The prognostic impact of human papillomavirus (HPV) in oropharyngeal cancer is generally acknowledged, and HPV‐status is assessed routinely in clinical practice. Paradoxically, while the oral cavity seems the predilection site for productive HPV‐infections, figures on HPV‐attribution in oral cavity squamous cell carcinoma (OCSCC) differ widely, and prognostic impact is uncertain. Major obstacles are the lack of reproducible assays to detect HPV in nonoropharyngeal cancers, the relatively small cohorts studied and consequently the shortfall of convincing data. In our study, we used a validated, nucleic acid‐based workflow to assess HPV‐prevalence in a consecutive cohort of 1016 OCSCCs, and investigated its prognostic impact. In parallel, we analyzed p16‐immunohistochemistry (p16‐IHC) as surrogate marker for transforming HPV‐infection and independent prognosticator. All OCSCC‐patients diagnosed between 2008 and 2014 at two Dutch university medical centers were included (N = 1069). Formalin‐fixed, paraffin‐embedded (FFPE)‐samples of 1016 OCSCCs could be retrieved. Punch biopsies were taken from the tumor area in the FFPE‐blocks and tested for HPV. P16‐IHC was performed on 580 OCSCCs, including all HPV‐positive tumors. From 940 samples (92.5%), nucleic acids were of sufficient quality for HPV‐testing. In total, 21 (2.2%) OCSCCs were HPV DNA‐positive. All HPV DNA‐positive tumors were E6 mRNA‐positive and considered as true HPV‐positive. There was no difference in survival between HPV‐positive and HPV‐negative OCSCCs. In total, 46 of 580 (7.9%) OCSCCs were p16‐immunopositive, including all HPV‐positive tumors. Survival was comparable in p16‐positive and p16‐negative OCSCCs. To conclude, HPV‐prevalence is very low in OCSCC and neither HPV‐status nor p16‐status affects outcome. Based on these data, determining HPV‐status in OCSCC seems irrelevant for clinical management.

ABSTRACT In oral-cancer, the number of tumor-infiltrating lymphocytes (TILs) associates with improved survival, yet the prognostic value of the cellular composition and localization of TILs is not defined. We quantified densities, localizations, and cellular networks of lymphocyte populations in 138 patients with T1-T2 primary oral-tongue squamous cell carcinoma treated with surgical resections without any perioperative (chemo)radiotherapy, and correlated outcomes to overall survival (OS). Multiplexed in-situ immunofluorescence was performed for DAPI, CD4, CD8, CD20, and pan-cytokeratin using formalin-fixed paraffin-embedded sections, and spatial distributions of lymphocyte populations were assessed in the tumor and stroma compartments at the invasive margin (IM) as well as the center of tumors. We observed a high density of CD4, CD8, and CD20 cells in the stroma compartment at the IM, but neither lymphocyte densities nor networks as single parameters associated with OS. In contrast, assessment of two contextual parameters within the stroma IM region of tumors, i.e., the number of CD20 cells within 20 µm radii of CD20 and CD4 cells, termed the CD20 Cluster Score, yielded a highly significant association with OS (HR 0.38; p = .003). Notably, the CD20 Cluster Score significantly correlated with better OS and disease-free survival in multivariate analysis (HR 0.34 and 0.47; p = .001 and 0.019) as well as with lower local recurrence rate (OR: 0.13; p = .028). Taken together, our study showed that the presence of stromal B-cell clusters at IM, in the co-presence of CD4 T-cells, associates with good prognosis in early oral-tongue cancer patients.

