Noncanonical HPV carcinogenesis drives radiosensitization of head and neck tumors.

We analyzed transcriptional data from 104 HPV+ (Human papillomavirus) HNSCC (head and neck squamous cell carcinoma) tumors together with two publicly available sources to identify highly robust transcriptional programs (modules) which could be detected consistently despite heterogeneous sequencing and quantification methodologies. Among 22 modules identified, we found a single module that naturally subclassifies HPV+ HNSCC tumors based on a bimodal pattern of gene expression, clusters all atypical features of HPV+ HNSCC biology into a single subclass, and predicts patient outcome in four independent cohorts. The subclass-defining gene set was strongly correlated with Nuclear factor kappa B (NF-κB) target expression. Tumors with high expression of this NF-κB module were rarely associated with activating PIK3CA alterations or viral integration, and also expressed higher levels of HPHPV E2 and had decreased APOBEC mutagenesis. Alternatively, they harbored inactivating alterations of key regulators of NF-κB, TNF receptor associated factor 3 (TRAF3), and cylindromatosis (CYLD), as well as retinoblastoma protein (RB1). HPV+ HNSCC cells in culture with experimental depletion of TRAF3 or CYLD displayed increased expression of the subclass-defining genes, as well as robust radio-sensitization, thus recapitulating both the tumor transcriptional state and improved treatment response observed in patient data. Across all gene sets investigated, methylation to expression correlations were the strongest for the subclass-defining, NF-κB-related genes. Increased tumor-infiltrating CD4+ T cells and increased Estrogen receptors alpha (ERα) expression were identified in NF-κB active tumors. Based on the relatively high rates of cure in HPV+ HNSCC, deintensification of therapy to reduce treatment-related morbidity is being studied at many institutions. Tumor subclassification based on oncogenic subtypes may help guide the selection of therapeutic intensity or modality for patients with HPV+ HNSCC.

Human Immunodeficiency Virus Is Associated With Poor Overall Survival Among Patients With Head and Neck Cancer.

BACKGROUND: Head and neck squamous cell cancer (HNSCC) occurs at higher rates among persons with HIV (PWH). This study compares the impact of sociodemographic and clinicopathologic characteristics on outcomes among PWH-HNSCC compared with HNSCC patients without HIV. METHODS: Patient data from HNSCC individuals were collected at a single academic hospital center between 2002 and 2018. Forty-eight patients with HIV (HIV-HNSCC) and 2894 HNSCC patients without HIV were included. Multivariate analysis determined predictors of survival using Cox proportional hazards regression model. HIV-positive and -negative tumors were analyzed by quantitative immunofluorescence for expression of CD4, CD8, CD20 and PD-L1. RESULTS: HIV-HNSCC patients had a lower median overall survival than HNSCC patients without HIV (34 [18-84] vs 94 [86-103] months; P < .001). In multivariate analysis that included age, sex, race/ethnicity, stage, site, tobacco use, time to treatment initiation, and insurance status, HIV was an independent predictor of poorer survival, with a hazard ratio of 1.98 (95% CI: 1.32-2.97; P < .001). PWH with human papillomavirus (HPV)-positive oropharyngeal tumors also had worse prognosis than HPV-positive oropharyngeal tumors in the population without HIV (P < .001). The tumor microenvironment among HIV-HNSCC patients revealed lower intratumoral CD8 infiltration among HIV+ HPV+ tumors compared with HIV- HPV+ tumors (P = .04). CONCLUSIONS: HIV-HNSCC patients had worse prognosis than the non-HIV population, with HIV being an independent predictor of poor clinical outcomes when accounting for important sociodemographic and clinicopathologic factors. Our findings highlight differences in tumor biology that require further detailed characterization in large cohorts and increased inclusion of PWH in immunotherapy trials.

A phase 2 study of neoadjuvant chemotherapy plus durvalumab in resectable locally advanced head and neck squamous cell carcinoma.

