A druggable cascade links methionine metabolism to epigenomic reprogramming in squamous cell carcinoma.

Upper aerodigestive squamous cell carcinoma (UASCC) is a common and aggressive malignancy with few effective therapeutic options. Here, we investigate amino acid metabolism in this cancer, surprisingly noting that UASCC exhibits the highest methionine level across all human cancers, driven by its transporter LAT1. We show that LAT1 is also expressed at the highest level in UASCC, transcriptionally activated by UASCC-specific promoter and enhancers, which are directly coregulated by SCC master regulators TP63/KLF5/SREBF1. Unexpectedly, unbiased bioinformatic screen identifies EZH2 as the most significant target downstream of the LAT1-methionine pathway, directly linking methionine metabolism to epigenomic reprogramming. Importantly, this cascade is indispensable for the survival and proliferation of UASCC patient-derived tumor organoids. In addition, LAT1 expression is closely associated with cellular sensitivity to inhibition of the LAT1-methionine-EZH2 axis. Notably, this unique LAT1-methionine-EZH2 cascade can be targeted effectively by either pharmacological approaches or dietary intervention in vivo. In summary, this work maps a unique mechanistic cross talk between epigenomic reprogramming with methionine metabolism, establishes its biological significance in the biology of UASCC, and identifies a unique tumor-specific vulnerability which can be exploited both pharmacologically and dietarily.

Single-Cell Molecular Profiling of Head and Neck Squamous Cell Carcinoma Reveals Five Dysregulated Signaling Pathways Associated With Circulating Tumor Cells.

OBJECTIVES: To determine the dysregulated signaling pathways of head and neck squamous cell carcinoma associated with circulating tumor cells (CTCs) via single-cell molecular characterization. INTRODUCTION: Head and neck squamous cell carcinoma (HNSCC) has a significant global burden and is a disease with poor survival. Despite trials exploring new treatment modalities to improve disease control rates, the 5 year survival rate remains low at only 60%. Most cancer malignancies are reported to progress to a fatal phase due to the metastatic activity derived from treatment-resistant cancer cells, regarded as one of the most significant obstacles to develope effective cancer treatment options. However, the molecular profiles of cancer cells have not been thoroughly studied. METHODS: Here, we examined in-situ HNSCC tumors and pairwisely followed up with the downstream circulating tumor cells (CTCs)-based on the surrogate biomarkers to detect metastasis that is established in other cancers - not yet being fully adopted in HNSCC treatment algorithms. RESULTS: Specifically, we revealed metastatic HNSCC patients have complex CTCs that could be defined through gene expression and mutational gene profiling derived from completed single-cell RNASeq (scRNASeq) that served to confirm molecular pathways inherent in these CTCs. To enhance the reliability of our findings, we cross-validated those molecular profiles with results from previously published studies. CONCLUSION: Thus, we identified 5 dysregulated signaling pathways in CTCs to derive HNSCC biomarker panels for screening HNSCC in situ tumors.

ObjectivesInvestigating the dysregulated signaling pathways of head and neck squamous cell carcinoma (HNSCC) linked with circulating tumor cells (CTCs) using single-cell molecular characterization.IntroductionHNSCC poses a significant global health burden with poor survival rates despite advancements in treatment. Metastatic activity from treatment-resistant cancer cells remains a major challenge in developing effective treatments. However, the molecular profiles of cancer cells, particularly CTCs, are not well-understood.MethodsWe analyzed in-situ HNSCC tumors and corresponding CTCs using surrogate biomarkers to detect metastasis, a technique not widely used in HNSCC treatment protocols.ResultsOur study revealed complex CTCs in metastatic HNSCC patients characterized by gene expression and mutational gene profiling via single-cell RNASeq (scRNASeq). These profiles confirmed molecular pathways inherent in CTCs, further validated by previous research.ConclusionThrough our research, we identified five dysregulated signaling pathways in CTCs, suggesting potential biomarker panels for HNSCC screening in situ tumors.

Reciprocal inhibition between TP63 and STAT1 regulates anti-tumor immune response through interferon-γ signaling in squamous cancer.

