Application of liquid biopsy as multi-functional biomarkers in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is a molecularly heterogeneous disease, with a 5-year survival rate that still hovers at ~60% despite recent advancements. The advanced stage upon diagnosis, limited success with effective targeted therapy and lack of reliable biomarkers are among the key factors underlying the marginally improved survival rates over the decades. Prevention, early detection and biomarker-driven treatment adaptation are crucial for timely interventions and improved clinical outcomes. Liquid biopsy, analysis of tumour-specific biomarkers circulating in bodily fluids, is a rapidly evolving field that may play a striking role in optimising patient care. In recent years, significant progress has been made towards advancing liquid biopsies for non-invasive early cancer detection, prognosis, treatment adaptation, monitoring of residual disease and surveillance of recurrence. While these emerging technologies have immense potential to improve patient survival, numerous methodological and biological limitations must be overcome before their implementation into clinical practice. This review outlines the current state of knowledge on various types of liquid biopsies in HNSCC, and their potential applications for diagnosis, prognosis, grading treatment response and post-treatment surveillance. It also discusses challenges associated with the clinical applicability of liquid biopsies and prospects of the optimised approaches in the management of HNSCC.

Phase I study of nab-paclitaxel-based induction followed by nab-paclitaxel-based concurrent chemotherapy and re-irradiation in previously treated head and neck squamous cell carcinoma.

BACKGROUND: Recurrent head and neck squamous cell carcinoma (HNSCC) is associated with poor overall survival (OS). Prior studies suggested incorporation of nab-paclitaxel (A) may improve outcomes in recurrent HNSCC. METHODS: This Phase I study evaluated induction with carboplatin and A followed by concomitant FHX (infusional 5-fluorouracil, hydroxyurea and twice-daily radiation therapy administered every other week) plus A with cohort dose escalation ranging from 10-100 mg/m2 in recurrent HNSCC. The primary endpoint was maximally tolerated dose (MTD) and dose-limiting toxicity (DLT) of A when given in combination with FHX (AFHX). RESULTS: Forty-eight eligible pts started induction; 28 pts started AFHX and were evaluable for toxicity. Two DLTs occurred (both Grade 4 mucositis) at a dose level 20 mg/m2. No further DLTs were observed with subsequent dose escalation. The MTD and recommended Phase II dose (RP2D) of A was 100 mg/m2. CONCLUSIONS: In this Phase I study, the RP2D of A with FHX is 100 mg/m2 (AFHX). The role of re-irradiation with immunotherapy warrants further investigation. CLINICAL TRIAL INFORMATION: This clinical trial was registered with ClinicalTrials.gov identifier: NCT01847326.

Ultrasensitive detection of tumor-specific mutations in saliva of patients with oral cavity squamous cell carcinoma.

BACKGROUND: Oral cavity squamous cell carcinoma (OCSCC) is the most common head and neck malignancy. Although the survival rate of patients with advanced-stage disease remains approximately 20% to 60%, when detected at an early stage, the survival rate approaches 80%, posing a pressing need for a well validated profiling method to assess patients who have a high risk of developing OCSCC. Tumor DNA detection in saliva may provide a robust biomarker platform that overcomes the limitations of current diagnostic tests. However, there is no routine saliva-based screening method for patients with OCSCC. METHODS: The authors designed a custom next-generation sequencing panel with unique molecular identifiers that covers coding regions of 7 frequently mutated genes in OCSCC and applied it on DNA extracted from 121 treatment-naive OCSCC tumors and matched preoperative saliva specimens. RESULTS: By using stringent variant-calling criteria, mutations were detected in 106 tumors, consistent with a predicted detection rate ≥88%. Moreover, mutations identified in primary malignancies were also detected in 93% of saliva samples. To ensure that variants are not errors resulting in false-positive calls, a multistep analytical validation of this approach was performed: 1) re-sequencing of 46 saliva samples confirmed 88% of somatic variants; 2) no functionally relevant mutations were detected in saliva samples from 11 healthy individuals without a history of tobacco or alcohol; and 3) using a panel of 7 synthetic loci across 8 sequencing runs, it was confirmed that the platform developed is reproducible and provides sensitivity on par with droplet digital polymerase chain reaction. CONCLUSIONS: The current data highlight the feasibility of somatic mutation identification in driver genes in saliva collected at the time of OCSCC diagnosis.

Diagnostic tests for oral cancer and potentially malignant disorders in patients presenting with clinically evident lesions.

