcinoma cells overexpressing TIGAR have higher oxygen consumption rates and ATP levels when exposed to glutamine, lactate, or the combination of glutamine and lactate. Coculture of TIGAR overexpressing carcinoma cells and fibroblasts compared with control cocultures induce more pronounced glycolytic differences between carcinoma and fibroblast cells. Carcinoma cells overexpressing TIGAR have reduced glucose uptake and lactate production. Conversely, fibroblasts in coculture with TIGAR overexpressing carcinoma cells induce HIF (hypoxia-inducible factor) activation with increased glucose uptake, increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and lactate dehydrogenase-A expression. We also studied the effect of this enzyme on tumor growth. TIGAR overexpression in carcinoma cells increases tumor growth in vivo with increased proliferation rates. However, a catalytically inactive variant of TIGAR did not induce tumor growth. Therefore, TIGAR expression in breast carcinoma cells promotes metabolic compartmentalization and tumor growth with a mitochondrial metabolic phenotype with lactate and glutamine catabolism. Targeting TIGAR warrants consideration as a potential therapy for breast cancer.

Orbital Involvement by NUT Midline Carcinoma.

A 32-year-old female presented with a sino-orbital lesion that proved to be a NUT midline carcinoma. This is only the third case of orbital involvement by this aggressive lesion. The clinical, radiographic, and histopathologic features of NUT midline carcinoma are discussed, as well as its management options.

Sinonasal Oxalosis due to Fungal Rhinosinusitis: A Unique Case of a Destructive Pseudotumor.

Oxalosis refers to the accumulation of calcium oxalate crystals in various organs and tissues, most commonly due to Aspergillus infection involving the lung or sinonasal tract. Both invasive and noninvasive forms of fungal rhinosinusitis can be associated with calcium oxalate crystal deposition. Here, we report a unique case of sinonasal oxalosis presenting as a destructive lesion in the absence of invasive fungal disease. Due to the clinical and pathologic significance of calcium oxalate crystals as seen in this patient, specimens from the sinonasal tract should be evaluated for the presence of these crystals, which may be a surrogate marker for fungal infection and may also independently cause tissue destruction.

MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression.

Mesenchymal-epithelial plasticity driving cancer progression in cancer-associated fibroblasts (CAFs) is undetermined. This work identifies a subgroup of CAFs in human breast cancer exhibiting mesenchymal-to-epithelial transition (MET) or epithelial-like profile with high miR-200c expression. MiR-200c overexpression in fibroblasts is sufficient to drive breast cancer aggressiveness. Oxidative stress in the tumor microenvironment induces miR-200c by DNA demethylation. Proteomics, RNA-seq and functional analyses reveal that miR-200c is a novel positive regulator of NFκB-HIF signaling via COMMD1 downregulation and stimulates pro-tumorigenic inflammation and glycolysis. Reprogramming fibroblasts toward MET via miR-200c reduces stemness and induces a senescent phenotype. This pro-tumorigenic profile in CAFs fosters carcinoma cell resistance to apoptosis, proliferation and immunosuppression, leading to primary tumor growth, metastases, and resistance to immuno-chemotherapy. Conversely, miR-200c inhibition in fibroblasts restrains tumor growth with abated oxidative stress and an anti-tumorigenic immune environment. This work determines the mechanisms by which MET in CAFs via miR-200c transcriptional enrichment with DNA demethylation triggered by oxidative stress promotes cancer progression. CAFs undergoing MET trans-differentiation and senescence coordinate heterotypic signaling that may be targeted as an anti-cancer strategy.

How far are we off? Analyzing the accuracy of surgical margin relocation in the head and neck.

BACKGROUND: Positive surgical margin rates remain high in head and neck cancer surgery. Relocation is challenging given the complex, three-dimensional (3D) anatomy. METHODS: Prospective, multi-institutional study to determine accuracy of head and neck surgeons and pathologists relocating margins on virtual 3D specimen models using written descriptions from pathology reports. Using 3D models of 10 head and neck surgical specimens, each participant relocated 20 mucosal margins (10 perpendicular, 10 shave). RESULTS: A total of 32 participants, 23 surgeons and 9 pathologists, marked 640 margins. Of the 320 marked perpendicular margins, 49.7% were greater than 1 centimeter from the true margin with a mean relocation error of 10.2 mm. Marked shave margins overlapped with the true margin a mean 54% of the time, with no overlap in 44 of 320 (13.8%) shave margins. CONCLUSIONS: Surgical margin relocation is imprecise and challenging even for experienced surgeons and pathologists. New communication technologies are needed.