BACKGROUND: Patients with locally advanced head and neck squamous cell cancer (HNSCC) are treated with surgery followed by adjuvant (chemo) radiotherapy or definitive chemoradiation, but recurrence rates are high. Immune checkpoint blockade improves survival in patients with recurrent/metastatic HNSCC; however, the role of chemo-immunotherapy in the curative setting is not established. METHODS: This phase 2, single-arm, multicenter study evaluated neoadjuvant chemo-immunotherapy with carboplatin, nab-paclitaxel, and durvalumab in patients with resectable locally advanced HNSCC. The primary end point was a hypothesized pathologic complete response rate of 50%. After chemo-immunotherapy and surgical resection, patients received study-defined, pathologic risk adapted adjuvant therapy consisting of either durvalumab alone (low risk), involved field radiation plus weekly cisplatin and durvalumab (intermediate risk), or standard chemoradiation plus durvalumab (high risk). RESULTS: Between December 2017 and November 2021, 39 subjects were enrolled at three centers. Oral cavity was the most common primary site (69%). A total of 35 of 39 subjects underwent planned surgical resection; one subject had a delay in surgery due to treatment-related toxicity. The most common treatment-related adverse events were cytopenias, fatigue, and nausea. Post treatment imaging demonstrated an objective response rate of 57%. Pathologic complete response and major pathologic response were achieved in 29% and 49% of subjects who underwent planned surgery, respectively. The 1-year progression-free survival was 83.8% (95% confidence interval, 67.4%-92.4%). CONCLUSIONS: Neoadjuvant carboplatin, nab-paclitaxel, and durvalumab before surgical resection of HNSCC were safe and feasible. Although the primary end point was not met, encouraging rates of pathologic complete response and clinical to pathologic downstaging were observed.

Characteristics and outcomes associated with anxiety and depression in a head and neck cancer survivorship cohort.

PURPOSE: To assess the prevalence and predictors of mental health disorders (MDHs) among head and neck squamous cell carcinoma (HNSCC) survivors, and the association with health-related quality of life (HRQOL), pain, and survival outcomes. MATERIALS AND METHODS: This was a retrospective, cross-sectional study of HNSCC survivors surveyed at an outpatient oncology clinic from May 2012 through July 2016. RESULTS: Among 198 HNSCC survivors, 21% reported a MHD. Female sex (OR 6.60, 95% CI 2.08 to 20.98; p = 0.001) and Medicare insurance status (OR 4.95, 95% CI 1.52 to 16.11; p = 0.008) were significant predictors of reporting a MHD in the fully adjusted model. Patients reporting a MHD reported significantly worse pain (p < 0001) and worse HRQOL on the PROMIS Physical (p < 0.001), PROMIS Mental (p < 0.001), and FACT-GP (p < 0.026) questionnaires. Diagnosis of a MHD was not correlated with 5-year OS (74% vs. 84%; p = 0.087). CONCLUSION: Initiatives for early identification and intervention of MHDs as part of survivorship initiatives may engender clinically meaningful outcomes in head and neck cancer.

Genomic heterogeneity and copy number variant burden are associated with poor recurrence-free survival and 11q loss in human papillomavirus-positive squamous cell carcinoma of the oropharynx.

BACKGROUND: Human papillomavirus-positive (HPV+) squamous cell carcinoma of the oropharynx (OPSCC) is the most prevalent HPV-associated malignancy in the United States. Favorable treatment outcomes have led to increased interest in treatment de-escalation to reduce treatment morbidity as well as the development of prognostic markers to identify appropriately low-risk patients. Intratumoral genomic heterogeneity and copy number alteration burden have been demonstrated to be predictive of poor outcomes in many other cancers; therefore, we sought to determine whether intratumor heterogeneity and genomic instability are associated with poor outcomes in HPV+ OPSCC. METHODS: Tumor heterogeneity estimates were made based on targeted exome sequencing of 45 patients with HPV+ OPSCC tumors. Analysis of an additional cohort of HPV+ OPSCC tumors lacking matched normal sequencing allowed copy number analysis of 99 patient tumors. RESULTS: High intratumorally genomic heterogeneity and high numbers of copy number alterations were strongly associated with worse recurrence-free survival. Tumors with higher heterogeneity and frequent copy number alterations were associated with loss of distal 11q, which encodes key genes related to double-strand break repair, including ATM and MRE11A. CONCLUSIONS: Both intratumor genomic heterogeneity and high-burden copy number alterations are strongly associated with poor recurrence-free survival in patients with HPV+ OPSCC. The drivers of genomic instability and heterogeneity in these tumors remains to be elucidated. However, 11q loss and defective DNA double-strand break repair have been associated with genomic instability in other solid tumors. Copy number alteration burden and intratumoral heterogeneity represent promising avenues for risk stratification of patients with HPV+OPSCC.