BACKGROUND: Squamous cell carcinoma is the most common form of malignancy of the oral cavity, and is often proceeded by oral potentially malignant disorders (OPMD). Early detection of oral cavity squamous cell carcinoma (oral cancer) can improve survival rates. The current diagnostic standard of surgical biopsy with histology is painful for patients and involves a delay in order to process the tissue and render a histological diagnosis; other diagnostic tests are available that are less invasive and some are able to provide immediate results. This is an update of a Cochrane Review first published in 2015. OBJECTIVES: Primary objective: to estimate the diagnostic accuracy of index tests for the detection of oral cancer and OPMD, in people presenting with clinically evident suspicious and innocuous lesions. SECONDARY OBJECTIVE: to estimate the relative accuracy of the different index tests. SEARCH METHODS: Cochrane Oral Health's Information Specialist searched the following databases: MEDLINE Ovid (1946 to 20 October 2020), and Embase Ovid (1980 to 20 October 2020). The US National Institutes of Health Ongoing Trials Register (ClinicalTrials.gov) and the World Health Organization International Clinical Trials Registry Platform were also searched for ongoing trials to 20 October 2020. No restrictions were placed on the language or date of publication when searching the electronic databases. We conducted citation searches, and screened reference lists of included studies for additional references. SELECTION CRITERIA: We selected studies that reported the diagnostic test accuracy of the following index tests when used as an adjunct to conventional oral examination in detecting OPMD or oral cavity squamous cell carcinoma: vital staining (a dye to stain oral mucosa tissues), oral cytology, light-based detection and oral spectroscopy, blood or saliva analysis (which test for the presence of biomarkers in blood or saliva). DATA COLLECTION AND ANALYSIS: Two review authors independently screened titles and abstracts for relevance. Eligibility, data extraction and quality assessment were carried out by at least two authors, independently and in duplicate. Studies were assessed for methodological quality using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2). Meta-analysis was used to combine the results of studies for each index test using the bivariate approach to estimate the expected values of sensitivity and specificity. MAIN RESULTS: This update included 63 studies (79 datasets) published between 1980 and 2020 evaluating 7942 lesions for the quantitative meta-analysis. These studies evaluated the diagnostic accuracy of conventional oral examination with: vital staining (22 datasets), oral cytology (24 datasets), light-based detection or oral spectroscopy (24 datasets). Nine datasets assessed two combined index tests. There were no eligible diagnostic accuracy studies evaluating blood or salivary sample analysis. Two studies were classed as being at low risk of bias across all domains, and 33 studies were at low concern for applicability across the three domains, where patient selection, the index test, and the reference standard used were generalisable across the population attending secondary care. The summary estimates obtained from the meta-analysis were: - vital staining: sensitivity 0.86 (95% confidence interval (CI) 0.79 to 0.90) specificity 0.68 (95% CI 0.58 to 0.77), 20 studies, sensitivity low-certainty evidence, specificity very low-certainty evidence; - oral cytology: sensitivity 0.90 (95% CI 0.82 to 0.94) specificity 0.94 (95% CI 0.88 to 0.97), 20 studies, sensitivity moderate-certainty evidence, specificity moderate-certainty evidence; - light-based: sensitivity 0.87 (95% CI 0.78 to 0.93) specificity 0.50 (95% CI 0.32 to 0.68), 23 studies, sensitivity low-certainty evidence, specificity very low-certainty evidence; and - combined tests: sensitivity 0.78 (95% CI 0.45 to 0.94) specificity 0.71 (95% CI 0.53 to 0.84), 9 studies, sensitivity very low-certainty evidence, specificity very low-certainty evidence. AUTHORS' CONCLUSIONS: At present none of the adjunctive tests can be recommended as a replacement for the currently used standard of a surgical biopsy and histological assessment. Given the relatively high values of the summary estimates of sensitivity and specificity for oral cytology, this would appear to offer the most potential. Combined adjunctive tests involving cytology warrant further investigation. Potentially eligible studies of blood and salivary biomarkers were excluded from the review as they were of a case-control design and therefore ineligible. In the absence of substantial improvement in the tests evaluated in this updated review, further research into biomarkers may be warranted.

Clinical assessment for the detection of oral cavity cancer and potentially malignant disorders in apparently healthy adults.