Squamous cell carcinomas (SCCs) are common and aggressive malignancies. Immune check point blockade (ICB) therapy using PD-1/PD-L1 antibodies has been approved in several types of advanced SCCs. However, low response rate and treatment resistance are common. Improving the efficacy of ICB therapy requires better understanding of the mechanism of immune evasion. Here, we identify that the SCC-master transcription factor TP63 suppresses interferon-γ (IFNγ) signaling. TP63 inhibition leads to increased CD8+ T cell infiltration and heighten tumor killing in in vivo syngeneic mouse model and ex vivo co-culture system, respectively. Moreover, expression of TP63 is negatively correlated with CD8+ T cell infiltration and activation in patients with SCC. Silencing of TP63 enhances the anti-tumor efficacy of PD-1 blockade by promoting CD8+ T cell infiltration and functionality. Mechanistically, TP63 and STAT1 mutually suppress each other to regulate the IFNγ signaling by co-occupying and co-regulating their own promoters and enhancers. Together, our findings elucidate a tumor-extrinsic function of TP63 in promoting immune evasion of SCC cells. Over-expression of TP63 may serve as a biomarker predicting the outcome of SCC patients treated with ICB therapy, and targeting TP63/STAT/IFNγ axis may enhance the efficacy of ICB therapy for this deadly cancer.

Activation of Epstein-Barr Virus' Lytic Cycle in Nasopharyngeal Carcinoma Cells by NEO212, a Conjugate of Perillyl Alcohol and Temozolomide.

The Epstein-Barr virus (EBV) is accepted as a primary risk factor for certain nasopharyngeal carcinoma (NPC) subtypes, where the virus persists in a latent stage which is thought to contribute to tumorigenesis. Current treatments are sub-optimal, and recurrence occurs in many cases. An alternative therapeutic concept is aimed at triggering the lytic cycle of EBV selectively in tumor cells as a means to add clinical benefit. While compounds able to stimulate the lytic cascade have been identified, their clinical application so far has been limited. We are developing a novel anticancer molecule, NEO212, that was generated by covalent conjugation of the alkylating agent temozolomide (TMZ) to the naturally occurring monoterpene perillyl alcohol (POH). In the current study, we investigated its potential to trigger the lytic cycle of EBV in NPC cells in vitro and in vivo. We used the established C666.1 cell line and primary patient cells derived from the brain metastasis of a patient with NPC, both of which harbored latent EBV. Upon treatment with NEO212, there was an increase in EBV proteins Zta and Ea-D, key markers of the lytic cycle, along with increased levels of CCAAT/enhancer-binding protein homologous protein (CHOP), a marker of endoplasmic reticulum (ER) stress, followed by the activation of caspases. These effects could also be confirmed in tumor tissue from mice implanted with C666.1 cells. Towards a mechanistic understanding of these events, we used siRNA-mediated knockdown of CHOP and inclusion of anti-oxidant compounds. Both approaches blocked lytic cycle induction by NEO212. Therefore, we established a sequence of events, where NEO212 caused reactive oxygen species (ROS) production, which triggered ER stress and elevated the levels of CHOP, which was required to stimulate the lytic cascade of EBV. Inclusion of the antiviral agent ganciclovir synergistically enhanced the cytotoxic impact of NEO212, pointing to a potential combination treatment for EBV-positive cancers which should be explored further. Overall, our study establishes NEO212 as a novel agent able to stimulate EBV's lytic cycle in NPC tumors, with implications for other virus-associated cancers.

Epigenomic analyses identify FOXM1 as a key regulator of anti-tumor immune response in esophageal adenocarcinoma.

Unlike most cancer types, the incidence of esophageal adenocarcinoma (EAC) has rapidly escalated in the western world over recent decades. Using whole genome bisulfite sequencing (WGBS), we identify the transcription factor (TF) FOXM1 as an important epigenetic regulator of EAC. FOXM1 plays a critical role in cellular proliferation and tumor growth in EAC patient-derived organoids and cell line models. We identify ERBB2 as an upstream regulator of the expression and transcriptional activity of FOXM1. Unexpectedly, gene set enrichment analysis (GSEA) unbiased screen reveals a prominent anti-correlation between FOXM1 and immune response pathways. Indeed, syngeneic mouse models show that FOXM1 inhibits the infiltration of CD8+ T cells into the tumor microenvironment. Consistently, FOXM1 suppresses CD8+ T cell chemotaxis in vitro and antigen-dependent CD8+ T cell killing. This study characterizes FOXM1 as a significant EAC-promoting TF and elucidates its novel function in regulating anti-tumor immune response.