Predictive capacity of immune-related adverse events and cytokine profiling in neoadjuvant immune checkpoint inhibitor trials for head and neck squamous cell carcinoma.

OBJECTIVES: Certain low-level immune-related adverse events (irAEs) have been associated with survival benefits in patients with various solid tumors on immune checkpoint inhibitors (ICIs). We aimed to investigate the association between irAEs and response to neoadjuvant ICIs in patients with head and neck squamous cell carcinoma (HNSCC) and to identify differences in circulating cytokine levels based on irAE status. METHODS: This was a retrospective cohort study including three neoadjuvant clinical trials from July 2017 to January 2022: NCT03238365 (nivolumab ± tadalafil), NCT03854032 (nivolumab ± BMS986205), NCT03618654 (durvalumab ± metformin). The presence and type of irAEs, pathologic treatment response, and survival were compared. Canonical linear discriminant analysis (LDA) was performed to identify combinations of circulating cytokines predictive of irAEs using plasma sample multiplex assay. RESULTS: Of 113 participants meeting inclusion criteria, 32 (28.3%) developed irAEs during treatment or follow-up. Positive p16 status was associated with irAEs (odds ratio [OR] 2.489; 95% CI 1.069-6.119; p = 0.043). irAEs were associated with pathologic treatment response (OR 3.73; 95% CI 1.34-10.35; p = 0.011) and with higher OS in the combined cohort (HR 0.319; 95% CI 0.113-0.906; p = 0.032). Patients with irAEs within the nivolumab cohort had significant elevations of select cytokines pre-treatment. Canonical LDA identified key drivers of irAEs among all trials, which were highly predictive of future irAE status. CONCLUSIONS: irAEs are associated with response to neoadjuvant ICI therapy in HNSCC and can serve as clinical indicators for improved clinical outcomes. irAEs can be predicted by concentrations of several circulating cytokines prior to treatment.

SMARCB1-Retained and SMARCB1-Deficient SNUC are Genetically Distinct: A Pilot Study Using RNA Sequencing.

Background Understanding the genetic basis for the molecular classification of sinonasal undifferentiated carcinoma (SNUC) based on SMARCB1 may improve our understating regarding the nature of the disease. The objective of the study was to compare the genetic profile of SMARCB1-retained (SR-SNUC) and SMARCB1-deficient SNUC (SD-SNUC). Methods Formalin-fixed, paraffin-embedded tissue from treatment-naive patients with SNUC were selected. Three cases of SR-SNUC, four cases of SD-SNUC, and four samples of nontumor tissue (control samples) were selected. Ribonucleic acid (RNA) sequencing was performed. Results SR-SNUC had a higher number of variants (1 variant for every 15,000 bases) compared with SD-SNUC (1 variant every 29,000 bases). The ratio of missense to silent mutation ratio was higher for SR-SNUC (0.8) as compared with SD-SNUC (0.7). Approximately 1,500 genes were differentially expressed between SR-SNUC and SD-SNUC. The genes that had a higher expression in SR-SNUC included TPD52L1, B3GNT3, GFY, TJP3, ELL3, CYP4F3, ALDH3B2, CKMT1B, VIPR1, SLC7A5, PPP2R2C, UPK3B, MUC1, ELF5, STY7, and H2AC14. The gene that had a higher expression in SD-SNUC was ZFHX4. Most of these genes were related to either protein translation or immune regulation. The most common ( n = 3, 75%) mechanisms of loss of SMARCB1 gene in SD-SNUC was loss of heterozygosity. Conclusion RNA sequencing is a viable and informative approach for genomic profiling of archival SNUC samples. Both SR-SNUC and SD-SNUC were noted to have distinct genetic profiles underlying the molecular classification of these diseases.

Nivolumab and ipilimumab in combination with radiotherapy in patients with high-risk locally advanced squamous cell carcinoma of the head and neck.