BACKGROUND: The early detection of oral cavity squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMD), followed by appropriate treatment, may improve survival and reduce the risk for malignant transformation respectively. This is an update of a Cochrane Review first published in 2013. OBJECTIVES: To estimate the diagnostic test accuracy of conventional oral examination, vital rinsing, light-based detection, mouth self-examination, remote screening, and biomarkers, used singly or in combination, for the early detection of OPMD or OSCC in apparently healthy adults. SEARCH METHODS: Cochrane Oral Health's Information Specialist searched the following databases: Cochrane Oral Health's Trials Register (to 20 October 2020), MEDLINE Ovid (1946 to 20 October 2020), and Embase Ovid (1980 to 20 October 2020). The US National Institutes of Health Trials Registry (ClinicalTrials.gov) and the World Health Organization International Clinical Trials Registry Platform were searched for ongoing trials. No restrictions were placed on the language or date of publication when searching the electronic databases. We conducted citation searches, and screened reference lists of included studies for additional references. SELECTION CRITERIA: We selected studies that reported the test accuracy of any of the aforementioned tests in detecting OPMD or OSCC during a screening procedure. Diagnosis of OPMD or OSCC was provided by specialist clinicians or pathologists, or alternatively through follow-up. DATA COLLECTION AND ANALYSIS: Two review authors independently screened titles and abstracts for relevance. Eligibility, data extraction, and quality assessment were carried out by at least two authors independently and in duplicate. Studies were assessed for methodological quality using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2). We reported the sensitivity and specificity of the included studies. We provided judgement of the certainty of the evidence using a GRADE assessment. MAIN RESULTS: We included 18 studies, recruiting 72,202 participants, published between 1986 and 2019. These studies evaluated the diagnostic test accuracy of conventional oral examination (10 studies, none new to this update), mouth self-examination (four studies, two new to this update), and remote screening (three studies, all new to this update). One randomised controlled trial of test accuracy directly evaluated conventional oral examination plus vital rinsing versus conventional oral examination alone. There were no eligible studies evaluating light-based detection or blood or salivary sample analysis (which tests for the presence of biomarkers for OPMD and OSCC). Only one study of conventional oral examination was judged as at overall low risk of bias and overall low concern regarding applicability. Given the clinical heterogeneity of the included studies in terms of the participants recruited, setting, prevalence of the target condition, the application of the index test and reference standard, and the flow and timing of the process, the data could not be pooled within the broader categories of index test. For conventional oral examination (10 studies, 25,568 participants), prevalence in the test accuracy sample ranged from 1% to 51%. For the seven studies with prevalence of 10% or lower, a prevalence more comparable to the general population, the sensitivity estimates were variable, and ranged from 0.50 (95% confidence interval (CI) 0.07 to 0.93) to 0.99 (95% CI 0.97 to 1.00); the specificity estimates were more consistent and ranged from 0.94 (95% CI 0.88 to 0.97) to 0.99 (95% CI 0.98 to 1.00). We judged the overall certainty of the evidence to be low, and downgraded for inconsistency and indirectness. Evidence for mouth self-examination and remote screening was more limited. We judged the overall certainty of the evidence for these index tests to be very low, and downgraded for imprecision, inconsistency, and indirectness. We judged the evidence for vital rinsing (toluidine blue) as an adjunct to conventional oral examination compared to conventional oral examination to be moderate, and downgraded for indirectness as the trial was undertaken in a high-risk population. AUTHORS' CONCLUSIONS: There is a lack of high-certainty evidence to support the use of screening programmes for oral cavity cancer and OPMD in the general population. Frontline screeners such as general dentists, dental hygienists, other allied professionals, and community healthcare workers should remain vigilant for signs of OPMD and OSCC.

Nondestructive, multiplex three-dimensional mapping of immune infiltrates in core needle biopsy.

Enumeration of tumor-infiltrating lymphocytes (TILs) in H&E stained tissue sections has demonstrated limited value in predicting immune responses to cancer immunotherapy, likely reflecting the diversity of cell types and immune activation states among tumor infiltrates. Multiparametric flow cytometry enables robust phenotypic and functional analysis to distinguish suppression from activation, but tissue dissociation eliminates spatial context. Multiplex methods for immunohistochemistry (IHC) are emerging, but these interrogate only a single tissue section at a time. Here, we report transparent tissue tomography (T3) as a tool for three-dimensional (3D) imaging cytometry in the complex architecture of the tumor microenvironment, demonstrating multiplexed immunofluorescent analysis in core needle biopsies. Using T3 imaging, image processing and machine learning to map CD3+CD8+ cytotoxic T cells (CTLs) in whole core needle biopsies from Her2+ murine mammary tumors and human head and neck surgical specimens revealed marked inhomogeneity within single needle cores, confirmed by serial section IHC. Applying T3 imaging cytometry, we discovered a strong spatial correlation between CD3+CD8+ CTLs and microvasculature in the EGFR+ parenchyma, revealing significant differences among head and neck cancer patients. These results show that T3 offers simple and rapid access to three-dimensional and quantitative maps of the tumor microenvironment and immune infiltrate, offering a new diagnostic tool for personalized cancer immunotherapy.

Histological features and prognostic significance of treatment effect in lymph node metastasis in head and neck squamous cell carcinoma.