Free Flap Outcomes for Head and Neck Surgery in Patients with COVID-19.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) affects the vascular system, subjecting patients to a hypercoagulable state. This is of particular concern for the success of microvascular free flap reconstruction. This study aims to report head and neck free flap complications in patients with COVID-19 during the perioperative period. We believe these patients are more likely to experience flap complications given the hypercoagulable state. METHODS: This is a multi-institutional retrospective case series of patients infected with COVID-19 during the perioperative period for head and neck free flap reconstruction from March 2020 to January 2022. RESULTS: Data was collected on 40 patients from 14 institutions. Twenty-one patients (52.5%) had a positive COVID-19 test within 10 days before surgery and 7 days after surgery. The remaining patients had a positive test earlier than 10 days before surgery. A positive test caused a delay in surgery for 16 patients (40.0%) with an average delay of 44.7 days (9-198 days). Two free flap complications (5.0%) occurred with no free flap deaths. Four patients (10.0%) had surgical complications and 10 patients had medical complications (25.0%). Five patients (12.5%) suffered from postoperative COVID-19 pneumonia. Three deaths were COVID-19-related and one from cancer recurrence during the study period. CONCLUSION: Despite the heightened risk of coagulopathy in COVID-19 patients, head and neck free flap reconstructions in patients with COVID-19 are not at higher risk for free flap complications. However, these patients are at increased risk of medical complications. LEVEL OF EVIDENCE: 4 Laryngoscope, 2023.

Comprehensive analyses of partially methylated domains and differentially methylated regions in esophageal cancer reveal both cell-type- and cancer-specific epigenetic regulation.

BACKGROUND: As one of the most common malignancies, esophageal cancer has two subtypes, squamous cell carcinoma and adenocarcinoma, arising from distinct cells-of-origin. Distinguishing cell-type-specific molecular features from cancer-specific characteristics is challenging. RESULTS: We analyze whole-genome bisulfite sequencing data on 45 esophageal tumor and nonmalignant samples from both subtypes. We develop a novel sequence-aware method to identify large partially methylated domains (PMDs), revealing profound heterogeneity at both methylation level and genomic distribution of PMDs across tumor samples. We identify subtype-specific PMDs that are associated with repressive transcription, chromatin B compartments and high somatic mutation rate. While genomic locations of these PMDs are pre-established in normal cells, the degree of loss is significantly higher in tumors. We find that cell-type-specific deposition of H3K36me2 may underlie genomic distribution of PMDs. At a smaller genomic scale, both cell-type- and cancer-specific differentially methylated regions (DMRs) are identified for each subtype. Using binding motif analysis within these DMRs, we show that a cell-type-specific transcription factor HNF4A maintains the binding sites that it generates in normal cells, while establishing new binding sites cooperatively with novel partners such as FOSL1 in esophageal adenocarcinoma. Finally, leveraging pan-tissue single-cell and pan-cancer epigenomic datasets, we demonstrate that a substantial fraction of cell-type-specific PMDs and DMRs identified here in esophageal cancer are actually markers that co-occur in other cancers originating from related cell types. CONCLUSIONS: These findings advance our understanding of DNA methylation dynamics at various genomic scales in normal and malignant states, providing novel mechanistic insights into cell-type- and cancer-specific epigenetic regulations.

Comparison of Free Flap Outcomes at a University Hospital versus County Hospital Setting for Head and Neck Reconstruction