Treatment deintensification in human papillomavirus-positive oropharynx cancer: Outcomes from the National Cancer Data Base.

BACKGROUND: The growing epidemic of human papillomavirus-positive (HPV+) oropharyngeal cancer and the favorable prognosis of this disease etiology have led to a call for deintensified treatment for some patients with HPV+ cancers. One of the proposed methods of treatment deintensification is the avoidance of chemotherapy concurrent with definitive/adjuvant radiotherapy. To the authors' knowledge, the safety of this form of treatment de-escalation is unknown and the current literature in this area is sparse. The authors investigated outcomes after various treatment combinations stratified by American Joint Committee on Cancer (AJCC) eighth edition disease stage using patients from the National Cancer Data Base. METHODS: A retrospective study of 4443 patients with HPV+ oropharyngeal cancer in the National Cancer Data Base was conducted. Patients were stratified into AJCC eighth edition disease stage groups. Multivariate Cox regressions as well as univariate Kaplan-Meier analyses were conducted. RESULTS: For patients with stage I disease, treatment with definitive radiotherapy was associated with diminished survival compared with chemoradiotherapy (hazard ratio [HR], 1.798; P = .029), surgery with adjuvant radiotherapy (HR, 2.563; P = .002), or surgery with adjuvant chemoradiotherapy (HR, 2.427; P = .001). For patients with stage II disease, compared with treatment with chemoradiotherapy, patients treated with a single-modality (either surgery [HR, 2.539; P = .009] or radiotherapy [HR, 2.200; P = .030]) were found to have poorer survival. Among patients with stage III disease, triple-modality therapy was associated with improved survival (HR, 0.518; P = .024) compared with treatment with chemoradiotherapy. CONCLUSIONS: Deintensification of treatment from chemoradiotherapy to radiotherapy or surgery alone in cases of HPV+ AJCC eighth edition stage I or stage II disease may compromise patient safety. Treatment intensification to triple-modality therapy for patients with stage III disease may improve survival in this group. Cancer 2018;124:717-26. © 2017 American Cancer Society.

TRAF3/CYLD mutations identify a distinct subset of human papillomavirus-associated head and neck squamous cell carcinoma.

BACKGROUND: The incidence of human papillomavirus (HPV)-associated (HPV-positive) head and neck squamous cell carcinoma (HNSCC) of the oropharynx has dramatically increased over the last decade and continues to rise. Newly diagnosed HPV-positive HNSCCs in the United States currently outnumber any other HPV-associated cancers, including cervical cancer. Despite introduction of the HPV vaccine, the epidemic of HPV-positive HNSCC is expected to continue for approximately 60 years. Compared with patients who have tobacco-associated HNSCC, those who have HPV-positive HNSCC have better overall survival and response to treatment. Current treatment, including chemotherapy and radiation therapy, is associated with lifelong morbidity, and there are limited treatments and no curative options for patients who develop recurrent metastatic disease. Therapeutic de-escalation (decreased radiation dose) is being tested through clinical trials; however, those studies select patients based solely on tumor and patient smoking characteristics. Mechanisms of HPV-driven carcinogenesis in HNSCC are not well understood, which limits new therapeutic strategies and hinders the appropriate selection of patients for de-escalation therapy. METHODS: The authors analyzed HNSCC data from The Cancer Genome Atlas to identify molecular characteristics that correlate with outcomes and integration status of the HPV genome. RESULTS: The current investigations identified a subset of HPV-positive HNSCCs with mutations in the genes TRAF3 (tumor necrosis factor receptor-associated factor 3) and CYLD (cylindromatosis lysine 63 deubiquitinase). Defects in TRAF3 and CYLD correlated with the activation of transcriptional factor nuclear factor κB, episomal HPV status of tumors, and improved patient survival. CONCLUSIONS: Defects in TRAF3/CYLD were accompanied with the activation of nuclear factor κB signaling and maintenance of episomal HPV in tumors, suggesting that these mutations may support an alternative mechanism of HPV tumorigenesis in head and neck tumors. Cancer 2017;123:1778-1790. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