AIMS AND OBJECTIVES: Cervical lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) is common. Pre-operative chemoradiotherapy (preCRT) and postoperative chemoradiotherapy (postCRT) is frequently employed in such patients. The prognostic value of viable SCC, treatment effect or no SCC in resected lymph nodes in patients who received or did not receive preCRT and postCRT was investigated. METHODS AND RESULTS: Resected cervical lymph nodes from 146 patients with HNSCC were evaluated for viable SCC, treatment effect or no SCC. Immunostains for Ki67, cyclin D1, caspase 3 and H2AFX were performed on viable SCC or nucleate keratin debris. Clinical and histological data were correlated with tumour recurrence or persistence. Patients with nucleate keratin debris in lymph nodes had outcomes similar to those with diffuse treatment effect and no SCC. Viable tumour in lymph nodes was associated with worse prognosis in patients who received preCRT (P = 0.01). This relative worsening of prognosis was not observed in patients with oropharyngeal SCC or recurrent disease. Lower proliferation index in lymph node SCC was associated with preCRT and with worse outcomes (P = 0.0002). Overall, patients who received preCRT or postCRT had outcomes not significantly different from those who did not. CONCLUSION: The presence of viable SCC in cervical lymph nodes has prognostic import when taken in context with the patient's history. Viable SCC in lymph nodes was significantly associated with worse outcome among patients with non-oropharyngeal SCC who received preCRT. Nucleate keratin debris should not be considered viable SCC in lymph nodes.

The transcriptional profile of coronary arteritis in Kawasaki disease.

BACKGROUND: Kawasaki Disease (KD) can cause potentially life-threatening coronary arteritis in young children, and has a likely infectious etiology. Transcriptome profiling is a powerful approach to investigate gene expression in diseased tissues. RNA sequencing of KD coronary arteries could elucidate the etiology and the host response, with the potential to improve KD diagnosis and/or treatment. METHODS: Deep RNA sequencing was performed on KD (n = 8) and childhood control (n = 7) coronary artery tissues, revealing 1074 differentially expressed mRNAs. Non-human RNA sequences were subjected to a microbial discovery bioinformatics platform, and microbial sequences were analyzed by Metastats for association with KD. RESULTS: T lymphocyte activation, antigen presentation, immunoglobulin production, and type I interferon response were significantly upregulated in KD arteritis, while the tumor necrosis factor α pathway was not differentially expressed. Transcripts from known infectious agents were not specifically associated with KD coronary arteritis. CONCLUSIONS: The immune transcriptional profile in KD coronary artery tissues has features of an antiviral immune response such as activated cytotoxic T lymphocyte and type I interferon-induced gene upregulation. These results provide new insights into the pathogenesis of KD arteritis that can guide selection of new immunomodulatory therapies for high-risk KD patients, and provide direction for future etiologic studies.

Radiation-induced equilibrium is a balance between tumor cell proliferation and T cell-mediated killing.

Local failures following radiation therapy are multifactorial, and the contributions of the tumor and the host are complex. Current models of tumor equilibrium suggest that a balance exists between cell birth and cell death due to insufficient angiogenesis, immune effects, or intrinsic cellular factors. We investigated whether host immune responses contribute to radiation-induced tumor equilibrium in animal models. We report an essential role for immune cells and their cytokines in suppressing tumor cell regrowth in two experimental animal model systems. Depletion of T cells or neutralization of IFN-γ reversed radiation-induced equilibrium, leading to tumor regrowth. We also demonstrate that PD-L1 blockade augments T cell responses, leading to rejection of tumors in radiation-induced equilibrium. We identify an active interplay between tumor cells and immune cells that occurs in radiation-induced tumor equilibrium and suggest a potential role for disruption of the PD-L1/PD-1 axis in increasing local tumor control.

Diagnostic tests for oral cancer and potentially malignant disorders in patients presenting with clinically evident lesions.

