PD-1 blockade synergizes with intratumoral vaccination of a therapeutic HPV protein vaccine and elicits regression of tumor in a preclinical model.

INTRODUCTION: The human papillomavirus (HPV) encoded oncoproteins E6 and E7 are constitutively expressed in HPV-associated cancers, making them logical therapeutic targets. Intramuscular immunization of patients with HPV16 L2E7E6 fusion protein vaccine (TA-CIN) is well tolerated and induces HPV-specific cellular immune responses. Efficacy of PD-1 immune checkpoint blockade correlates with the level of tumor-infiltrating CD8 + T cells, yet most patients lack significant tumor infiltration of immune cells making immune checkpoint blockade suboptimal. We hypothesized that intratumoral vaccination with TA-CIN could increase the number of tumor-infiltrating CD8 + T cells, synergize with PD-1 blockade and result in better control of tumors compared with either PD-1 blockade or vaccination alone. METHODS: We examined the immunogenicity and antitumor effects of intratumoral vaccination with TA-CIN alone or in combination with PD-1 blockade in the TC-1 syngeneic murine tumor model expressing HPV16 E6/E7. RESULTS: Intratumoral vaccination with TA-CIN induced stronger antigen-specific CD8 + T cell responses and antitumor effects. Intratumoral TA-CIN vaccination generated a systemic immune response that was able to control distal TC-1 tumors. Furthermore, intratumoral TA-CIN vaccination induced tumor infiltration of antigen-specific CD8 + T cells. Knockout of Batf3 abolished antigen-specific CD8 + T cell responses and antitumor effects of intratumoral TA-CIN vaccination. Finally, PD-1 blockade synergizes with intratumoral TA-CIN vaccination resulting in significantly enhanced antigen-specific CD8 + T cell responses and complete regression of tumors, whereas either alone failed to control established TC-1 tumor. CONCLUSIONS: Our results provide rationale for future clinical testing of intratumoral TA-CIN vaccination in combination with PD-1 blockade for the control of HPV16-associated tumors.

NF-κB and stat3 transcription factor signatures differentiate HPV-positive and HPV-negative head and neck squamous cell carcinoma.

Using high-throughput analyses and the TRANSFAC database, we characterized TF signatures of head and neck squamous cell carcinoma (HNSCC) subgroups by inferential analysis of target gene expression, correcting for the effects of DNA methylation and copy number. Using this discovery pipeline, we determined that human papillomavirus-related (HPV+) and HPV- HNSCC differed significantly based on the activity levels of key TFs including AP1, STATs, NF-κB and p53. Immunohistochemical analysis confirmed that HPV- HNSCC is characterized by co-activated STAT3 and NF-κB pathways and functional studies demonstrate that this phenotype can be effectively targeted with combined anti-NF-κB and anti-STAT therapies. These discoveries correlate strongly with previous findings connecting STATs, NF-κB and AP1 in HNSCC. We identified five top-scoring pair biomarkers from STATs, NF-κB and AP1 pathways that distinguish HPV+ from HPV- HNSCC based on TF activity and validated these biomarkers on TCGA and on independent validation cohorts. We conclude that a novel approach to TF pathway analysis can provide insight into therapeutic targeting of patient subgroup for heterogeneous disease such as HNSCC.

Study protocol: randomised controlled trial of conditioned open-label placebo (COLP) for perioperative pain management in patients with head and neck cancer.

INTRODUCTION: Patients with head and neck cancer have a substantial risk of chronic opioid dependence following surgery due to pain and psychosocial consequences from both the disease process and its treatments. Conditioned open-label placebos (COLPs) have been effective for reducing the dose of active medication required for a clinical response across a wide range of medical conditions. We hypothesise that the addition of COLPs to standard multimodal analgesia will be associated with reduced baseline opioid consumption by 5 days after surgery in comparison to standard multimodal analgesia alone in patients with head and neck cancer. METHODS AND ANALYSIS: This randomised controlled trial will evaluate the use of COLP for adjunctive pain management in patients with head and neck cancer. Participants will be randomised with 1:1 allocation to either the treatment as usual or COLP group. All participants will receive standard multimodal analgesia, including opioids. The COLP group will additionally receive conditioning (ie, exposure to a clove oil scent) paired with active and placebo opioids for 5 days. Participants will complete surveys on pain, opioid consumption and depression symptoms through 6 months after surgery. Average change in baseline opioid consumption by postoperative day 5 and average pain levels and opioid consumption through 6 months will be compared between groups. ETHICS AND DISSEMINATION: There remains a demand for more effective and safer strategies for postoperative pain management in patients with head and neck cancer as chronic opioid dependence has been associated with decreased survival in this patient population. Results from this study may lay the groundwork for further investigation of COLPs as a strategy for adjunctive pain management in patients with head and neck cancer. This clinical trial has been approved by the Johns Hopkins University Institutional Review Board (IRB00276225) and is registered on the National Institutes of Health Clinical Trials Database. TRIAL REGISTRATION NUMBER: NCT04973748.

