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

Abstract Background In head and neck cancers, the number of intra-tumoral lymphocytes associates with improved survival. The impact of the exact cellular composition and localization of these lymphocytes, however, is less well studied. In the current study, we assessed the prognostic values of density, localization and cellular networks of defined lymphocyte populations in early-stage oral tongue cancer. Methods Patient with T1-T2, primary oral tongue squamous cell carcinoma and treated with surgical resections and without any peri-operative (chemo) radiotherapy were included in a discovery cohort (n = 47). Multiplexed in-situ immunofluorescent staining was performed using FFPE sections for CD4, CD8, CD20, pan-cytokeratin and cellular nuclei (DAPI); and spatial distributions of 3 lymphocyte populations were assessed in the tumour and stromal compartments, both at the invasive margin (IM) and the center of tumours (CT). Using algorithm-based pathology and nearest neighbor analysis (NNA), we have computed cellular densities and networks for lymphocytes and related these immune parameters to overall survival (OS). Findings were validated using another cohort of patients with identical clinical characteristics (n = 91). Results In our discovery cohort, we observed a high stromal density of CD20-positive B cells at IM but not CT, which correlated with OS (p = 0.005, HR 0.225). NNA demonstrated that survival benefit particularly related to the number of CD20 cells in the vicinity of CD4 cells and the frequency of B cells touching each other. The prognostic value of B cell-rich areas was validated in a second cohort, but only for those patients with low stromal densities of CD4 T cells (in accordance to discovery cohort, p = 0.007, HR = 0.275). Conclusion Our study highlights the prognosis of B cell-rich areas in early-stage oral-tongue cancer patients, particularly in the context of low intra-tumoral CD4 T cell densities. Legal entity responsible for the study The authors. Funding Erasmus MC, Rotterdam, the Netherlands and HRH Princess Chulabhorn College of Medical Science, Bangkok, Thailand. Disclosure All authors have declared no conflicts of interest.

The sentinel lymph node (SLN) biopsy is a highly accurate staging procedure and the most important prognostic factor in melanoma patients. The European Organisation for Research and Treatment of Cancer (EORTC) Melanoma Group aimed to design an updated evolved SLN protocol for the histopathological workup and reporting. We herein recommend extending the distance between steps according to the short axis dimension of the lymph node and optimise both conventional sectioning and staining procedures including immunohistochemistry. We also provide guidance on the description of the spatial localisation of melanoma deposits in a SLN. The histopathological features to be reported include the following: presence or absence of the metastasis, the intranodal location of the metastasis (subcapsular, parenchymal, combined, extensive confluent and extensive multifocal), the number of the metastatic deposits (1, 2-5, 6-10, 11-20 and >20), the maximum dimension of the largest metastasis (indicating its site) and the presence of extracapsular extension and of naevus cells. This updated EORTC protocol is expected to clarify and simplify the existing procedures, ensuring a reasonable workload for the laboratory and for the pathologists resulting in cost saving with no loss, and possible increase, in accuracy.

Lentigo maligna (LM) is a melanoma in situ and the incidence is still rising in The Netherlands. LM is mostly located in the face, therefore radical surgical removal, which is the first choice of treatment, can be challenging in this delicate anatomical region. Staged excision is considered a useful alternative. The initial diagnosis of clinically suspicious LM is usually based on just one or a few biopsies, which may lead to reclassification into lentigo maligna melanoma (LMM) based on histological evaluation of the excision specimen. This article is protected by copyright. All rights reserved.