BACKGROUND: Oral squamous cell carcinoma is the most common form of malignancy of the lip and oral cavity, often being proceeded by potentially malignant disorders (PMD). Early detection can reduce the malignant transformation of PMD and can improve the survival rate for oral cancer. The current standard of scalpel biopsy with histology is painful for patients and involves a delay whilst histology is completed; other tests are available that are unobtrusive and provide immediate results. PRIMARY OBJECTIVE: To estimate the diagnostic accuracy of index tests for the detection of oral cancer and PMD of the lip and oral cavity, in people presenting with clinically evident lesions. SECONDARY OBJECTIVE: To estimate the relative accuracy of the different index tests. SEARCH METHODS: The electronic databases were searched on 30 April 2013. We searched MEDLINE (OVID) (1946 to April 2013) and four other electronic databases (the Cochrane Diagnostic Test Accuracy Studies Register, the Cochrane Oral Health Group's Trials Register, EMBASE (OVID) and MEDION (Ovid)). There were no restrictions on language in the searches of the electronic databases. We conducted citation searches and screened reference lists of included studies for additional references. SELECTION CRITERIA: We selected studies that reported the diagnostic test accuracy of the following index tests when used as an adjunct to conventional oral examination in detecting PMD or oral squamous cell carcinoma of the lip or oral cavity: vital staining, oral cytology, light-based detection and oral spectroscopy, blood or saliva analysis (which test for the presence of biomarkers in blood or saliva). DATA COLLECTION AND ANALYSIS: Two review authors independently screened titles and abstracts for relevance. Eligibility, data extraction and quality assessment were carried out by at least two authors, independently and in duplicate. Studies were assessed for methodological quality using QUADAS-2. Meta-analysis was used to combine the results of studies for each index test using the bivariate approach to estimate the expected values of sensitivity and specificity. MAIN RESULTS: We included 41 studies, recruiting 4002 participants, in this review. These studies evaluated the diagnostic accuracy of conventional oral examination with: vital staining (14 studies), oral cytology (13 studies), light-based detection or oral spectroscopy (13 studies). Six studies assessed two combined index tests. There were no eligible diagnostic accuracy studies evaluating blood or salivary sample analysis.The summary estimates for vital staining obtained from the meta-analysis were sensitivity of 0.84 (95% CI 0.74 to 0.90) with specificity of 0.70 (0.59 to 0.79), with 14 studies were included in the meta-analysis. For cytology, sensitivity was 0.91 (0.81 to 0.96) and specificity was 0.91 (0.81 to 0.95) with 12 studies included in the meta-analysis. For light-based detection, sensitivity was 0.91 (0.77 to 0.97) and specificity was 0.58 (0.22 to 0.87) with 11 studies included in the meta-analysis. The relative test accuracy was assessed by adding covariates to the bivariate analysis, no difference in model fit was observed. AUTHORS' CONCLUSIONS: The overall quality of the included studies was poor. None of the adjunctive tests can be recommended as a replacement for the currently used standard of a scalpel biopsy and histological assessment. Given the relatively high values of the summary estimates of sensitivity and specificity for cytology, this would appear to offer the most potential. Combined adjunctive tests involving cytology warrant further investigation.

Expression of the EPHB4 receptor tyrosine kinase in head and neck and renal malignancies--implications for solid tumors and potential for therapeutic inhibition.

Solid malignancies are often characterized by overexpression of various receptor tyrosine kinases (RTKs) against which many targeted therapies are currently in use and in active development. EPHB4 has recently emerged as a frequently overexpressed RTK in many types of cancer. Here, we demonstrate expression patterns of EPHB4 in two solid malignancies: squamous cell carcinoma of the head and neck (HNSCC) and renal cell carcinoma (RCC), by immunohistochemical analysis. We demonstrate the first association between EPHB4 expression and progression of HNSCC from normal tissue to dysplasia and to cancer. Interestingly, most RCC subtypes exhibited expression patterns that were opposite from that found in HNSCC, possibly owing to their unique biology and high degree of organ and tumor vasculature. Taken together, these results suggest a possible role for EPHB4 as a therapeutic target in these malignancies.

Switching anti-EGFR antibody re-sensitizes head and neck cancer patient following acquired resistance to cetuximab.

Cetuximab induces responses in about 13% of head and neck squamous cell carcinomas (HNSCC). We describe the molecular mechanism of acquired resistance to cetuximab, which could be overcome by switching to a different anti-EGFR antibody. Biopsies were collected at three different time points: before the start of cetuximab (PRE-cetux), at acquired resistance to cetuximab (AR-cetux), and at acquired resistance to duligotuzumab (AR-duligo). Biopsies were analyzed using tumor and normal whole-exome sequencing, RNASeq, and targeted panel sequencing with ultra-deep coverage to generate differential mutation and expression profiles. WES and targeted sequencing analysis identified an EGFR p.G465R extracellular domain mutation in AR-cetux biopsy. Furthermore, RNASeq confirmed the expression of this mutation in the tumor tissue. This mutation prevented the binding of cetuximab to EGFR and was not present in PRE-cetux and AR-duligo biopsies, suggesting a potential mechanism of acquired resistance to cetuximab. Molecular dynamic simulations confirmed that duligotuzumab effectively binds EGFR with a p.G465R mutation. Interestingly, the p.G465R mutation improved the stability of the duligotuzumab-EGFR complex as compared to the wild-type EGFR. This is the first report of an EGFR ECD mutation associated with acquired resistance to cetuximab, posing a need for further validation. We suggest appropriate serial mutational profiling to identify ECD mutations should be considered for select patients with initial cetuximab benefit.

Neoadjuvant Nivolumab Plus Chemotherapy Followed By Response-Adaptive Therapy for HPV+ Oropharyngeal Cancer: OPTIMA II Phase 2 Open-Label Nonrandomized Controlled Trial.