Background Inadequate resection margins in oral cavity squamous cell carcinoma have an adverse effect on patient outcome. Intraoperative assessment provides immediate feedback enabling the surgeon to achieve adequate resection margins. The goal of this study was to evaluate the value of specimen-driven intraoperative assessment by comparing the margin status in the period before and the period after the introduction of specimen-driven assessment as a standard of care (period 2010–2012 vs period 2013–2017). Methods A cohort of patients surgically treated for oral squamous cell carcinoma at the Erasmus MC Cancer Institute, Rotterdam, between 2010–2012 was studied retrospectively and compared to results of a prospectively collected cohort between 2013–2017. The frequency, type and results of intraoperative assessment of resection margins were analyzed. Results One hundred seventy-four patients were included from 2010–2012, 241 patients were included from 2013–2017. An increase in the frequency of specimen-driven assessment was seen between the two periods, from 5% in 2010–2012 to 34% in 2013–2017. When performing specimen-driven assessment, 16% tumor-positive resection margins were found in 2013–2017, compared to 43% tumor-positive resection margins overall in 2010–2012. We found a significant reduction of inadequate resection margins for specimen-driven intraoperative assessment (p < 0.001). Also, tumor recurrence significantly decreased, and disease-specific survival improved when performing specimen-driven intraoperative assessment. Conclusions Specimen-driven intraoperative assessment improves resection margins and consequently, the outcome of oral cancer patients. We advocate this method as standard of care.

Abstract With an incidence of 350.000 new cases per year, cancer of the oral cavity ranks among the 10 most common solid organ cancers. Most of these cancers are squamous cell carcinomas. Five‐year survival is about 50%. It has been shown that clear resection margins (>5 mm healthy tissue surrounding the resected tumor) have a significant positive effect on locoregional control and survival. It is not uncommon that the resection margins of oral tumors are inadequate. However, when providing the surgeon with intraoperative feedback on the resection margin status, it is expected that obtaining adequate resection margins is improved. In this respect, it has been shown that specimen‐driven intraoperative assessment of resection margins is superior to defect‐driven intraoperative assessment of resection margins. In this concise report, it is described how a specimen‐driven approach can increase the rate of adequate resections of oral cavity squamous cell carcinoma as well as that it is discussed how intraoperative assessment can be further improved with regard to the surgical treatment of oral cavity squamous cell carcinoma.

Background: Oral premalignant lesions (OPLs) represent the most common oral precancerous conditions. One of the major challenges in this field is the identification of OPLs at higher risk for oral squamous cell cancer (OSCC) development, by discovering molecular pathways deregulated in the early steps of malignant transformation. Analysis of deregulated levels of single genes and pathways has been successfully applied to head and neck squamous cell cancers (HNSCC) and OSCC with prognostic/predictive implications. Exploiting the availability of gene expression profile and clinical follow-up information of a well-characterized cohort of OPL patients, we aim to dissect tissue OPL gene expression to identify molecular clusters/signatures associated with oral cancer free survival (OCFS). Materials and methods: The gene expression data of 86 OPL patients were challenged with: an HNSCC specific 6 molecular subtypes model (Immune related: HPV related, Defense Response and Immunoreactive; Mesenchymal, Hypoxia and Classical); one OSCC-specific signature (13 genes); two metabolism-related signatures (3 genes and signatures raised from 6 metabolic pathways associated with prognosis in HNSCC and OSCC, respectively); a hypoxia gene signature. The molecular stratification and high versus low expression of the signatures were correlated with OCFS by Kaplan–Meier analyses. The association of gene expression profiles among the tested biological models and clinical covariates was tested through variance partition analysis. Results: Patients with Mesenchymal, Hypoxia and Classical clusters showed an higher risk of malignant transformation in comparison with immune-related ones (log-rank test, p = 0.0052) and they expressed four enriched hallmarks: “TGF beta signaling” “angiogenesis”, “unfolded protein response”, “apical junction”. Overall, 54 cases entered in the immune related clusters, while the remaining 32 cases belonged to the other clusters. No other signatures showed association with OCFS. Our variance partition analysis proved that clinical and molecular features are able to explain only 21% of gene expression data variability, while the remaining 79% refers to residuals independent of known parameters. Conclusions: Applying the existing signatures derived from HNSCC to OPL, we identified only a protective effect for immune-related signatures. Other gene expression profiles derived from overt cancers were not able to identify the risk of malignant transformation, possibly because they are linked to later stages of cancer progression. The availability of a new well-characterized set of OPL patients and further research is needed to improve the identification of adequate prognosticators in OPLs.