The prognostic value of extranodal extension in human papillomavirus-associated oropharyngeal squamous cell carcinoma.

BACKGROUND: Extranodal (or extracapsular) extension (ENE) is an adverse prognostic factor in patients with head and neck cancers who undergo primary surgery. However, the significance of ENE in human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is not well established, and single-institution studies have not established that ENE predicts inferior outcome. The authors investigated the prognostic value of ENE in HPV-positive patients who underwent primary surgery and whether adjuvant chemoradiation improved overall survival (OS) compared with radiation alone in ENE-positive patients. METHODS: Patients who underwent primary surgery for pathologic T1 (pT1) through pT4 tumors, pathologic N1 (pN1) through pN3 lymph node status, HPV-positive OPSCC were identified in the National Cancer Data Base from 2010 through 2012. Features associated with ENE were analyzed. Univariable and multivariable Cox regression analyses identified predictors of OS. The effect of adjuvant treatment on OS in ENE-positive cohort was also evaluated. RESULTS: In total, 1043 patients met inclusion criteria, among whom 43.5% were ENE-positive. Of the ENE-positive patients who had treatment details available, 72% received concurrent chemoradiotherapy, 16% received radiotherapy, and 12% received no adjuvant treatment. After a median follow-up of 28.4 months, ENE was associated with worse 3-year OS (89.3% vs 93.6%; P = .01). On multivariable analysis that included involved lymph nodes, only ENE, lymphovascular invasion, pT3/pT4 tumors, and Charlson-Deyo score were associated with worse OS. Among ENE-positive patients, there was no difference in 3-year OS between those who received adjuvant concurrent chemoradiotherapy versus radiotherapy alone (89.6% vs 89.3%, respectively; P = .55). Propensity score-matched comparison revealed similar results. CONCLUSIONS: ENE is associated with inferior OS in patients with HPV-positive OPSCC. However, OS was not better with adjuvant chemoradiotherapy compared with radiotherapy alone in ENE-positive patients. The current findings support the need for prospective studies of adjuvant chemoradiation in HPV-positive patients with ENE. Cancer 2017;123:2762-72. © 2017 American Cancer Society.

Characterization of HPV and host genome interactions in primary head and neck cancers.

Previous studies have established that a subset of head and neck tumors contains human papillomavirus (HPV) sequences and that HPV-driven head and neck cancers display distinct biological and clinical features. HPV is known to drive cancer by the actions of the E6 and E7 oncoproteins, but the molecular architecture of HPV infection and its interaction with the host genome in head and neck cancers have not been comprehensively described. We profiled a cohort of 279 head and neck cancers with next generation RNA and DNA sequencing and show that 35 (12.5%) tumors displayed evidence of high-risk HPV types 16, 33, or 35. Twenty-five cases had integration of the viral genome into one or more locations in the human genome with statistical enrichment for genic regions. Integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and both inter- and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Cancers with integrated vs. nonintegrated HPV displayed different patterns of DNA methylation and both human and viral gene expressions. Together, these data provide insight into the mechanisms by which HPV interacts with the human genome beyond expression of viral oncoproteins and suggest that specific integration events are an integral component of viral oncogenesis.

Proposing prognostic thresholds for lymph node yield in clinically lymph node-negative and lymph node-positive cancers of the oral cavity.