Abstract Introduction Patients at public county hospitals often have poorer access to healthcare with advanced disease on presentation. These factors, along with limited resources at county hospitals, may have an impact on outcomes for patients requiring complex head and neck reconstruction. Objectives To delineate differences in the frequency of complications in two different care settings, a public county hospital and a private university hospital. Methods Retrospective review of otolaryngology patients at a university hospital compared with a publicly-funded county hospital. The main outcome measure was major complications including total flap loss or unplanned reoperation in 30 days. Secondary outcome measures included medical complications, partial flap loss, and unplanned hospital readmission in 30 days. Results In the county hospital sample (n = 58) free flap failure or reoperation occurred in 20.7% of the patients, and minor complications, in 36.2% of the patients. In the university hospital sample (n = 65) flap failure or reoperation occurred in 9.2% of the patients, and minor complications, in 12.3% of the patients. Patients at the private hospital who had surgery in the oropharynx were least likely to have minor complications. Conclusions Patients at the county hospital had a higher but not statistically significant difference in flap failure and reoperation than those at a university hospital, although the county hospital experienced more minor postoperative complications. This is likely multifactorial, and may be related to poorer access to primary care preoperatively, malnutrition, poorly controlled or undiagnosed medical comorbidities, and differences in hospital resources.

Comparison of Free Flap Outcomes at a University Hospital versus County Hospital Setting for Head and Neck Reconstruction.

Introduction Patients at public county hospitals often have poorer access to healthcare with advanced disease on presentation. These factors, along with limited resources at county hospitals, may have an impact on outcomes for patients requiring complex head and neck reconstruction. Objective To delineate differences in the frequency of complications in two different care settings, a public county hospital and a private university hospital. Methods Retrospective review of otolaryngology patients at a university hospital compared with a publicly-funded county hospital. The main outcome measure was major complications including total flap loss or unplanned reoperation in 30 days. Secondary outcome measures included medical complications, partial flap loss, and unplanned hospital readmission in 30 days. Results In the county hospital sample ( n = 58) free flap failure or reoperation occurred in 20.7% of the patients, and minor complications, in 36.2% of the patients. In the university hospital sample ( n = 65) flap failure or reoperation occurred in 9.2% of the patients, and minor complications, in 12.3% of the patients. Patients at the private hospital who had surgery in the oropharynx were least likely to have minor complications. Conclusion Patients at the county hospital had a higher but not statistically significant difference in flap failure and reoperation than those at a university hospital, although the county hospital experienced more minor postoperative complications. This is likely multifactorial, and may be related to poorer access to primary care preoperatively, malnutrition, poorly controlled or undiagnosed medical comorbidities, and differences in hospital resources.

A novel role of master regulator transcription factor in anti-tumor immunity and cancer immunotherapy.

Gene expression network in cancer cells is orchestrated by a small number of master regulator transcription factors (MRTFs), which play a prominent role in regulating cancer intrinsic hallmarks, such as sustaining proliferative signaling, evading growth suppressors, resisting cell death, etc. A new study reports a new role of one MRTF, KLF5, in regulating tumor microenvironment in an extrinsic manner. These findings not only reveal novel mechanistic underpinnings of tumor evasion from immune destruction but also broaden our understanding of the transcriptional deregulation in cancer biology.

Opioid Sparing Multimodal Analgesia for Transoral Robotic Surgery: Improved Analgesia and Narcotic Use Reduction.

Objective: To compare postoperative pain scores and opioid consumption in patients after transoral robotic surgery (TORS). Study Design: Single institution retrospective cohort study. Setting: TORS was performed at a single academic tertiary care center. Methods: This study compared traditional opioid-based and opioid-sparing multimodal analgesia (MMA) regimens in patients with oropharyngeal and supraglottic malignancy after TORS. Data were obtained from the electronic health records from August 2016 to December 2021. The average postoperative pain scores and total opioid consumption in morphine milligram equivalents were calculated for postoperative days (PODs) 0 to 3. The secondary objectives were to quantify and characterize opioid prescriptions upon hospital discharge. Results: A total of 114 patients were identified for this study, 58 patients in the non-MMA cohort and 56 in the MMA cohort. Postoperative pain levels in the MMA cohort were statistically lower on POD 0 (p = 0.001), POD 1 (p = 0.001), and POD 3 (p = 0.004). Postoperative opioid consumption decreased significantly in the MMA cohort from 37.7 to 10.8 mg on POD 0 (p = 0.002), 65.9 to 19.9 mg on POD 1 (p < 0.001), 36.0 to 19.3 mg on POD 2 (p = 0.02), and 45.4 to 13.8 mg on POD 3 (p = 0.02). The number of patients discharged from the hospital with a prescription for narcotics was significantly lower in the MMA cohort (71.4%) compared with the non-MMA cohort (98.3%) (p < 0.001). Conclusion: Implementation of our MMA pain protocol reduced pain levels and narcotic consumption in the immediate postoperative period.