Surgeon Volume and Laryngectomy Outcomes.

OBJECTIVE: To examine the relationship between surgeon volume and operative morbidity and mortality for laryngectomy. DATA SOURCES: The Nationwide Inpatient Sample was used to identify 45,156 patients who underwent laryngectomy procedures for laryngeal or hypopharyngeal cancer between 2001 and 2011. Hospital and surgeon laryngectomy volume were modeled as categorical variables. METHODS: Relationships between hospital and surgeon volume and mortality, surgical complications, and acute medical complications were examined using multivariable regression. RESULTS: Higher-volume surgeons were more likely to operate at large, teaching, nonprofit hospitals and were more likely to treat patients who were white, had private insurance, hypopharyngeal cancer, low comorbidity, admitted electively, and to perform partial laryngectomy, concurrent neck dissection, and flap reconstruction. Surgeons treating more than 5 cases per year were associated with lower odds of medical and surgical complications, with a greater reduction in the odds of complications with increasing surgical volume. Surgeons in the top volume quintile (>9 cases/year) were associated with a decreased odds of in-hospital mortality (OR = 0.09 [0.01-0.74]), postoperative surgical complications (OR = 0.58 [0.45-0.74]), and acute medical complications (OR = 0.49 [0.37-0.64]). Surgeon volume accounted for 95% of the effect of hospital volume on mortality and 16%-47% of the effect of hospital volume on postoperative morbidity. CONCLUSION: There is a strong volume-outcome relationship for laryngectomy, with reduced mortality and morbidity associated with higher surgeon and higher hospital volumes. Observed associations between hospital volume and operative morbidity and mortality are mediated by surgeon volume, suggesting that surgeon volume is an important component of the favorable outcomes of high-volume hospital care. Laryngoscope, 133:834-840, 2023.

Feasibility of clinical evaluation of individuals with increased risk for HPV-associated oropharynx cancer.

BACKGROUND: Human papillomavirus-associated oropharynx squamous cell carcinoma (HPV-OPSCC) has no known pre-malignant lesion. While vaccination offers future primary prevention, there is current interest in secondary prevention. The feasibility of clinical evaluation of individuals at increased risk for HPV-OPSCC is unclear. METHODS: Individuals with risk factors for HPV-OPSCC were enrolled in a prospective study (MOUTH). Participants positive for biomarkers associated with HPV-OPSCC were eligible for a clinical evaluation which comprised a head and neck examination and imaging with ultrasound and/or magnetic resonance imaging (MRI). This study was designed to evaluate feasibility of clinical evaluation in a screening study. RESULTS: Three hundred and eighty-four participants were eligible for clinical evaluation. Of the 384, 204 (53%) completed a head and neck examination or imaging. Of these, 66 (32%) completed MRI (n = 51) and/or ultrasound (n = 64) studies. CONCLUSIONS: Clinical evaluations, including head and neck examination and imaging, are feasible in the context of a screening study for HPV-OPSCC.

Node Count as a Quality Indicator in Surgically Treated Mucosal Head and Neck Squamous Cell Cancer.