BACKGROUND: Prognostic lymph node yield thresholds have been identified and incorporated into treatment guidelines for multiple cancer sites, but not for oral cancer. The objective of this study was to identify optimal thresholds in elective and therapeutic neck dissection for oral cavity cancers. METHODS: Patients with oral cavity cancers in the National Cancer Database (NCDB) were stratified into clinically lymph node-negative (cN0) and clinically lymph node-positive (cN+) cohorts to reflect the differing surgical management for these diseases. Univariate and multivariate analyses were performed to assess the relation between lymph node yield and overall survival, adjusting for other prognostic factors. Thresholds derived from the NCDB were validated in the Surveillance, Epidemiology, and End Results database. RESULTS: In patients with cN0 cancers of the oral cavity from the NCDB, those who had <16 lymph nodes had significantly decreased survival. The proportion of positive lymph nodes was higher for patients who had ≥16 lymph nodes (27.2% vs 16.3% for < 16 lymph nodes; P < .001). This threshold was validated in 2715 lymph node-negative cancers from SEER, with a mortality hazard ratio of 0.825 for ≥ 16 lymph nodes (95% confidence interval, 0.764-0.950; P = .004). In patients with cN + oral cavity cancers from the NCDB, groups with <26 lymph nodes had significantly decreased survival. This threshold was validated in 1903 lymph node-positive cancers from SEER, with a mortality hazard ratio of 0.791 (95% confidence interval, 0.692-0.903; P = .001). Academic centers, higher volume centers, and geographic location predicted higher lymph node yields. CONCLUSIONS: More extensive neck dissection (≥16 lymph nodes in cN0, ≥ 26 lymph nodes in cN+) was associated with better survival. Further evaluation of practice patterns in lymph node yield may represent an opportunity for improved quality of care. Cancer 2016;122:3624-31. © 2016 American Cancer Society.

Treatment trends and survival effects of chemotherapy for hypopharyngeal cancer: Analysis of the National Cancer Data Base.

BACKGROUND: The current study was performed to characterize trends and survival outcomes for chemotherapy in the definitive and adjuvant treatment of hypopharyngeal cancer in the United States. METHODS: A total of 16,248 adult patients diagnosed with primary hypopharyngeal cancer without distant metastases between 1998 and 2011 were identified in the National Cancer Data Base. The association between treatment modality and overall survival was analyzed using Kaplan-Meier survival curves and 5-year survival rates. A multivariate Cox regression analysis was performed on a subset of 3357 cases to determine the treatment modalities that predict improved survival when controlling for demographic and clinical factors. RESULTS: There was a significant increase in the use of chemotherapy with radiotherapy both as definitive treatment (P<.001) and as adjuvant chemoradiotherapy with surgery (P=.001). This was accompanied by a decrease in total laryngectomy/pharyngectomy rates (P<.001). Chemoradiotherapy was associated with improved 5-year survival compared with radiotherapy alone in the definitive setting (31.8% vs 25.2%; log rank P<.001). Similarly, in multivariateanalysis, definitive radiotherapy was found to be associated with compromised survival compared with definitive chemoradiotherapy (hazard ratio, 1.51; P<.001). CONCLUSIONS: Survival analysis revealed that overall 5-year survival rates were higher for chemoradiotherapy compared with radiotherapy alone in the definitive setting, but were comparable between surgery with chemoradiotherapy and surgery with radiotherapy. Cancer 2016;122:1853-60. © 2016 American Cancer Society.

LZAP, a putative tumor suppressor, selectively inhibits NF-kappaB.

LZAP has been reported to inhibit cellular proliferation and clonogenic growth. Here, we report that decreased LZAP expression promoted cellular transformation, xenograft tumor growth, and xenograft tumor vascularity. Loss of LZAP also increased cellular invasion, and MMP-9 expression dependent on NF-kappaB. LZAP directly bound to RelA, impaired serine 536 phosphorylation of RelA, increased HDAC association with RelA, inhibited basal and stimulated NF-kappaB transcriptional activity, and was found at the promoter of selective NF-kappaB-responsive genes. LZAP protein levels were markedly decreased in 32% of primary HNSCCs (n = 28) and decreased LZAP levels in primary HNSCC correlated with increased expression of the NF-kappaB-regulated genes IL-8 and IkappaBalpha. In aggregate, these data support a role of LZAP in NF-kappaB regulation and tumor suppression.