Investigation of early neoplastic transformation and premalignant biology using genetically engineered organoid models.

Organoid modeling is a powerful, robust and efficient technology faithfully preserving physiological and pathological characteristics of tissues of origin. Recently, substantial advances have been made in applying genetically engineered organoid models to study early tumorigenesis and premalignant biology. These efforts promise to identify novel avenues for early cancer detection, intervention and prevention. Here, we highlight significant advancements in the functional characterization of early genomic and epigenomic events during neoplastic evolution using organoid modeling, discuss the application of the lineage-tracing methodology in organoids to study cancer cells-of-origin, and review future opportunities for further development and improvement of organoid modeling of cancer precursors.

Human papillomavirus detection in oral rinses and history of tonsillectomy in U.S. adults.

PURPOSE: This study aims to elucidate any relationship between prior tonsillectomy and the presence of oropharyngeal HPV DNA found in screening mouth rinses. MATERIALS AND METHODS: A cross sectional study was conducted using the 2011-2014 National Health and Nutrition Examination Survey (NHANES). Participants between 40 and 69 were included in the study and medical, surgical, and sexual health history were recorded. Multivariable analyses were conducted to examine factors associated with HPV prevalence in oral rinse samples. RESULTS: A total of 4825 participants were recorded with 21.1 % having a history of tonsillectomy. In the no tonsillectomy group, 8.6 % of respondents had a positive oral rinse for HPV, while 7.2 % of those with a tonsillectomy had a positive rinse sample. There was no association between age and HPV prevalence (OR = 1.04, 95 % CI: [1.00-1.07]). When controlling for demographics, medical history, and sexual behaviors, tonsillectomy history was not shown to have an association with HPV (OR = 0.86, 95 % CI: [0.53-1.40]). However, men, Hispanics, smokers, and those with higher lifetime sexual partners had increased odds of having a positive HPV oral rinse sample which was statistically significant. CONCLUSION: Our data showed that a history of tonsillectomy was not significantly associated with the presence of HPV in an oral rinse. However, a significant relationship was seen between the presence of HPV in oral rinses and certain demographic factors such as male gender, Hispanic race, smoking history, and increased sexual partners.

Variation in compensatory strategies as a function of target constriction degree in post-glossectomy speech.

Individuals who have undergone treatment for oral cancer oftentimes exhibit compensatory behavior in consonant production. This pilot study investigates whether compensatory mechanisms utilized in the production of speech sounds with a given target constriction location vary systematically depending on target manner of articulation. The data reveal that compensatory strategies used to produce target alveolar segments vary systematically as a function of target manner of articulation in subtle yet meaningful ways. When target constriction degree at a particular constriction location cannot be preserved, individuals may leverage their ability to finely modulate constriction degree at multiple constriction locations along the vocal tract.

An Exploration of Online Support Community Participation Among Patients With Vestibular Disorders.

OBJECTIVES/HYPOTHESIS: To formally document online support community (OSC) use among patients with vestibular symptoms and gain an appreciation for the perceived influence of participation on psychosocial outcomes and the impact on medical decision-making. STUDY DESIGN: Self reported internet-based questionnaire. METHODS: The Facebook search function was paired with a comprehensive list of vestibular diagnoses to systematically collect publicly available information on vestibular OSCs. Next, a survey was designed to gather clinicodemographic information, OSC characteristics, participation measures, perceived outcomes, and influence on medical decision-making. The anonymous instrument was posted to two OSCs that provide support for patients with general vestibular symptoms. RESULTS: Seventy-three OSCs were identified with >250,000 cumulative members and >10,000 posts per month. The survey was completed by 549 participants, a cohort of primarily educated middle-aged (median = 50, interquartile range 40-60), non-Hispanic white (84%), and female (89%) participants. The participants' most cited initial motivation and achieved goal of participants was to hear from others with the same diagnosis (89% and 88%, respectively). Daily users and those who reported seeing ≥5 providers before receiving a diagnosis indicated that OSC utilization significantly influenced their requested medical treatments (72% daily vs. 61% nondaily, P = .012; 61% <5 providers vs. 71% ≥5 providers P = .019, respectively). Most participants agreed that OSC engagement provides emotional support (74%) and helps to develop coping strategies (68%). Membership of ≥1 year was associated with a higher rate of learned coping skills (61% membership <1-year vs. 71% ≥1-year P = .016). CONCLUSIONS: The use of OSCs is widespread among vestibular diagnoses. A survey of two OSCs suggests these groups provide a significant source of peer support and can influence users' ability to interface with the medical system. LEVEL OF EVIDENCE: NA Laryngoscope, 132:1835-1842, 2022.