INTRODUCTION: A yield of ≥18 nodes from neck dissection has been shown to be associated with improved locoregional recurrence rates and survival. We sought to determine factors associated with lymph node yields below this threshold. MATERIALS AND METHODS: A retrospective review of patients who underwent neck dissection as part of definitive surgical treatment for mucosal head and neck squamous cell carcinoma (SCC) between January 2015 and December 2018 at an academic tertiary referral center was performed. Patients with a history of prior radiation or neck dissection were excluded. RESULTS: There were 412 neck dissections performed in 323 patients. Specimens containing <18 nodes decreased from 16.2% in 2015-2016 to 7.4% of neck dissections in 2017-2018. The proportion of neck dissections removing <3 levels decreased from 9.1% of neck dissections in 2015-2016 to 4.0% in 2017-2018. Multivariable regression analysis demonstrated that dissection of ≥3 levels (OR = 0.2 [0.1-0.4]) and neck dissection in 2017-2018 compared to 2015-2016 (OR = 0.4 [0.2-0.8]) were significantly associated with a lower odds of <18 nodes. Stage, site, race, sex, human papillomavirus status, positive nodes, surgeon volume, and pathologist volume were not associated with neck dissection specimens with <18 nodes, after controlling for all other variables. CONCLUSIONS: Increased recognition of the importance of node count as a quality indicator, and the extent of neck dissection is associated with increased nodal yield from neck dissection. These data suggest that node count can be used as a quality measure of neck dissection for mucosal SCC. LEVEL OF EVIDENCE: 4 Laryngoscope, 133:2160-2165, 2023.

Peri-Operative Pain and Opioid Use in Opioid-Naïve Patients Following Inpatient Head and Neck Surgery.

Pain management is an important consideration for Head and Neck Cancer (HNC) patients as they are at an increased risk of developing chronic opioid use, which can negatively impact both quality of life and survival outcomes. This retrospective cohort study aimed to evaluate pain, opioid use and opioid prescriptions following HNC surgery. Participants included patients undergoing resection of a head and neck tumor from 2019-2020 at a single academic center with a length of admission (LOA) of at least 24 h. Exclusion criteria were a history of chronic pain, substance-use disorder, inability to tolerate multimodal analgesia or a significant post-operative complication. Subjects were compared by primary surgical site: Neck (neck dissection, thyroidectomy or parotidectomy), Mucosal (resection of tumor of upper aerodigestive tract, excluding oropharynx), Oropharyngeal (OP) and Free flap (FF). Average daily pain and total daily opioid consumption (as morphine milligram equivalents, MME) and quantity of opioids prescribed at discharge were compared. A total of 216 patients met criteria. Pain severity and daily opioid consumption were comparable across groups on post-operative day 1, but both metrics were significantly greater in the OP group on the day prior to discharge (DpDC) (5.6 (1.9-8.6), p < 0.05; 49 ± 44 MME/day, p < 0.01). The quantity of opioids prescribed at discharge was associated with opioid consumption on the DpDC only in the Mucosal and FF groups, which had longer LOA (6-7 days) than the Neck and OP groups (1 day, p < 0.001). Overall, 65% of patients required at least one dose of an opioid on the DpDC, yet 76% of patients received a prescription for an opioid medication at discharge. A longer LOA (aOR = 0.82, 95% CI: 0.63-0.98) and higher Charlson Comorbidity Index (aOR = 0.08, 95% CI: 0.01-0.48) were negatively associated with receiving an opioid prescription at the time of discharge despite no opioid use on the DpDC, respectively. HNC patients, particularly those with shorter LOA, may be prescribed opioids in excess of their post-operative needs, highlighting the need the for improved pain management algorithms in this patient population. Future work aims to use prospective surveys to better define post-operative and outpatient pain and opioid requirements following HNC surgery.

Association of Tumor Site With the Prognosis and Immunogenomic Landscape of Human Papillomavirus-Related Head and Neck and Cervical Cancers.

