Intraoperative Techniques That Define the Mucosal Margins of Oral Cancer In-Vivo: A Systematic Review.

BACKGROUND: This systematic review investigates techniques for determining adequate mucosal margins during the resection of oral squamous cell carcinoma (SCC). The primary treatment involves surgical removal with ≥5 mm margins, highlighting the importance of accurate differentiation between SCC and dysplasia during surgery. METHODS: A comprehensive Embase and PubMed literature search was performed. Studies underwent quality assessment using QUADAS-2. RESULTS: After the full-text screening and exclusion of studies exhibiting high bias, eight studies were included, focusing on three margin visualization techniques: autofluorescence, iodine staining, and narrow-band imaging (NBI). Negative predictive value (NPV) was calculable across the studies, though reference standards varied. Results indicated NPVs for autofluorescence, iodine, and NBI ranging from 61% to 100%, 92% to 99%, and 86% to 100%, respectively. Autofluorescence did not significantly enhance margins compared to white light-guided surgery, while iodine staining demonstrated improvement for mild or moderate dysplasia. NBI lacked comparison with a white light-guided surgery cohort. CONCLUSIONS: We recommend studying and comparing the diagnostic accuracy of iodine staining and NBI in larger cohorts of patients with oral SCC, focusing on discriminating between SCC and (severe) dysplasia. Furthermore, we advise reporting the diagnostic accuracy alongside the treatment effects to improve the assessment of these techniques.

Differences in the association of time to treatment initiation and survival according to various head and neck cancer sites in a nationwide cohort.

OBJECTIVES: To assess whether there are differences in the effects of time to treatment interval (TTI) on patient survival for head and neck cancer (HNC) sites in order to provide evidence that can support decision-making regarding prioritizing treatment. MATERIALS AND METHODS: Patients in the Netherlands with a first primary HNC without distant metastasis between 2010 and 2014 were included for analysis (N = 10,486). TTI was defined as the time from pathologic diagnosis to the start of initial treatment. Overall survival (OS), cox regression analyses and cubic spline hazard models were calculated and visualized. RESULTS: Overall, the hazard of dying was higher (HR = 1.003; 95 % CI 1.001-1.005) with each additional day until treatment initiation. The pattern, as visualized in cubic spline graphs, differed by site the hazard increased more steeply with increasing TTI for oral cavity cancer. For oropharyngeal and laryngeal cancer, a slight increase commenced after a longer TTI than for oral cavity cancer, while there was hardly an increase in hazard with increasing TTI for hypopharyngeal cancer. CONCLUSION: The relationship between longer TTI and decreased survival was confirmed, but slight variations in the pattern of the hazard of dying by TTI by tumour site were observed. These findings could support decisions on prioritizing treatment. However, other aspects such as extent of treatment and quality of life should be investigated further so this can also be included.

Comparative transcriptomics of porcine liver-resident CD8αdim, liver CD8αhigh and circulating blood CD8αhigh NK cells reveals an intermediate phenotype of liver CD8αhigh NK cells.

Liver-resident NK (lrNK) cells have been studied in humans as well as in mice. Unfortunately, important differences have been observed between murine and human lrNK cells, complicating the extrapolation of data obtained in mice to man. We previously described two NK cell subsets in the porcine liver: A CD8αhigh subset, with a phenotype much like conventional CD8αhigh NK cells found in the peripheral blood, and a specific liver-resident CD8αdim subset which phenotypically strongly resembles human lrNK cells. These data suggest that the pig might be an attractive model for studying lrNK cell biology. In the current study, we used RNA-seq to compare the transcriptome of three porcine NK cell populations: Conventional CD8αhigh NK cells from peripheral blood (cNK cells), CD8αhigh NK cells isolated from the liver, and the liver-specific CD8αdim NK cells. We found that highly expressed transcripts in the CD8αdim lrNK cell population mainly include genes associated with the (adaptive) immune response, whereas transcripts associated with cell migration and extravasation are much less expressed in this subset compared to cNK cells. Overall, our data indicate that CD8αdim lrNK cells show an immature and anti-inflammatory phenotype. Interestingly, we also observed that the CD8αhigh NK cell population that is present in the liver appears to represent a population with an intermediate phenotype. Indeed, while the transcriptome of these cells largely overlaps with that of cNK cells, they also express transcripts associated with liver residency, in particular CXCR6. The current, in-depth characterization of the transcriptome of porcine liver NK cell populations provides a basis to use the pig model for research into liver-resident NK cells.