Alpha-2 Heremans Schmid Glycoprotein (AHSG) modulates signaling pathways in head and neck squamous cell carcinoma cell line SQ20B.

This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-ß was examined to determine whether levels of the TGF-ß binding AHSG influenced the effect of TGF-ß on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-ß influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis.

Trends and variations in the use of adjuvant therapy for patients with head and neck cancer.

BACKGROUND: The National Comprehensive Cancer Network guidelines recommend that patients with surgically resected head and neck cancers that have adverse pathologic features should receive adjuvant therapy in the form of radiotherapy (RT) or chemoradiation (CRT). To the authors' knowledge, the current study is the first analysis of temporal trends and use patterns of adjuvant therapy for these patients. METHODS: Patients with head and neck cancer and adverse pathologic features were identified in the National Cancer Data Base (1998-2011). Data were analyzed using chi-square, Student t, and log-rank tests; multivariate logistic regression; and Cox multivariate regression. RESULTS: A total of 73,088 patients were identified: 41.5% had received adjuvant RT, 33.5% had received adjuvant CRT, and 25.0% did not receive any adjuvant therapy. From 1998 to 2011, the increase in the use of adjuvant CRT was greatest for patients with oral cavity (6-fold) and laryngeal (5-fold) cancers. Multivariate analysis demonstrated that Medicare/Medicaid insurance (odds ratio [OR], 1.05; 95% confidence interval [95% CI], 1.01-1.11), distance ≥34 miles from the cancer center (OR, 1.66; 95% CI, 1.59-1.74), and academic (OR, 1.26; 95% CI, 1.20-1.31) and high-volume (OR, 1.10; 95% CI, 1.05-1.15) centers were independently associated with patients not receiving adjuvant therapy. Receipt of adjuvant therapy was found to be independently associated with improved overall survival (hazard ratio, 0.84; 95% CI, 0.81-0.86). CONCLUSIONS: Approximately 25% of patients are not receiving National Comprehensive Cancer Network guideline-directed adjuvant therapy. Patient-level and hospital-level factors are associated with variations in the receipt of adjuvant therapy. Further evaluation of these differences in practice patterns is needed to standardize practice and potentially improve the quality of care. Cancer 2014;120:3353-3360. © 2014 American Cancer Society.

Global stability of plasma proteomes for mass spectrometry-based analyses.

Peptide-based mass spectrometry approaches, such as multiple reaction monitoring, provide a powerful means to measure candidate protein biomarkers in plasma. A potential confounding problem is the effect of preanalytical variables, which may affect the integrity of proteins and peptides. Although some blood proteins undergo rapid physiological proteolysis ex vivo, the stability of most plasma proteins to preanalytical variables remains largely unexplored. We applied liquid chromatography-tandem mass spectrometry shotgun proteomics and multiple reaction monitoring analyses to characterize the stability of proteins at the peptide level in plasma. We systematically evaluated the effects of delay in plasma preparation at different temperatures, multiple freeze-thaw cycles and erythocyte hemolysis on peptide and protein inventories in prospectively collected human plasma. Time course studies indicated few significant changes in peptide and protein identifications, semitryptic peptides and methionine-oxidized peptides in plasma from blood collected in EDTA plasma tubes and stored for up to a week at 4 °C or room temperature prior to plasma isolation. Similarly, few significant changes were observed in similar analyses of plasma subjected to up to 25 freeze-thaw cycles. Hemolyzed samples produced no significant differences beyond the presence of hemoglobin proteins. Finally, paired comparisons of plasma and serum samples prepared from the same patients also yielded few significant differences, except for the depletion of fibrinogen in serum. Blood proteins thus are broadly stable to preanalytical variables when analyzed at the peptide level. Collection protocols to generate plasma for multiple reaction monitoring-based analyses may have different requirements than for other analyses directed at intact proteins.

De-Escalated Therapy and Early Treatment of Recurrences in HPV-Associated Head and Neck Cancer: The Potential for Biomarkers to Revolutionize Personalized Therapy.