Importance: Human papillomavirus (HPV)-positive status in patients with oropharyngeal squamous cell carcinoma (OPSCC) is associated with improved survival compared with HPV-negative status. However, it remains controversial whether HPV is associated with improved survival among patients with nonoropharyngeal and cervical squamous cell tumors. Objective: To investigate differences in the immunogenomic landscapes of HPV-associated tumors across anatomical sites (the head and neck and the cervix) and their association with survival. Design, Setting, and Participants: This cohort study used genomic and transcriptomic data from the Cancer Genome Atlas (TCGA) for 79 patients with OPSCC, 435 with nonoropharyngeal head and neck squamous cell carcinoma (non-OP HNSCC), and 254 with cervical squamous cell carcinoma and/or endocervical adenocarcinoma (CESC) along with matched clinical data from TCGA. The data were analyzed from November 2020 to March 2021. Main Outcomes and Measures: Positivity for HPV was classified by RNA-sequencing reads aligned with the HPV reference genome. Gene expression profiles, immune cell phenotypes, cytolytic activity scores, and overall survival were compared by HPV tumor status across multiple anatomical sites. Results: The study comprised 768 patients, including 514 (66.9%) with HNSCC (380 male [73.9%]; mean [SD] age, 59.5 [10.8] years) and 254 (33.1%) with CESC (mean [SD] age, 48.7 [14.1] years). Human papillomavirus positivity was associated with a statistically significant improvement in overall survival for patients with OPSCC (adjusted hazard ratio [aHR], 0.06; 95% CI, 0.02-0.17; P < .001) but not for those with non-OP HNSCC (aHR, 0.64; 95% CI, 0.31-1.27; P = .20) or CESC (aHR, 0.50; 95% CI, 0.15-1.67; P = .30). The HPV-positive OPSCCs had increased tumor immune infiltration and immunomodulatory receptor expression compared with HPV-negative OPSCCs. Compared with HPV-positive non-OP HNSCCs, HPV-positive OPSCCs showed greater expression of immune-related metrics including B cells, T cells, CD8+ T cells, T-cell receptor diversity, B-cell receptor diversity, and cytolytic activity scores, independent of tumor variant burden. The immune-related metrics were similar when comparing HPV-positive non-OP HNSCCs and HPV-positive CESCs with their HPV-negative counterparts. The 2-year overall survival rate was significantly higher for patients with HPV-positive OPSCC compared with patients with HPV-negative OPSCC (92.0% [95% CI, 84.8%-99.9%] vs 45.8% [95% CI, 28.3%-74.1%]; HR, 0.10 [95% CI, 0.03-0.30]; P = .009). Conclusions and Relevance: In this cohort study, tumor site was associated with the immune landscape and survival among patients with HPV-related tumors despite presumed similar biologic characteristics. These tumor site-related findings provide insight on possible outcomes of HPV positivity for tumors in oropharyngeal and nonoropharyngeal sites and a rationale for the stratification of HPV-associated tumors by site and the subsequent development of strategies targeting immune exclusion in HPV-positive nonoropharyngeal cancer.

The mouse atherosclerosis locus at chromosome 10 (Ath11) acts early in lesion formation with subcongenic strains delineating 2 narrowed regions.

OBJECTIVE: Ath11, an atherosclerosis susceptibility locus on proximal chromosome 10 (0 to 21 cM) revealed in a cross between apolipoprotein E deficient C57BL/6 (B6) and FVB mice, was recently confirmed in congenic mice. The objectives of this study were to assess how Ath11 affects lesion development and morphology, to determine aortic gene expression in congenics, and to narrow the congenic interval. METHODS AND RESULTS: Assessing lesion area over time in congenic mice showed that homozygosity for the FVB allele increased lesion area at 6 weeks persisting through to 24 weeks of age. Staining of aortic root sections at 16 weeks did not reveal obvious differences between congenics. Aortic expression-array analysis at 6 weeks revealed 97 genes that were >2-fold regulated, including 1 gene in the quantitative trait locus interval, Aldh8a1, and 2 gene clusters regulated by Hnf4alpha and Esr1. Analysis of lesion area in 11 subcongenic strains revealed 2 narrowed regions, 10a (21 genes), acting in females, and 10b (7 genes), acting in both genders. CONCLUSIONS: Ath11 appears to act early in lesion formation, with significant effects on aortic gene expression. This quantitative trait locus is genetically complex, containing a female-specific region 10a from 0 to 7.3 megabases (21 genes) and a gender-independent region 10b from 20.1 to 21.9 megabases (7 genes).

The Role of Preoperative Briefing and Postoperative Debriefing in Surgical Education.

OBJECTIVE: To study the impact of a new preoperative briefing and postoperative debriefing tool on the perceived quality of surgical education and to assess attitudes of residents and attendings regarding this tool. DESIGN: Surrounding introduction and use of the tool (JHFIRE: Joint Huddles for Improving Resident Education), perceived quality of surgical education was assessed with pre- and postintervention System for Evaluation of Teaching Qualities (SETQ) surveys. Additionally, a postintervention Likert survey regarding the JHFIRE tool itself was completed by residents and faculty. SETTING: Johns Hopkins University Department of Otolaryngology-Head and Neck Surgery, a tertiary care academic institution. PARTICIPANTS: All residents and attendings who used the tool were invited to participate. 40 participants (13 residents, 27 attendings) completed the preintervention SETQ. 11 participants (3 residents, 7 attendings, 1 unspecified) completed the postintervention SETQ. For postintervention qualitative assessment of the tool itself, 12 participants (3 residents, 7 attendings, 2 unspecified) provided feedback. RESULTS: The tool was well-received with large subjective benefit in improving resident surgical education. A total of 88% thought that the time spent on the debriefings was "just right" and 91% planned to make the debriefings a regular part of operative performance assessments. Despite this overwhelmingly positive feedback, there was no overall difference in pre- and postintervention SETQ scores for climate of surgical education in the Department (4.25 ± 0.55 vs. 4.10 ± 0.88, p = 0.63). CONCLUSIONS: Introduction of 4 item preoperative briefing and 4 item postoperative debriefing checklists was welcomed by both residents and faculty for its ability to shape surgical education in the operating room into a guided discovery model of hands-on education. Overall SETQ scores did not change, but most participants found value in the tool and plan to continue its use.