Jaw Bone Invasion of Oral Squamous Cell Carcinoma Is Associated with Osteoclast Count and Expression of Its Regulating Proteins in Patients and Organoids.

AIMS: Oral squamous cell carcinoma (OSCC) frequently invades the jaw. The exact mechanism of bone invasion remains unclear. This study investigates (premature) osteoclasts and the expression of its differentiation regulating proteins RANKL, OPG and RANK in patients with OSCC. METHODS: Resection specimens from OSCC patients were divided into NI group (No Invasion), E group (Erosion) or I group (bone Invasion). Tissue sections were stained with Cathepsin K (osteoclast-counting), RANKL, OPG and RANK. The staining intensity was scored on different regions of the tumor: front, center, back and normal mucosa. Immunohistochemistry and qPCR for RANKL/OPG/RANK were performed on five head and neck squamous cell carcinoma (HNSCC) organoids. RESULTS: The mean number of osteoclasts (I group) and premature osteoclasts (E group) was significantly higher compared to the NI group (p = 0.003, p = 0.036). RANKL expression was significantly higher in the tumor front and tumor center compared to normal mucosa (all groups). In the I group, RANKL and RANK expression was significantly higher in the tumor front compared to the tumor back and there was a trend of higher RANKL expression in the tumor front compared to the E group and NI group. qPCR showed a 20-43 times higher RANKL mRNA expression in three out of five tumor organoids compared to a normal squamous cell organoid line. There was no correlation between protein and mRNA expression in the HNSCC organoids. CONCLUSIONS: These findings suggest that OSCCs induce bone invasion by stimulating osteoclast activation by regulating the production of RANKL and RANK proteins.

Alphaherpesvirus-mediated remodeling of the cellular transcriptome results in depletion of m6A-containing transcripts.

The mechanisms by which viruses regulate host mRNAs during infection are still poorly understood. Several host transcripts that encode proteins that contribute to the anti-viral response contain the N6-methyladenosine nucleotide (m6A). In this study, we investigated if and how viruses from different (sub) families specifically affect m6A-containing host transcripts. Systematic analysis of host transcriptomes after infection with diverse types of viruses showed that m6A-methylated transcripts are selectively downregulated during infection with Sendai virus, African swine fever virus and the alphaherpesviruses herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV). Focusing on PRV and HSV-1, we found that downregulation of m6A-methylated transcripts depends on the YTHDF family of m6A-binding proteins, and correlates with localization of these proteins to enlarged P-bodies. Knockdown of YTHDF proteins in primary cells reduced PRV protein expression and increased expression of antiviral interferon-stimulated genes, suggesting that virus-induced depletion of host m6A-containing transcripts constitutes an immune evasion strategy.

Treatment of buccal mucosal carcinomas: A survey amongst head and neck surgeons in the Netherlands.

Objective: Currently, there is no up-to-date guideline for the treatment of buccal mucosal squamous cell carcinoma (BMSCC) in the Netherlands. A questionnaire was used to investigate the opinions of Dutch head and neck surgeons on BMSCC of the cheek treatment. Methods: A questionnaire was sent to all 91 head and neck surgeons in the Netherlands. Their opinions on surgical tumor-free margins, through-and-through defects, and indications for local adjuvant therapy were questioned. Results: The response rate was 51%. To prevent a through-and-through defect, 67% of the surgeons would accept a deep clinical (macroscopic) margin of ≤5 mm. The less adverse histological characteristics a tumor has, the less consensus there is amongst the surgeons for local adjuvant treatment in case of close margins. Conclusion: There is no consensus amongst Dutch head and neck surgeons about the optimal treatment for BMSCC of the cheek. There are different opinions on acceptable resection margins, indications for a through-and-through defect, and indications for adjuvant treatment. BMSCC of the cheek treatment should be more uniform and less surgeon dependent. Level of evidence: N/A.