Human papillomavirus-associated (HPV+) head and neck squamous cell carcinoma (HNSCC) is the most common HPV-associated cancer in the United States, with a rapid increase in incidence over the last two decades. The burden of HPV+ HNSCC is likely to continue to rise, and given the long latency between infection and the development of HPV+ HNSCC, it is estimated that the effect of the HPV vaccine will not be reflected in HNSCC prevalence until 2060. Efforts have begun to decrease morbidity of standard therapies for this disease, and its improved characterization is being leveraged to identify and target molecular vulnerabilities. Companion biomarkers for new therapies will identify responsive tumors. A more basic understanding of two mechanisms of HPV carcinogenesis in the head and neck has identified subtypes of HPV+ HNSCC that correlate with different carcinogenic programs and that identify tumors with good or poor prognosis. Current development of biomarkers that reliably identify these two subtypes, as well as biomarkers that can detect recurrent disease at an earlier time, will have immediate clinical application.

The Toxicological Effects of e-Cigarette Use in the Upper Airway: A Scoping Review.

OBJECTIVE: While evidence continues to emerge on the negative health effects of electronic cigarettes (e-cigarettes) on the lungs, little is known regarding their deleterious effects on the upper airway. The purpose of this review is to summarize the toxicological effects of e-cigarettes, and their components, on the upper airway. DATA SOURCES: PubMed, SCOPUS, EMBASE databases. REVIEW METHODS: Systematic searches were performed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines from 2003 to 2023. Studies were included if they investigated the toxicological effects of e-cigarette exposure on human or animal upper airway tissue. Two authors independently screened, reviewed, and appraised all included articles. RESULTS: A total of 822 unique articles were identified, of which 53 met inclusion criteria and spanned subsites including the oral cavity (22/53 studies), nasal cavity/nasopharynx (13/53), multiple sites (10/53), larynx (5/53), trachea (2/53), and oropharynx (1/53). The most commonly observed consequences of e-cigarette use on the upper airway included: proinflammatory (15/53 studies), histological (13/53), cytotoxicity (11/53), genotoxicity (11/53), and procarcinogenic (6/53). E-cigarette humectants independently induced toxicity at multiple upper airway subsites, however, effects were generally amplified when flavoring(s) and/or nicotine were added. Across almost all studies, exposure to cigarette smoke exhibited increased toxicity in the upper airway compared with exposure to e-cigarette vapor. CONCLUSION: Current data suggest that while e-cigarettes are generally less harmful than traditional cigarettes, they possess a distinct toxicological profile that is enhanced upon the addition of flavoring(s) and/or nicotine. Future investigations into underexamined subsites, such as the oropharynx and hypopharynx, are needed to comprehensively understand the effects of e-cigarettes on the upper airway.

Pediatric Robotic Laryngeal Cleft Repair.

OBJECTIVE: Compare surgical and swallow outcomes in robotic versus traditional laryngeal cleft (LC) repairs. STUDY DESIGN: Retrospective cohort study. SETTING: Tertiary care pediatric hospital. METHODS: Pediatric patients who underwent robotic or traditional (open or endoscopic) LC repair between 2010 and 2021 were identified. Patient characteristics, operative times, adverse events, hospital length of stay (LOS), and modified barium swallow study (MBSS) results were compared. RESULTS: Eighteen robotic and thirty traditional LC repairs were identified. Mean surgical (149 vs 111 min, P < .05) and OR times (207 vs 139 min, P < .002) were increased for robotic type I LC repairs, but were similar for type II and III LC. Mean hospital LOS was increased for robotic type I LC repairs (2.6 vs 1.2 days, P < .006), but was decreased for type II (4 vs 12.2 days) and type III (4.3 vs 94.5 days) LC. Postoperative MBSS results were improved for robotic type I LC repairs at 12 months (82% vs 43%, P = .05), and trended toward improvement at 6 months for type II (75% vs 22%), and type III (67% vs 50%) LC repairs, although significance was limited for type II and III LC due to the number of subjects. A robotic approach was used successfully to revise all recurrent LC that failed traditional repairs. CONCLUSION: Robotic type 1 LC repairs demonstrated increased operative times and hospital LOS but improved postoperative swallow outcomes compared to traditional approaches may be particularly useful in cases of recurrent clefts.