Preoperative Briefings and Postoperative Debriefings to Increase Resident Operative Autonomy and Performance.

OBJECTIVE: To implement the use of standardized preoperative briefings and postoperative debriefings for surgical cases involving residents in an effort to improve resident autonomy and skill acquisition. DESIGN: Prospective longitudinal study. SETTING: Johns Hopkins Department of Otolaryngology-Head and Neck Surgery. PARTICIPANTS: Resident and attending physicians. RESULTS: Joint Huddles for Improving Resident Education (JHFIRE) tool was created and successfully implemented by 19 residents and 17 faculty members. Over the course of three data collection periods spanning an academic year, overall scores improved though not statistically significantly in the metrics of Zwisch autonomy, Resident Performance, and Objective Structured Assessment of Technical Skills (OSATS) scores. Female residents were scored significantly higher by attendings than their male counterparts in the assessment of baseline Resident Performance. CONCLUSIONS: (1) JHFIRE tool implemented a standardized preoperative briefing and postoperative debriefing to improve communication and resident skill acquisition; (2) The tool was accepted and utilized throughout an academic year; (3) Zwisch, Resident Performance, and OSATS scores improved though not significantly.

An Immunogenomic Investigation of Oral Cavity Squamous Cell Carcinoma in Patients Aged 45 Years and Younger.

OBJECTIVES/HYPOTHESIS: To investigate differences in the immunogenomic landscape among young patients presenting with oral cavity squamous cell carcinoma (OCSCC). STUDY DESIGN: Retrospective database review. METHODS: Normalized messenger mRNA expression data were downloaded from The Cancer Genome Atlas (TCGA) database. OCSCC patients were categorized into young and older age groups with a cutoff of 45 years. Human papillomavirus-positive tumors were excluded. Cell fractions, marker expression, and mutational load were compared between age groups using the Wilcoxon rank sum test. Adjustment for multiple comparisons was performed using the Benjamini-Hochberg method, with a false discovery rate of 0.05. RESULTS: Two hundred forty-five OCSCC tumors were included; 21 (8.6%) were young (37.1 ± 7.5 years) and 224 (91.4%) were older (64.5 ± 10.3 years). There was no significant difference between groups in the fraction of B and T lymphocytes, macrophages, monocytes, natural killers, and dendritic cells. Cytolytic activity score was decreased in young patients (8.33 vs. 18.9, P = .023). Additionally, young patients had significantly lower expression of immunomodulatory markers of immune activation, including PD-1 (PDCD1, P = .003), CTLA4 (P = .025), TIGIT (P = .002), GITR (TNFRSF18, P = .005), OX40 (TNFRSF4, P = .009), LAG-3 (P < .001), and TIM-3 (HAVCR2, P = .002). Young patients had a significantly lower number of single nucleotide variant-derived neoantigens (26.2 vs. 60.6, P < .001). CONCLUSIONS: OCSCC patients aged 45 years and younger appear to have an attenuated immune response that may be related to a lower frequency of immunogenic mutations. This may contribute to the pathogenesis of these tumors, and ultimately help inform personalized immune-based therapeutic strategies for young patients with OCSCC. LEVEL OF EVIDENCE: NA Laryngoscope, 131:304-311, 2021.

Nutritional Status as a Predictive Biomarker for Immunotherapy Outcomes in Advanced Head and Neck Cancer.