Importance: Immune checkpoint inhibitors improve survival in recurrent and/or metastatic head and neck cancer, yet their role in curative human papillomavirus-positive oropharyngeal cancer (HPV+ OPC) remains undefined. Neoadjuvant nivolumab and chemotherapy followed by response-adaptive treatment in HPV+ OPC may increase efficacy while reducing toxicity. Objective: To determine the deep response rate and tolerability of the addition of neoadjuvant nivolumab to chemotherapy followed by response-adapted locoregional therapy (LRT) in patients with HPV+ OPC. Design, Setting, and Participants: This phase 2 nonrandomized controlled trial conducted at a single academic center enrolled 77 patients with locoregionally advanced HPV+ OPC from 2017 to 2020. Data analyses were performed from February 10, 2021, to January 9, 2023. Interventions: Addition of nivolumab to neoadjuvant nab-paclitaxel and carboplatin (studied in the first OPTIMA trial) followed by response-adapted LRT in patients with HPV+ OPC stages III to IV. Main Outcomes and Measures: Primary outcome was deep response rate to neoadjuvant nivolumab plus chemotherapy, defined as the proportion of tumors with 50% or greater shrinkage per the Response Evaluation Criteria in Solid Tumors 1.1. Secondary outcomes were progression-free survival (PFS) and overall survival (OS). Swallowing function, quality of life, and tissue- and blood-based biomarkers, including programmed death-ligand 1 (PD-L1) expression and circulating tumor HPV-DNA (ctHPV-DNA), were also evaluated. Results: The 73 eligible patients (median [range] age, 61 [37-82] years; 6 [8.2%] female; 67 [91.8%] male) started neoadjuvant nivolumab and chemotherapy. Deep responses were observed in 51 patients (70.8%; 95% CI, 0.59-0.81). Subsequent risk- and response-adaptive therapy was assigned as follows: group A, single-modality radiotherapy alone or transoral robotic surgery (28 patients); group B, intermediate-dose chemoradiotherapy of 45 to 50 Gray (34 patients); and group C, regular-dose chemoradiotherapy of 70 to 75 Gray (10 patients). Two-year PFS and OS were 90.0% (95% CI, 0.80-0.95) and 91.4% (95% CI, 0.82-0.96), respectively. By response-adapted group, 2-year PFS and OS for group A were 96.4% and 96.4%, and group B, 88.0% and 91.0%, respectively. Lower enteral feeding rates and changes in weight, as well as improved swallowing, were observed among patients who received response-adapted LRT. Pathologic complete response rate among patients who underwent transoral robotic surgery was 67.0%. PD-L1 expression was nonsignificantly higher for deeper responses and improved PFS, and ctHPV-DNA clearance was significantly associated with improved PFS. Conclusions and Relevance: This phase 2 nonrandomized controlled trial found that neoadjuvant nivolumab and chemotherapy followed by response-adapted LRT is feasible and has favorable tolerability, excellent OS, and improved functional outcomes in HPV+ OPC, including among patients with high-risk disease. Moreover, addition of nivolumab may benefit high PD-L1 expressors, and sensitive dynamic biomarkers (eg, ctHPV-DNA) are useful for patient selection. Trial Registration: ClinicalTrials.gov Identifier: NCT03107182.

Integrins α4 and αM, collagen1A1, and matrix metalloproteinase 7 are upregulated in acute Kawasaki disease vasculopathy.

BACKGROUND: Kawasaki disease (KD) can result in fatal coronary artery (CA) aneurysms, especially if left untreated. Our recent studies of its vascular pathology revealed subacute/chronic vasculitis that begins early in the illness with the proliferation of smooth muscle cell-derived myofibroblasts in a complex extracellular matrix (ECM). We hypothesized that a dysregulation of specific ECM and adhesion molecules occurs in KD CAs. METHODS: Gene expression profiling for ECM and adhesion molecules was performed on six acute KD and eight control CAs using a targeted real-time PCR array approach. RESULTS: Integrins α4 and αM (ITGA4, ITGAM), collagen type I, α1 (COL1A1), and matrix metalloproteinase 7 (MMP7) were significantly upregulated in KD CAs as compared with controls. Immunohistochemistry with anti-ITGAM antibodies revealed expression on inflammatory cells within the CA wall in patients with KD but not in controls. CONCLUSION: Integrins ITGA4 and ITGAM are upregulated in KD vasculopathy, probably promoting inflammatory recruitment that stimulates smooth muscle cell transition to myofibroblasts and their proliferation. MMP7 probably enhances myofibroblast proliferation and luminal lesion expansion, and overexpression of COL1A1 may lead to CA stenosis. Identification of the molecular pathogenesis of KD vasculopathy may lead to the development of circulating biomarkers and to directed therapeutic interventions.