Evidence for AJUBA-catenin-CDH4-linked differentiation resistance of mesenchymal stem cells implies tumorigenesis and progression of head and neck squamous cell carcinoma: a single-cell transcriptome approach.

An increasing number of reports indicate that mesenchymal stem cells (MSCs) play an essential role in promoting tumorigenesis and progression of head and neck squamous cell carcinoma (HNSCC). However, the molecular mechanisms underlying this process remain unclear. Using the MSC model system, this study analyzes the molecular pathway by which differentiation resistant MSCs promote HNSCC. MSCs were cultured in osteogenic differentiation media and harvested on days 12 and 19. Cells were stained for cell differentiation analysis using Alizarin Red. The osteogenesis-resistant MSCs (OR-MSCs) and MSC-differentiation-derived osteoblasts (D-OSTBs) were identified and subjected to the single-cell transcriptome analysis. Gene-specific analyses of these two sub-populations were performed for the patterns of differential expression. A total of 1 780 differentially expressed genes were determined to distinguish OR-MSCs significantly from D-OSTB. Notably, AJUBA, ß-catenin, and CDH4 expression levels were upregulated considerably within the OR-MSCs compared to D-OSTBs. To confirm their clinical relevance, a survey of a clinical cohort revealed a high correlation among the expression levels of AJUBA, ß-catenin and CDH4. The results shed new light that OR-MSCs participate in the development of HNSCC via a pathway mediated by AJUBA, ß-catenin, CDH4, and CTNNB1, thereby implying that MSC-based therapy is a promising therapeutic approach in the management of HNSCC.

Complexity of vocal tract shaping in glossectomy patients and typical speakers: A principal component analysis.

The glossectomy procedure, involving surgical resection of cancerous lingual tissue, has long been observed to affect speech production. This study aims to quantitatively index and compare complexity of vocal tract shaping due to lingual movement in individuals who have undergone glossectomy and typical speakers using real-time magnetic resonance imaging data and Principal Component Analysis. The data reveal that (i) the type of glossectomy undergone largely predicts the patterns in vocal tract shaping observed, (ii) gross forward and backward motion of the tongue body accounts for more change in vocal tract shaping than do subtler movements of the tongue (e.g., tongue tip constrictions) in patient data, and (iii) fewer vocal tract shaping components are required to account for the patients' speech data than typical speech data, suggesting that the patient data at hand exhibit less complex vocal tract shaping in the midsagittal plane than do the data from the typical speakers observed.

Outcomes of Combined Antegrade-Retrograde Dilations for Radiation-Induced Esophageal Strictures in Head and Neck Cancer Patients.

The purpose of this study is to analyze outcomes of combined antegrade-retrograde dilations (CARD). This retrospective study was conducted on 14 patients with a history of head and neck cancer, treated with radiation therapy that was complicated by either complete or near-complete esophageal stenosis. All patients had minimal oral intake and depended on a gastrostomy tube for nutrition. Swallow function before and after CARD was assessed using the Functional Oral Intake Scale, originally developed for stroke patients and applied to head and neck cancer patients. Patients undergoing CARD demonstrated a quantifiable improvement in swallow function (p = 0.007) that persisted at last known follow-up (p = 0.015) but only a minority (23.1%) achieved oral intake sufficient to obviate the need for tube feeds. Complication rates were 24% per procedure or 36% per patient, almost all complications required procedural intervention, and all complications occurred in patients with complete stenosis. Our study suggests further caution when considering CARD, careful patient selection, and close post-operative monitoring.