The potential of the Crystal Cam handheld gamma-camera for preoperative and intraoperative sentinel lymph node localization in early-stage oral cancer.

PURPOSE: Evaluating the Crystal Cam handheld gamma-camera for preoperative and intraoperative sentinel lymph node (SLN) localization in early-stage oral cancer. METHODS: The handheld gamma-camera was used complementary to conventional gamma-probe guidance for intraoperative SLN localization in 53 early-stage oral cancer patients undergoing SLN biopsy. In 36 of these patients, a blinded comparison was made between preoperative handheld gamma-camera and lymphoscintigraphy outcomes. Of those, the reliability for marking the SLN's location using both handheld gamma-camera and a 57Co-penpoint marker was evaluated in 15 patients. RESULTS: In the entire cohort, the handheld gamma-camera preoperatively detected 116/122 (95%) of SLNs identified by lymphoscintigraphy. In those patients where the observer was blinded for lymphoscintigraphy (n = 36), 71/77 (92%) SLNs were correctly identified by handheld gamma-camera. Overlooked SLNs by handheld gamma-camera were mainly located near the injection site. The SLN's marked location by handheld gamma-camera and 57Co-penpoint marker was considered accurate in 42/43 (98%) SLNs. The intraoperative use of the handheld gamma-camera led to the extirpation of 16 additional 'hot' lymph nodes in 14 patients, 4 of which harbored metastases, and prevented 2 patients (4%) from being erroneously staged negative for nodal metastasis. In those with follow-up ≥ 24 months or false-negative outcomes < 24 months following SLNB, a sensitivity of 82% and negative predictive value of 93% was obtained. CONCLUSION: The Crystal Cam handheld gamma-camera offers reliable preoperative and intraoperative SLN localization and might reduce the risk of missing a malignant SLN during surgery. Detecting SLNs near the injection site by handheld gamma-camera remains challenging.

Patient-derived head and neck cancer organoids allow treatment stratification and serve as a tool for biomarker validation and identification.

BACKGROUND: Organoids are in vitro three-dimensional structures that can be grown from patient tissue. Head and neck cancer (HNC) is a collective term used for multiple tumor types including squamous cell carcinomas and salivary gland adenocarcinomas. METHODS: Organoids were established from HNC patient tumor tissue and characterized using immunohistochemistry and DNA sequencing. Organoids were exposed to chemo- and radiotherapy and a panel of targeted agents. Organoid response was correlated with patient clinical response. CRISPR-Cas9-based gene editing of organoids was applied for biomarker validation. FINDINGS: A HNC biobank consisting of 110 models, including 65 tumor models, was generated. Organoids retained DNA alterations found in HNC. Comparison of organoid and patient response to radiotherapy (primary [n = 6] and adjuvant [n = 15]) indicated potential for guiding treatment options in the adjuvant setting. In organoids, the radio-sensitizing potential of cisplatin and carboplatin could be validated. However, cetuximab conveyed radioprotection in most models. HNC-targeted treatments were tested on 31 models, indicating possible novel treatment options with the potential for treatment stratification in the future. Activating PIK3CA mutations did not predict alpelisib response in organoids. Protein arginine methyltransferase 5 (PRMT5) inhibitors were identified as a potential treatment option for cyclin-dependent kinase inhibitor 2A (CDKN2A) null HNC. CONCLUSIONS: Organoids hold potential as a diagnostic tool in personalized medicine for HNC. In vitro organoid response to radiotherapy (RT) showed a trend that mimics clinical response, indicating the predictive potential of patient-derived organoids. Moreover, organoids could be used for biomarker discovery and validation. FUNDING: This work was funded by Oncode PoC 2018-P0003.