Proceedings of the North American Society of Head and Neck Pathology Companion Meeting, New Orleans, LA, March 12, 2023: Oral Cavity Dysplasia: Why Does Histologic Grading Continue to be Contentious?

Head and neck squamous cell carcinoma (HNSCC) is the world's 6th most common malignancy. Oral cavity SCC (OCSCC) represents approximately one third of the HNSCC cases diagnosed annually in the United States. Despite therapeutic advances, OCSCC is frequently lethal, with a modest 5-year survival. Because OCSCC is often preceded by premalignant lesions, it is an ideal disease for screening initiatives. The conventional visual and tactile exam (CVTE), coupled with a tissue biopsy, remains the gold standard. However, CVTE alone cannot reliably differentiate between reactive/inflammatory and dysplastic lesions. Further, the histologic diagnosis of dysplasia is subjective in nature and a highly imperfect predictor of malignant transformation. This prognostic uncertainty creates a significant clinical management dilemma-watchful waiting with increased patient psychological and economic burdens versus unnecessary aggressive treatment. As such, the development and validation of novel diagnostic platforms such as Artificial Intelligence (AI) and prognostic molecular biomarkers may help address these critical unmet clinical needs.

Inflammatory Tongue Conditions and Risk of Oral Tongue Cancer Among the US Elderly Individuals.

PURPOSE: The incidence of oral tongue cancers has increased since the 1980s among US men and women for unknown reasons. We investigated associations of inflammatory tongue conditions with risk of cancers of the oral tongue, other oral cavity, and oropharynx among the US elderly individuals (age 65 years or older). METHODS: We conducted a case-control study (2,534 oral tongue cancers, 6,832 other oral cavity cancers, 9,373 oropharyngeal cancers, and 200,000 controls) within the SEER-Medicare data set (1992-2013). Medicare records were used to identify patients with clinically diagnosed inflammatory tongue conditions (glossitis, benign migratory glossitis, median rhomboid glossitis, atrophic glossitis, glossodynia, other specified conditions [eg, atrophy and hypertrophy], and other unspecified conditions) and oral precancer (leukoplakia/erythroplakia). Only conditions preceding cancer/control selection by >12 months were included. RESULTS: The prevalence of inflammatory tongue conditions was significantly higher in patients with tongue cancer than controls (6.0% v 0.6%; odds ratios [ORs], adjusted for age, sex, race, Medicare utilization, and precancer, 5.8 [95% CI, 4.7 to 7.2]). This overall association primarily arose from glossitis, 5.6 (95% CI, 4.4 to 7.2); other specified conditions, 9.1 (95% CI, 5.5 to 15.2); and other unspecified conditions, 13.7 (95% CI, 8.0 to 23.7). These associations remained strongly elevated >5 years preceding tongue cancer (arguing against reverse causation), for conditions diagnosed by a specialist (arguing against misclassification), and among patients who received an oral biopsy (arguing against missed cancer). During 2013, an estimated 1 in 11 patients with oral tongue cancer had a preceding diagnosis of inflammatory tongue conditions. Associations of inflammatory tongue conditions were relatively weak for other oral cavity cancers (ORs, 1.8 [95% CI, 1.5 to 2.3]) and oropharyngeal cancer (OR, 1.3 [95% CI, 1.0 to 1.6]) and were observed only closest to cancer diagnosis. CONCLUSION: Inflammatory tongue conditions were associated with strongly increased risks of oral tongue cancers and preceded cancer diagnosis by several years, underscoring the need for increased clinical surveillance among patients with such apparently benign diagnoses.

SMYD3 represses tumor-intrinsic interferon response in HPV-negative squamous cell carcinoma of the head and neck.

Cancers often display immune escape, but the mechanisms are incompletely understood. Herein, we identify SMYD3 as a mediator of immune escape in human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC), an aggressive disease with poor response to immunotherapy with pembrolizumab. SMYD3 depletion induces upregulation of multiple type I interferon (IFN) response and antigen presentation machinery genes in HNSCC cells. Mechanistically, SMYD3 binds to and regulates the transcription of UHRF1, encoding for a reader of H3K9me3, which binds to H3K9me3-enriched promoters of key immune-related genes, recruits DNMT1, and silences their expression. SMYD3 further maintains the repression of immune-related genes through intragenic deposition of H4K20me3. In vivo, Smyd3 depletion induces influx of CD8+ T cells and increases sensitivity to anti-programmed death 1 (PD-1) therapy. SMYD3 overexpression is associated with decreased CD8 T cell infiltration and poor response to neoadjuvant pembrolizumab. These data support combining SMYD3 depletion strategies with checkpoint blockade to overcome anti-PD-1 resistance in HPV-negative HNSCC.