BACKGROUND: The combination of nivolumab and ipilimumab has been approved for the treatment of multiple solid tumors. This was a phase I study investigating definitive radioimmunotherapy (RIT) with nivolumab and ipilimumab for the treatment of locally advanced (LA) squamous cell carcinoma of the head and neck (SCCHN). METHODS: Patients with newly diagnosed, stage IVA-IVB SCCHN eligible for cisplatin-based chemotherapy received nivolumab (3 mg/kg every 2 weeks for a total of 17 doses) and ipilimumab (1 mg/kg every 6 weeks for a total of 6 doses) starting 2 weeks prior to radiotherapy. The primary endpoint was safety of definitive RIT. Secondary endpoints included progression-free survival (PFS) and overall survival (OS). Exploratory endpoints included the association of baseline programmed death-ligand 1 (PD-L1) expression as well as on-treatment changes in immune bias with treatment outcomes. RESULTS: Twenty-four patients were enrolled. With a median follow-up of 36.1 months, grade 3 or higher treatment-related adverse events were reported in 21 individuals (88%); 5 individuals developed in-field soft tissue ulceration during consolidation immunotherapy, resulting in one fatality. The 3-year PFS and OS rates were 74% (95% CI 58% to 94%) and 96% (95% CI 88% to 100%), respectively. PD-L1 combined positive score (CPS) did not correlate with death or disease progression. Decreases in extracellular vesicle PD-L1 within the concurrent RIT phase were associated with prolonged PFS (p=0.006). Also, interval decreases in circulating interleukin (IL)4, IL9, IL12, and IL17a during concurrent RIT were associated with subsequent ulceration. CONCLUSIONS: Definitive RIT with nivolumab and ipilimumab has sufficient clinical activity to support further development. Early changes in circulating biomarkers appear able to predict treatment outcomes as well as ensuing in-field soft tissue ulceration. TRIAL REGISTRATION NUMBER: NCT03162731.

CD8+ and FoxP3+ T-Cell Cellular Density and Spatial Distribution After Programmed Death-Ligand 1 Check Point Inhibition.

OBJECTIVES: To analyze CD8+ and FoxP3+ T-cell cellular density (CD) and intercellular distances (ID) in head and neck squamous cell carcinoma (HNSCC) samples from a neoadjuvant trial of durvalumab +/- metformin. METHODS: Paired pre- and post-treatment primary HNSCC tumor samples were stained for CD8+ and FoxP3+. Digital image analysis was used to determine estimated mean CD8+ and FoxP3+ CDs and CD8+-FoxP3+ IDs in the leading tumor edge (LTE) and tumor adjacent stroma (TAS) stratified by treatment arm, human papillomavirus (HPV) status, and pathologic treatment response. A subset of samples was characterized for T-cell related signatures using digital spatial genomic profiling. RESULTS: Post-treatment analysis revealed a significant decrease in FoxP3+ CD and an increase in CD8+ CDs in the TAS between patients receiving durvalumab and metformin versus durvlaumab alone. Both treatment arms demonstrated significant post-treatment increases in ID. Although HPV+ and HPV- had similar immune cell CDs in the tumor microenvironment, HPV+ pre-treatment samples had 1.60 times greater ID compared with HPV- samples, trending toward significance (p = 0.05). At baseline, pathologic responders demonstrated a 1.16-fold greater CD8+ CDs in the LTE (p = 0.045) and 2.28-fold greater ID (p = 0.001) than non-responders. Digital spatial profiling revealed upregulation of FoxP3+ and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) in the TAS (p = 0.006, p = 0.026) in samples from pathologic responders. CONCLUSIONS: Analysis of CD8+ and FoxP3+ detected population differences according to HPV status, pathologic response, and treatment. Greater CD8+-FoxP3+ ID was associated with pathologic response. CD8+ and FoxP3+ T-cell distributions may be predictive of response to immune checkpoint inhibition. CLINICALTRIALS: gov (Identifier NCT03618654). LEVEL OF EVIDENCE: 3 Laryngoscope, 133:1875-1884, 2023.

Variance in 3D anatomic localization of surgical margins based on conventional margin labeling in head and neck squamous cell carcinoma.

OBJECTIVE: In head and neck cancer (HNC), positive margins are strongly predictive of treatment failure. We sought to measure the accuracy of localization of margin sampling sites based on conventional anatomic labels using a digital 3D-model. METHODS: Preoperative CT scans for 9 patients with HNC treated operatively at our institution were imported into a multiplanar radiology software, which was used to render a digital 3D model of each tumor intended to represent the resection specimen. Surgical margin labels recorded during the operative case were collected from pathology records. Margin labels (N = 64) were presented to participating physicians.Participants were asked to mark the anatomic location of each surgical margin using the 3D-model and corresponding radiographic planes for reference.For each individual margin, the 3D coordinates of each participant's marker were used to calculate a mean localization point called the geometric centroid. Mean distance from individual markers to the centroid was compared between participantsand margin types. RESULTS: Amongst 7 surgeons, markers were placed a mean distance of 12.6 mm ([SD] = 7.5) from the centroid.Deep margins were marked with a greater mean distance than mucosal/skin margins (19.6 [24.8] mm vs. 15.3 [14.9] mm, p = 0.034). When asked to relocate a margin following re-resection, surgeons marked a point an average of 20.6 [12.4] mm from their first marker with a range of 3.9- 45.1 mm. CONCLUSIONS: Retrospective localization of conventionally labeled margins is an imprecise process with variability across the care team. Future interventions targeting margin documentation and communication may improve sampling precision.

Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers.

The most common cancers of the aerodigestive tract (ADT) are non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC). The tumor stroma plays an important role in ADT cancer development and progression, and contributes to the metabolic heterogeneity of tumors. Cancer-associated fibroblasts (CAFs) are the most abundant cell type in the tumor stroma of ADT cancers and exert pro-tumorigenic functions. Metabolically, glycolytic CAFs support the energy needs of oxidative (OXPHOS) carcinoma cells. Upregulation of the monocarboxylate transporter 4 (MCT4) and downregulation of isocitrate dehydrogenase 3α (IDH3α) are markers of glycolysis in CAFs, and upregulation of the monocarboxylate transporter 1 (MCT1) and the translocase of the outer mitochondrial membrane 20 (TOMM20) are markers of OXPHOS in carcinoma cells. It is unknown if glycolytic metabolism in CAFs is a driver of ADT cancer aggressiveness. In this study, co-cultures in vitro and co-injections in mice of ADT carcinoma cells with fibroblasts were used as experimental models to study the effects of fibroblasts on metabolic compartmentalization, oxidative stress, carcinoma cell proliferation and apoptosis, and overall tumor growth. Glycolytic metabolism in fibroblasts was modulated using the HIF-1α inhibitor BAY 87-2243, the antioxidant N-acetyl cysteine, and genetic depletion of MCT4. We found that ADT human tumors express markers of metabolic compartmentalization and that co-culture models of ADT cancers recapitulate human metabolic compartmentalization, have high levels of oxidative stress, and promote carcinoma cell proliferation and survival. In these models, BAY 87-2243 rescues IDH3α expression and NAC reduces MCT4 expression in fibroblasts, and these treatments decrease ADT carcinoma cell proliferation and increase cell death. Genetic depletion of fibroblast MCT4 decreases proliferation and survival of ADT carcinoma cells in co-culture. Moreover, co-injection of ADT carcinoma cells with fibroblasts lacking MCT4 reduces tumor growth and decreases the expression of markers of metabolic compartmentalization in tumors. In conclusion, metabolic compartmentalization with high expression of MCT4 in CAFs drives aggressiveness in ADT cancers.

Tadalafil Enhances Immune Signatures in Response to Neoadjuvant Nivolumab in Resectable Head and Neck Squamous Cell Carcinoma.

PURPOSE: We hypothesize that the addition of the phosphodiesterase-5 inhibitor tadalafil to the PD-1 inhibitor nivolumab, is safe and will augment immune-mediated antitumor responses in previously untreated squamous cell carcinoma of the head and neck (HNSCC). PATIENTS AND METHODS: We conducted a two-arm multi-institutional neoadjuvant randomized trial in any-stage resectable HNSCC (NCT03238365). Patients were stratified at randomization by human papillomavirus (HPV) status. Patients in both arms received nivolumab 240 mg intravenously on days 1 and 15 followed by surgery on day 28. Those in the combination therapy arm also received tadalafil 10 mg orally once daily for 4 weeks. Imaging, blood, and tumor were obtained pretreatment and posttreatment for correlative analysis. RESULTS: Neoadjuvant therapy was well-tolerated with no grade 3 to 5 adverse events and no surgical delays. Twenty-five of 46 (54%) evaluable patients had a pathologic treatment response of ≥20%, including three (7%) patients with a complete pathologic response. Regardless of HPV status, tumor proliferation rate was a negative predictor of response. A strong pretreatment T-cell signature in the HPV-negative cohort was a predictor of response. Tadalafil altered the immune microenvironment, as evidenced by transcriptome data identifying enriched B- and natural killer cell gene sets in the tumor and augmented effector T cells in the periphery. CONCLUSIONS: Preoperative nivolumab ± tadalafil is safe in HNSCC and results in more than 50% of the patients having a pathologic treatment response of at least 20% after 4 weeks of treatment. Pretreatment specimens identified HPV status-dependent signatures that predicted response to immunotherapy while posttreatment specimens showed augmentation of the immune microenvironment with the addition of tadalafil.