A closer look at the resection margins of buccal mucosa cancer: Their influence on local recurrence free survival.

BACKGROUND: The adequate surgical margin for local control of buccal mucosa squamous cell carcinoma (BMSCC) is under debate. This study investigates surgical margins and other factors associated with local recurrence free survival (LRFS) in a large cohort of BMSCC patients. METHODS: Multiple factors were evaluated retrospectively in 97 patients with BMSCC. Cox-regression and Kaplan-Meier curves were used for analysis. RESULTS: The local recurrence rate was 23%. The tumor-free margin was <5.0 mm in 89% of the patients and the deep margin was significantly more often inadequate. Multivariate analysis associated pT3-classification, former smokers, tumor-free margin status, and postoperative (chemo)radiation (PO(ch)RT) with local recurrence. Re-resections did not improve LRFS in patients with <5.0 mm tumor-free margins. CONCLUSIONS: Adequate tumor-free margins are pivotal for LRFS of BMSCC. PO(ch)RT, not re-resection, can improve LRFS in patients with <5.0 mm tumor-free margins.

Ultrasound-guided resection for squamous cell carcinoma of the buccal mucosa: A feasibility study.

BACKGROUND: Image-guided surgery could help obtain clear (≥5.0 mm) resection margins. This feasibility study investigated ultrasound-guided resection accuracy of buccal mucosa squamous cell carcinoma (BMSCC). METHODS: MRI and ultrasound measurements of tumor thickness were compared to histology in 13 BMSCC-patients. Ultrasound measured margins (at five locations) on the specimen were compared to the corresponding histological margins. RESULTS: Accuracy of in- and ex-vivo ultrasound (mean deviation from histology: 1.6 mm) for measuring tumor thickness was comparable to MRI (mean deviation from histology: 2.6 mm). The sensitivity to detect clear margins using ex-vivo ultrasound was low (48%). If an ex-vivo ultrasound cutoff of ≥7.5 mm would be used, the sensitivity would increase to 86%. CONCLUSIONS: Ultrasound-guided resection of BMSCC's is feasible. In- and ex-vivo ultrasound measure tumor thickness in BMSCC accurately. We recommend ≥7.5 mm resection margins on ex-vivo ultrasound to obtain histological clear margins. Additional research is required to establish the effect of 7.5 mm ultrasound cutoff.

Effects of a nutritional intervention on impaired behavior and cognitive function in an emphysematous murine model of COPD with endotoxin-induced lung inflammation.

One cluster of the extrapulmonary manifestations in chronic obstructive pulmonary disease (COPD) is related to the brain, which includes anxiety, depression and cognitive impairment. Brain-related comorbidities are related to worsening of symptoms and increased mortality in COPD patients. In this study, a murine model of COPD was used to examine the effects of emphysema and repetitive pulmonary inflammatory events on systemic inflammatory outcomes and brain function. In addition, the effect of a dietary intervention on brain-related parameters was assessed. Adult male C57Bl/6J mice were exposed to elastase or vehicle intratracheally (i.t.) once a week on three consecutive weeks. Two weeks after the final administration, mice were i.t. exposed to lipopolysaccharide (LPS) or vehicle for three times with a 10 day interval. A dietary intervention enriched with omega-3 PUFAs, prebiotic fibers, tryptophan and vitamin D was administered from the first LPS exposure onward. Behavior and cognitive function, the degree of emphysema and both pulmonary and systemic inflammation as well as blood-brain barrier (BBB) integrity and neuroinflammation in the brain were assessed. A lower score in the cognitive test was observed in elastase-exposed mice. Mice exposed to elastase plus LPS showed less locomotion in the behavior test. The enriched diet seemed to reduce anxiety-like behavior over time and cognitive impairments associated with the presented COPD model, without affecting locomotion. In addition, the enriched diet restored the disbalance in splenic T-helper 1 (Th1) and Th2 cells. There was a trend toward recovering elastase plus LPS-induced decreased expression of occludin in brain microvessels, a measure of BBB integrity, as well as improving expression levels of kynurenine pathway markers in the brain by the enriched diet. The findings of this study demonstrate brain-associated comorbidities - including cognitive and behavioral impairments - in this murine model for COPD. Although no changes in lung parameters were observed, exposure to the specific enriched diet in this model appeared to improve systemic immune disbalance, BBB integrity and derailed kynurenine pathway which may lead to reduction of anxiety-like behavior and improved cognition.