The association between pretreatment nutritional status and immunotherapy response in patients with advanced head and neck cancer is unclear. We retrospectively analyzed a cohort of 99 patients who underwent treatment with anti-PD-1 or anti-CTLA-4 antibodies (or both) for stage IV HNSCC between 2014 and 2020 at the Johns Hopkins Hospital. Patient demographics and clinical characteristics were retrieved from electronic medical records. Baseline prognostic nutritional index (PNI) scores and pretreatment body mass index (BMI) trends were calculated. Associations between PNI and BMI were correlated with overall survival (OS), progression-free survival (PFS), and immunotherapy response. In univariate analysis, there was a significant correlation between OS and PFS with baseline PNI (OS: HR: 0.464; 95% CI: 0.265-0.814; PFS: p = 0.007 and HR: 0.525; 95% CI: 0.341-0.808; p = 0.003). Poor OS was also associated with a greater decrease in pretreatment BMI trend (HR: 0.42; 95% CI: 0.229-0.77; p = 0.005). In multivariate analysis, baseline PNI but not BMI trend was significantly associated with OS and PFS (OS: log (HR) = -0.79, CI: -1.6, -0.03, p = 0.041; PFS: log (HR) = -0.78, CI: -1.4, -0.18, p = 0.011). In conclusion, poor pretreatment nutritional status is associated with negative post-immunotherapy outcomes.

Novel strategy using F1-congenic mice for validation of QTLs: studies at the proximal chromosome 10 atherosclerosis susceptibility locus.

OBJECTIVE: We have previously identified a quantitative trait locus (QTL) for atherosclerosis susceptibility on proximal chromosome 10 (Chr10) (Ath11) in independent crosses of FVB and C57BL/6 (B6) mice on the apolipoprotein E (ApoE-/-) and LDL receptor (LDLR-/-) deficient backgrounds. The aims of the current study were to (1) test a novel strategy for validating QTLs using interval-specific congenic strains that were heterozygous (F1) across the genome, (2) validate the Chr10 QTL, and (3) to assess whether the phenotype is transferable by bone marrow transplantation. METHODS AND RESULTS: We generated Chr10 (0 to 21 cM) interval-specific mice on the F1.ApoE-/- background by crossing congenic FVB.ApoE-/-Chr10(B6/FVB) with B6.ApoE-/-, and B6.ApoE-/-Chr10(B6/FVB) with FVB.ApoE-/- mice. Lesion size was significantly larger in the resultant F1.ApoE-/-Chr10(FVB/FVB) mice compared to F1.ApoE-/-Chr10(B6/FVB) and F1.ApoE-/-Chr10(B6/B6) mice, validating the Chr10 QTL. The effect of the congenic interval was more robust on the F1.ApoE-/- than on the FVB.ApoE-/- and B6.ApoE-/- backgrounds. Bone marrow transplantation in congenic mice showed that the effect of the proximal Chr10 interval was not transferable by bone marrow-derived cells. CONCLUSIONS: A novel strategy of congenic strains on an F1 background proved useful to validate an atherosclerosis susceptibility QTL on mouse proximal Chr10.

The protective effect of A20 on atherosclerosis in apolipoprotein E-deficient mice is associated with reduced expression of NF-kappaB target genes.

Up-regulation of inflammatory responses is considered a driving force of atherosclerotic lesion development. One key regulator of inflammation is the A20 (also called TNF-alpha-induced protein 3 or Tnfaip3) gene, which is responsible for NF-kappaB termination and maps to an atherosclerosis susceptibility locus revealed by quantitative trait locus-mapping studies at mouse proximal chromosome 10. In the current study, we examined the role of A20 in atherosclerotic lesion development. At the aortic root lesion size was found to be increased in C57BL/6 (BG) apolipoprotein E-deficient (ApoE(-/-)) mice haploinsufficient for A20, compared with B6 ApoE(-/-) controls that expressed A20 normally (60% in males and 23% in females; P < 0.001 and P < 0.05, respectively). In contrast, lesion size was found to be decreased in F(1) (B6 x FVB/N) mice overexpressing A20 by virtue of containing an A20 BAC transgene compared with nontransgenic controls (30% in males, P < 0.001, and 17% in females, P = 0.02). The increase in lesions in the A20 haploinsufficient mice correlated with increased expression of proatherosclerotic NF-kappaB target genes, such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and macrophage-colony-stimulating factor, and elevated plasma levels of NF-kappaB-driven cytokines. These findings suggest that A20 diminishes atherosclerosis by decreasing NF-kappaB activity, thereby modulating the proinflammatory state associated with lesion development.