Molecular characterization of the salivary adenoid cystic carcinoma immune landscape by anatomic subsites.

Adenoid cystic carcinoma (AdCC) is a slow-growing salivary gland malignancy that relapses frequently. AdCCs of the submandibular gland exhibit unique differences in prognosis and treatment response to adjuvant radiotherapy compared to other sites, yet the role of tumor anatomic subsite on gene expression and tumor immune microenvironment (TIME) composition remains unclear. We used 87 samples, including 48 samples (27 AdCC and 21 normal salivary gland tissue samples) from 4 publicly available AdCC RNA sequencing datasets, a validation set of 33 minor gland AdCCs, and 39 samples from an in-house cohort (30 AdCC and 9 normal salivary gland samples). RNA sequencing data were used for single sample gene set enrichment analysis and TIME deconvolution. Quantitative PCR and multiplex immunofluorescence were performed on the in-house cohort. Wilcoxon rank-sum, nonparametric equality-of-medians tests and linear regression models were used to evaluate tumor subsite differences. AdCCs of different anatomic subsites including parotid, submandibular, sublingual, and minor salivary glands differed with respect to expression of several key tumorigenic pathways. Among the three major salivary glands, the reactive oxygen species (ROS)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway signature was significantly underexpressed in AdCC of submandibular compared to parotid and sublingual glands while this association was not observed among normal glands. Additionally, the NRF2 pathway, whose expression was associated with favorable overall survival, was overexpressed in AdCCs of parotid gland compared to minor and submandibular glands. The TIME deconvolution identified differences in CD4+ T cell populations between AdCC of major and minor glands and natural killer (NK) cells among AdCC of minor, submandibular, and parotid glands while plasma cells were enriched in normal submandibular glands compared to other normal gland controls. Our data reveal key molecular differences in AdCC of different anatomic subsites. The ROS and NRF2 pathways are underexpressed in submandibular and minor AdCCs compared to parotid gland AdCCs, and NRF2 pathway expression is associated with favorable overall survival. The CD4+ T, NK, and plasma cell populations also vary by tumor subsites, suggesting that the observed submandibular AdCC tumor-intrinsic pathway differences may be responsible for influencing the TIME composition and survival differences.

Transoral robotic surgery for pediatric upper airway pathology: An institutional update.

OBJECTIVE: Provide an update on our institution's experience with utilizing transoral robotic surgery (TORS) in pediatric airway surgery and compare these results to surgery by traditional methods. METHODS: Pediatric patients who underwent TORS for treatment of upper airway pathology between 2010 and 2021 at our institution were retrospectively identified and compared to patients with the same or similar pathology who underwent a traditional (open or endoscopic) surgical approach over the same time period. Outcomes of interest included patient demographics, operative times, adverse events, hospital length of stay (LOS), and modified barium swallow (MBSS) results. RESULTS: Forty children (19M, 21F) underwent 46 TORS procedures. Mean age was 6.4 years (range: 6 days-17 years). Most commonly treated pathology included: laryngeal clefts (LC) (n = 18), lymphatic malformations (n = 9), and base of tongue masses (n = 7). Surgical time was decreased in traditional type I LC repairs (mean: 111 vs 149 min, P = 0.04) and lymphatic malformation excisions (59 vs 120 min, p = 0.005). Hospital LOS was increased in TORS type I LC repairs (2.6 vs 1.2 days, P = 0.04). Adverse event rate was similar between TORS and traditional cohorts (17 % vs 16 % cases, P = 0.9). Postoperative MBSS results were improved for TORS type I LC repairs at 6 months (70 % vs 33 %, P = 0.09) and 12 months (82 % vs 43 %, P = 0.05). CONCLUSIONS: Pediatric TORS is practical and safe and has comparable outcomes to traditional surgery. Robotic-assisted LC repair displayed improved postoperative swallow results versus traditional approaches and may be particularly useful in recurrent cases.