Proteomic Comparison of Three Wild-Type Pseudorabies Virus Strains and the Attenuated Bartha Strain Reveals Reduced Incorporation of Several Tegument Proteins in Bartha Virions.

Pseudorabies virus (PRV) is a member of the alphaherpesvirus subfamily and the causative agent of Aujeszky's disease in pigs. Driven by the large economic losses associated with PRV infection, several vaccines and vaccine programs have been developed. To this day, the attenuated Bartha strain, generated by serial passaging, represents the golden standard for PRV vaccination. However, a proteomic comparison of the Bartha virion to wild-type (WT) PRV virions is lacking. Here, we present a comprehensive mass spectrometry-based proteome comparison of the attenuated Bartha strain and three commonly used WT PRV strains: Becker, Kaplan, and NIA3. We report the detection of 40 structural and 14 presumed nonstructural proteins through a combination of data-dependent and data-independent acquisition. Interstrain comparisons revealed that packaging of the capsid and most envelope proteins is largely comparable in-between all four strains, except for the envelope protein pUL56, which is less abundant in Bartha virions. However, distinct differences were noted for several tegument proteins. Most strikingly, we noted a severely reduced incorporation of the tegument proteins IE180, VP11/12, pUS3, VP22, pUL41, pUS1, and pUL40 in Bartha virions. Moreover, and likely as a consequence, we also observed that Bartha virions are on average smaller and more icosahedral compared to WT virions. Finally, we detected at least 28 host proteins that were previously described in PRV virions and noticed considerable strain-specific differences with regard to host proteins, arguing that the potential role of packaged host proteins in PRV replication and spread should be further explored. IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha-an attenuated strain created by serial passaging-represents an exceptional success story in alphaherpesvirus vaccination. Here, we used mass spectrometry to analyze the Bartha virion composition in comparison to three established WT PRV strains. Many viral tegument proteins that are considered nonessential for viral morphogenesis were drastically less abundant in Bartha virions compared to WT virions. Interestingly, many of the proteins that are less incorporated in Bartha participate in immune evasion strategies of alphaherpesviruses. In addition, we observed a reduced size and more icosahedral morphology of the Bartha virions compared to WT PRV. Given that the Bartha vaccine strain elicits potent immune responses, our findings here suggest that differences in protein packaging may contribute to its immunogenicity. Further exploration of these observations could aid the development of efficacious vaccines against other alphaherpesvirus vaccines such as HSV-1/2 or EHV-1.

Alphaherpesvirus US3 protein-mediated inhibition of the m6A mRNA methyltransferase complex.

Chemical modifications of mRNA, the so-called epitranscriptome, represent an additional layer of post-transcriptional regulation of gene expression. The most common epitranscriptomic modification, N6-methyladenosine (m6A), is generated by a multi-subunit methyltransferase complex. We show that alphaherpesvirus kinases trigger phosphorylation of several components of the m6A methyltransferase complex, including METTL3, METTL14, and WTAP, which correlates with inhibition of the complex and a near complete loss of m6A levels in mRNA of virus-infected cells. Expression of the viral US3 protein is necessary and sufficient for phosphorylation and inhibition of the m6A methyltransferase complex. Although m6A methyltransferase complex inactivation is not essential for virus replication in cell culture, the consensus m6A methylation motif is under-represented in alphaherpesvirus genomes, suggesting evolutionary pressure against methylation of viral transcripts. Together, these findings reveal that phosphorylation can be associated with inactivation of the m6A methyltransferase complex, in this case mediated by the viral US3 protein.