Computed tomography performance in predicting extranodal extension in HPV-positive oropharynx cancer.

OBJECTIVES: To evaluate the performance characteristics of seven predetermined imaging features on pretreatment computed tomography (CT) in identifying extranodal extension (ENE) in cervical lymph node metastases from human papillomavirus-positive oropharyngeal carcinoma (HPV-OPC). STUDY DESIGN: Retrospective study. METHODS: Seventy-three patients with HPV-OPC who underwent primary surgery and cervical lymph node dissection were included. Preoperative contrast-enhanced CT (cCT) imaging was evaluated by two radiologists blinded to pathological results. Each cCT was scored for seven imaging features of interest: 1) indistinct capsular contours, 2) irregular nodal margins, 3) perinodal fat stranding, 4) perinodal fat planes, 5) nodal necrosis, 6) intranodal cysts, and 7) nodal matting. Logistic regression was employed to determine radiologist-specific odds ratios (OR) of predicting ENE for each imaging feature and radiologist-specific receiver operating characteristics (sensitivity [Sn], specificity [Sp], area under the curve [AUC], positive predictive value [PPV], negative predictive value [NPV]) for each imaging feature. RESULTS: Thirty-two (44%) patients had ENE-positive lymph nodes. The presence of irregular margins (ORA = 12.3, 95% confidence interval [CI]A = 2.3-65.9; ORB = 7.0, 95% CIB = 1.4-36.3) and absence of perinodal fat plane (ORA = 6.8, 95% CIA = 2.0-23.3; ORB = 14.2, 95% CIB = 1.7-120.5) were significantly associated with ENE for each radiologist. Irregular nodal margin status was most specific for ENE (SnA = 45%, SpA = 94%, AUCA = 69%, PPVA = 82%, NPVA = 73%; SnB = 28%, SpB = 95%, AUCB = 61%, PPVB = 80%, NPVB = 64%). Absence of perinodal fat plane was most sensitive for ENE (SnA = 87%, SpA = 50%, AUCA = 69%, PPVA = 59%, NPVA = 62%; SnB = 96%, SpB = 34%, AUCB = 65%, PPVB = 53%, NPVB = 63%). CONCLUSIONS: Of the seven imaging features hypothesized to be associated with ENE-status, the presence of irregular nodal margins and absence of perinodal fat plane were the most specific and sensitive features, respectively. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:1479-1486, 2020.

Structure-function analyses of candidate small molecule RPN13 inhibitors with antitumor properties.

We sought to design ubiquitin-proteasome system inhibitors active against solid cancers by targeting ubiquitin receptor RPN13 within the proteasome's 19S regulatory particle. The prototypic bis-benzylidine piperidone-based inhibitor RA190 is a michael acceptor that adducts Cysteine 88 of RPN13. In probing the pharmacophore, we showed the benefit of the central nitrogen-bearing piperidone ring moiety compared to a cyclohexanone, the importance of the span of the aromatic wings from the central enone-piperidone ring, the contribution of both wings, and that substituents with stronger electron withdrawing groups were more cytotoxic. Potency was further enhanced by coupling of a second warhead to the central nitrogen-bearing piperidone as RA375 exhibited ten-fold greater activity against cancer lines than RA190, reflecting its nitro ring substituents and the addition of a chloroacetamide warhead. Treatment with RA375 caused a rapid and profound accumulation of high molecular weight polyubiquitinated proteins and reduced intracellular glutathione levels, which produce endoplasmic reticulum and oxidative stress, and trigger apoptosis. RA375 was highly active against cell lines of multiple myeloma and diverse solid cancers, and demonstrated a wide therapeutic window against normal cells. For cervical and head and neck cancer cell lines, those associated with human papillomavirus were significantly more sensitive to RA375. While ARID1A-deficiency also enhanced sensitivity 4-fold, RA375 was active against all ovarian cancer cell lines tested. RA375 inhibited proteasome function in muscle for >72h after single i.p. administration to mice, and treatment reduced tumor burden and extended survival in mice carrying an orthotopic human xenograft derived from a clear cell ovarian carcinoma.

Risk factors for human papillomavirus-positive nonoropharyngeal squamous cell carcinoma.