Characterization of NOL7 gene point mutations, promoter methylation, and protein expression in cervical cancer.

NOL7 is a putative tumor suppressor gene localized to 6p23, a region with frequent loss of heterozygosity in a number of cancers, including cervical cancer (CC). We have previously demonstrated that reintroduction of NOL7 into CC cells altered the angiogenic phenotype and suppressed tumor growth in vivo by 95%. Therefore, to understand its mechanism of inactivation in CC, we investigated the genetic and epigenetic regulation of NOL7. NOL7 mRNA and protein levels were assessed in 13 CC cell lines and 23 consecutive CC specimens by real-time quantitative polymerase chain reaction, western blotting, and immunohistochemistry. Methylation of the NOL7 promoter was analyzed by bisulfite sequencing and mutations were identified through direct sequencing. A CpG island with multiple CpG dinucleotides spanned the 5' untranslated region and first exon of NOL7. However, bisulfite sequencing failed to identify persistent sites of methylation. Mutational sequencing revealed that 40% of the CC specimens and 31% of the CC cell lines harbored somatic mutations that may affect the in vivo function of NOL7. Endogenous NOL7 mRNA and protein expression in CC cell lines were significantly decreased in 46% of the CC cell lines. Finally, immunohistochemistry demonstrated strong NOL7 nucleolar staining in normal tissues that decreased with histologic progression toward CC. NOL7 is inactivated in CC in accordance with the Knudson 2-hit hypothesis through loss of heterozygosity and mutation. Together with evidence of its in vivo tumor suppression, these data support the hypothesis that NOL7 is the legitimate tumor suppressor gene located on 6p23.

Evidence-based clinical recommendations regarding screening for oral squamous cell carcinomas.

BACKGROUND: This article presents evidence-based clinical recommendations developed by a panel convened by the American Dental Association Council on Scientific Affairs. This report addresses the potential benefits and potential risks of screening for oral squamous cell carcinomas and the use of adjunctive screening aids to visualize and detect potentially malignant and malignant oral lesions. TYPES OF STUDIES REVIEWED: The panel members conducted a systematic search of MEDLINE, identifying 332 systematic reviews and 1,499 recent clinical studies. They selected 5 systematic reviews and 4 clinical studies to use as a basis for developing recommendations. RESULTS: The panel concluded that screening by means of visual and tactile examination to detect potentially malignant and malignant lesions may result in detection of oral cancers at early stages of development, but that there is insufficient evidence to determine if screening alters disease-specific mortality in asymptomatic people seeking dental care. CLINICAL IMPLICATIONS: The panel suggested that clinicians remain alert for signs of potentially malignant lesions or early-stage cancers while performing routine visual and tactile examinations in all patients, but particularly in those who use tobacco or who consume alcohol heavily. Additional research regarding oral cancer screening and the use of adjuncts is needed.

Effect of preradiation dental intervention on incidence of osteoradionecrosis in patients with head and neck cancer: A systematic review and meta-analysis.

BACKGROUND: The purpose of this systematic review was to examine whether dental intervention involving bone or soft-tissue manipulation preradiotherapy (pre-RT) is associated with lower rates of osteoradionecrosis of the jaw (ORNJ) in patients with head and neck cancer (HNC). TYPES OF STUDIES REVIEWED: The authors included relevant studies from MEDLINE, Embase, and Cochrane Library, including observational studies published from 2007 through 2021 and involving adults who underwent dental intervention pre-RT for HNC. Authors assessed evidence certainty by using the Grading of Recommendations Assessment, Development, and Evaluation approach. Random-effects models were used to calculate pooled relative risk estimates and hazard ratios. When meta-analysis was not possible, study-level measures of association and narrative summaries of the evidence were reported. RESULTS: Twenty-two studies were included. From the pooled, unadjusted analysis, patients undergoing pre-RT extractions may have a 55% increased risk of experiencing ORNJ (relative risk, 1.55; 95% CI, 0.85 to 2.86; very low certainty); the unadjusted pooled hazard ratio was 3.19 (95% CI, 0.99 to 10.31; very low certainty), corresponding to a possible increased hazard of developing ORNJ (very low certainty). Findings for other pre-RT procedures manipulating bone or tissue relied on limited, observational studies with low or very low certainty evidence. CONCLUSIONS: Mostly very low certainty evidence suggests that patients with HNC who need pre-RT dental intervention may have an increased risk of developing ORNJ compared with those who do not. PRACTICAL IMPLICATIONS: Maintaining optimal oral health may help reduce the need for urgent pre-RT dental treatment, potentially reducing ORNJ risk and minimizing delay of oncologic treatment in patients with HNC.