Application and accuracy of ultrasound-guided resections of tongue cancer.

OBJECTIVES: Surgical removal of squamous cell carcinoma of the tongue (SCCT) with tumour-free margin status (≥5 mm) is essential for loco-regional control. Inadequate margins (<5 mm) often indicate adjuvant treatment, which results in increased morbidity. Ultrasound (US)-guided SCCT resection may be a useful technique to achieve more adequate resection margins compared to conventional surgery. This study evaluates the application and accuracy of this technique. METHODS: Forty patients with SCCT were included in a consecutive US cohort. During surgery, the surgeon aimed for a 10-mm echographic resection margin, while the tumour border and resection plane were captured in one image. Ex-vivo US measurements of the resection specimen determined whether there was a need for an immediate re-resection. The margin status and the administration of adjuvant treatment were compared those of with a consecutive cohort of 96 tongue cancer patients who had undergone conventional surgery. A receiver operating characteristic analysis was done to assess the optimal margin of ex-vivo US measurements to detect histopathologically inadequate margins. RESULTS: In the US cohort, the frequency of free margin status was higher than in the conventional cohort (55% vs. 16%, p < 0.001), and the frequency of positive margins status (<1 mm) was lower (5% vs. 15%, respectively, p < 0.001). Adjuvant radiotherapy was halved (10% vs. 21%), and the need for re-resection was comparable (10% vs. 9%). A cut-off value of 8 mm for ex-vivo measurements prevented histopathologically inadequate margins in 76%. CONCLUSION: US-guided SCCT resections improve margin status and reduce the frequency of adjuvant radiotherapy.

Effect of targeted nutrient supplementation on physical activity and health-related quality of life in COPD: study protocol for the randomised controlled NUTRECOVER trial.

INTRODUCTION: Physical and mental health are often affected in chronic obstructive pulmonary disease (COPD) adversely affecting disease course and quality of life. Abnormalities in whole body and cellular energy metabolism, dietary and plasma nutrient status and intestinal permeability have been well established in these patients as systemic determinants of functional decline and underexplored treatable traits. The aim of this study is to investigate the efficacy of 1 year targeted nutrient supplementation on physical activity level and health-related quality of life in patients with COPD. METHODS AND ANALYSIS: This study is a single-centre randomised, placebo-controlled, double-blind trial in 166 patients with COPD recruited from multiple hospitals in the Netherlands. The intervention group will receive a multinutrient supplement, including vitamin D, tryptophan, long-chain polyunsaturated fatty acids and prebiotic dietary fibres as main components (94 kCal per daily dose). The control group will receive an isocaloric isonitrogenous placebo. Both groups will ingest one portion per day for at least 12 months and will additionally receive counselling on healthy lifestyle and medical adherence over the course of the study. Coprimary outcomes are physical activity assessed by triaxial accelerometry and health-related quality of life measured by the EuroQol-5 dimensions questionnaire. Secondary outcomes are cognitive function, psychological well-being, physical performance, patient-reported outcomes and the metabolic profile assessed by body composition, systemic inflammation, plasma nutrient levels, intestinal integrity and microbiome composition. Outcomes will be measured at baseline and after 12 months of supplementation. In case patients are hospitalised for a COPD exacerbation, a subset outcome panel will be measured during a 4-week recovery period after hospitalisation. ETHICS AND DISSEMINATION: This study was approved by the local Ethics Committee of Maastricht University. Subjects will be included after written informed consent is provided. Study outcomes will be disseminated through presentations at (inter)national conferences and through peer-reviewed journals. TRIAL REGISTRATION: NCT03807310.