BACKGROUND: Human papillomavirus (HPV)-positive oropharyngeal cancer (HPV-OPC) is distinct from HPV-unassociated head and neck cancer. However, whether risk factors for HPV-positive oropharyngeal and nonoropharyngeal squamous cell cancer are the same is unclear. METHODS: Incident cases of HPV-positive head and neck cell cancer and matched non-cancer controls were enrolled in a multi-institutional, prospective study examining risk factors, biomarkers, and survival. RESULTS: HPV-nonOPC (n = 20) were more likely to be ever smokers than controls (n = 80, OR 3.49, 95%CI 1.11-10.9) and HPV-OPC (n = 185, OR 3.28, 95%CI 1.10-10.2). Compared with HPV-OPC, HPV-nonOPC were less likely to have had over 3 oral sexual partners (OR 0.29, 95%CI 0.06-0.9), more likely to have multimorbidity (OR 3.30, 95%CI 1.04-10.5), and less likely to have antibodies to HPV16 E6 (90% vs 28%, OR 0.05, 95%CI 0.02-0.2). HPV-nonOPC had worse 4-year OS (77% vs 96%, P = .001) and RFS (69% vs 94%, P < .001) than HPV-OPC. CONCLUSIONS: HPV-positive nonoropharyngeal are distinct from HPV-positive oropharyngeal cancers.

The Impact of a Stepwise Approach to Primary Tumor Detection in Squamous Cell Carcinoma of the Neck With Unknown Primary.

OBJECTIVES/HYPOTHESIS: To examine the cumulative effect of diagnostic steps for primary tumor identification in patients with head and neck squamous cell carcinoma of unknown primary (HNSCCUP), including lingual tonsillectomy, and the impact of primary tumor identification on subsequent treatment. STUDY DESIGN: Retrospective analysis. METHODS: We reviewed the records of 110 patients diagnosed with HNSCCUP between 2003 and 2015. Results of diagnostic imaging (fluorodeoxyglucose-positron emission tomography/computed tomography [FDG-PET/CT]), tumor detection with direct laryngoscopy with biopsies, palatine tonsillectomy, and transoral robotic surgery (TORS) lingual tonsillectomy were recorded. Associations between demographic and treatment variables with overall survival (OS) and progression-free survival (PFS) were modeled with Cox proportional hazards models. RESULTS: FDG-PET/CT was suspicious for a primary site in 23/77 (30%) patients. Direct laryngoscopy identified a primary tumor in 34/110 patients (31%). Forty-seven patients underwent palatine tonsillectomy, which identified 17 primaries (36%), yielding a cumulative primary tumor identification of 51/110 (46%). Fourteen patients underwent TORS lingual tonsillectomy, which identified eight primaries (57%), resulting in a cumulative identification of 59/110 (53%). The detection rate increased from 28/63 (44%) to 31/47 (66%) after the addition of TORS lingual tonsillectomy to our institutional approach. Detection rates varied by HPV status. Primary tumor identification altered subsequent radiation planning, as patients with an identified primary tumor received radiation to a smaller volume of tissue than did those without an identified primary tumor. However, there was no significant association between primary tumor identification and OS or PFS. CONCLUSIONS: A stepwise approach to primary tumor identification identifies a primary tumor in a majority of patients. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:1610-1616, 2019.

Whole-Exome Sequencing of Salivary Gland Mucoepidermoid Carcinoma.

PURPOSE: Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy. To explore the genetic origins of MEC, we performed systematic genomic analyses of these tumors. EXPERIMENTAL DESIGN: Whole-exome sequencing and gene copy-number analyses were performed for 18 primary cancers with matched normal tissue. FISH was used to determine the presence or absence of the MECT1-MAML2 translocation in 17 tumors. RESULTS: TP53 was the most commonly mutated gene in MEC (28%), and mutations were found only in intermediate- and high-grade tumors. Tumors with TP53 mutations had more mutations overall than tumors without TP53 mutations (P = 0.006). POU6F2 was the second most frequently mutated gene, found in three low-grade MECs with the same in-frame deletion. Somatic alterations in IRAK1, MAP3K9, ITGAL, ERBB4, OTOGL, KMT2C, and OBSCN were identified in at least two of the 18 tumors sequenced. FISH analysis confirmed the presence of the MECT1-MAML2 translocation in 15 of 17 tumors (88%). CONCLUSIONS: Through these integrated genomic analyses, MECT1-MAML2 translocation and somatic TP53 and POU6F2 mutations appear to be the main drivers of MEC. Clin Cancer Res; 23(1); 283-8. ©2016 AACR.