Atopic dermatitis (AD) is a heterogeneous disease with various biological origins and clinical appearances. It is likely that different therapies or treatment intensities are not equally effective for all AD endotypes. The strongest genetic risk factor for AD is a null muta‐ tion in the filaggrin gene (FLG).1 Patients with eczema who carry a FLG null mutation are also prone to more persistent, severe eczema, and earlier onset of AD compared to patients without a FLG null mutation. Stratification of patients based on the FLG null endotype could enable more targeted treatment. Methods to determine FLG null mutations based on genotyping are time consuming and require specialized laboratory infrastructure, further complicated by the existence of over 50 different polymorphisms with widely varying prevalences between ethnic groups.2 In the stratum corneum (SC) filaggrin is enzymatically degraded into its constituting amino acids and their derivatives, together with specific salts and sugars collec‐ tively named natural moisturizing factor (NMF). Decreased NMF provides an accurate surrogate marker for the presence of FLG null polymorphisms.3 This can be measured rapidly and noninvasively by Raman spectroscopy in a clinically compatible test. We have assessed the potential of NMF as a novel clinical marker in AD by examining the association of clinically measured NMF val‐ ues with severity of AD, early onset of AD, and the co‐morbidities of AD: allergic sensitization, food allergy, bronchial hyperreactivity (BHR), asthma, and allergic rhinitis. Of 207 children with AD (0‐18 years of age), NMF values had been measured routinely during a visit to the pediatric atopy cen‐ ter KinderHaven‐Sophia Children's Hospital‐Erasmus MC University Medical Center Rotterdam in The Netherlands. The retrospective study protocol was approved by the medical ethics committee of Erasmus MC (MEC‐2016‐244). AD was diagnosed by a dermatologist according to the UK Working Party's Diagnostic Criteria for Atopic Dermatitis.4 NMF had been measured noninvasively on the palm of the hand by Raman spectroscopy using an in vivo Raman skin ana‐ lyzer (gen2‐SCA, RiverD International BV, Rotterdam). NMF values were classified as normal NMF (>1.14 arbitrary units) or decreased NMF (<0.995 arbitrary units), using a 0.07 confidence interval around the threshold of 1.07 as established by O’Regan et al.3 Patients with a NMF value between 0.995‐1.14 were excluded. The interval was the estimated 95% confidence interval, calculated as the standard error (SE) of the NMF value, averaged over the entire cohort, and multiplied by 1.96. Disease characteristics and comorbidity status were retrieved from the electronic medical patient files by two in‐ dependent researchers (see Appendix S1). Severity (mild to moderate or severe) of AD was measured by proxy of therapy based on the cri‐ teria as described by Wollenberg et al5 (Appendix S1). Associations between NMF status and the clinical parameters were tested by uni‐ variate and multivariate logistic regression models with adjustment for age and gender. Sixty‐seven out of 207 (32.4%) patients had decreased NMF. Figure 1 shows the distribution of disease severity in relation to the groups normal NMF and decreased NMF. Patients with decreased NMF had increased risk of severe AD, OR 2.12 (95% CI 1.02‐4.43), sensitization for food allergens, OR 2.27 (95%CI 1.21‐4.23), sensiti‐ zation for inhalation allergens, OR 2.22 (95%CI 1.13‐4.34), and food allergies, OR 2.79 (95% CI 1.33‐5.86; Table 1 and Table S1). Having decreased NMF did not show an association with early‐onset AD, allergic rhinitis, BHR, asthma and combined asthma, and/or BHR. In this retrospective study, we examined the associations between NMF values and the clinical parameters of the atopic syndrome. NMF