With great interest we have read the letter by De Giorgi et al. in which they express their disagreement with the conclusions of our study on the potential value of a Raman spectroscopy device in the clinical diagnosis of cutaneous melanoma. In our paper, we tested the diagnostic accuracy of Raman spectroscopy, expressed as sensitivity, specificity and number needed to treat, in a set of pigmented skin lesions that were deemed suspicious for melanoma by dermatologists. Whereas current clinical diagnosis and dermoscopy rely on recognition of morphological characteristics, Raman spectroscopy provides information about the molecular composition of pigmented skin lesions. Our results indicate that Raman spectroscopy constitutes a valuable diagnostic tool: all melanomas that were analyzed tested positive with Raman spectroscopy (sensitivity 100%), and the estimated number needed to treat was 2.7 (ratio between the number of lesions tested positive by Raman spectroscopy and the total confirmed melanoma). De Georgi et al. disagree with our conclusions which state that the diagnostic model based on Raman spectroscopy has enabled greater sensitivity and specificity in melanoma diagnosis, detecting all thin melanomas and reducing the number of unnecessary excisions by more than two-fold compared with the current clinical practice. They object to the fact that pigmented skin lesions were enrolled in the study after a dermatologist performed a clinical assessment and had excised lesions that were clinically suspicious for melanoma, and state that this does not reflect clinical practice. They furthermore state that such “lesion pre-selection frequently includes many melanomas that are easy to diagnose, and which often have an exceedingly high frequency of malignancies within the lesions examined, thus creating an “artificial” diagnostic setting compared to real practice”. De Giorgi et al. have misinterpreted the objective of our study and our data set. Our results are based on the use of Raman spectroscopy as an add-on to diagnose dermatologist-selected lesions. We do not want to by-pass the dermatologist. The selection of suspicious lesions by a dermatologist is part of the intended clinical practice. This must not be confused with a bias in the case series used. The sample set mostly consisted of difficult to classify lesions, including melanoma in situ and dysplastic nevi, deemed suspicious for melanoma based on visual inspection by dermatologists specialized in pigmented skin lesions. This selection of lesions was in line with the objective of our study; namely to investigate the diagnostic use of Raman spectroscopy as an adjunct technique to distinguish between melanoma and unnecessary diagnostic excisions. This is fully in line with the main conclusion drawn from the results regarding the diagnostic accuracy in an independent validation set, and the possible reduction of the number of unnecessary diagnostic excisions if the Raman instrument were used as an add-on to classify lesions considered suspicious by dermatologists. De Giorgi et al. also criticize the fact that amelanotic lesions were excluded from analysis. All lesions that were excised by the dermatologist for suspicion for melanoma were subjected to Raman spectroscopy. This included unpigmented lesions suspicious for amelanotic melanoma. After histopathological evaluation, the lesions diagnosed as non-melanocytic were excluded from analysis (basal cell carcinoma, seborrheic wart, lichenoid keratosis, dermatofibroma, haemangioma, scar), because the aim of the study at this stage was to distinguish between melanoma and non-melanoma melanocytic lesions. De Giorgi et al. furthermore state that only a melanoma left unexcised represents a clinically relevant false negative diagnosis and that in their experience this does not frequently occur, and likely limited to subjects harboring a clinically “featureless” tumor. We can only refer to the literature. The accuracy and reproducibility of melanocytic skin lesion diagnosis is poor, in particular among general practitioners, as has been demonstrated in several studies. More evidence-based studies are required to provide data about the role of Raman spectroscopy to improve clinical diagnosis of melanoma in different medical settings, including screening of inconspicuous melanocytic skin lesions. With this study we provide evidence that accurate diagnostic results can be obtained by Raman spectroscopy on pigmented skin lesions selected by dermatologists as suspicious for melanoma. We believe that these results represent an important step towards accurate clinical diagnosis of melanoma.

Abstract Background/purpose: To determine the efficacy and toxicity profile of a stereotactic body radiotherapy (SBRT) boost as a first line treatment in patients with oropharyngeal squamous cell carcinoma (OPSCC). Materials and methods: We performed a retrospective cohort study in 195 consecutive OPSCC patients with T1-small T3 disease, treated at Erasmus MC between 2009 and 2016 with a SBRT (3 × 5.5 Gy) boost after 46 Gy IMRT. Primary endpoints were disease-specific survival (DSS) and Grade ≥3 toxicity (Common Terminology Criteria). The Kaplan-Meier method and Cox regression model were applied to determine rates and risk factors. Results: The median follow-up was 4.3 years. Treatment compliance was high (100%). Rates of 5-year DSS and late grade ≥3 toxicity were 85% and 28%, respectively. Five-year overall survival was 67%. The most frequently observed toxicities were mucosal ulceration or soft tissue necrosis (n = 30, 5 year 18%), dysphagia or weight loss (n = 18, 5 year 12%) and osteoradionecrosis (n = 11, 5 year 9%). Current smoker status (hazard ratio [HR] = 2.9, p = .001) and Charlson Comorbidity Index ≥2 (HR = 1.9, p = .03) were was associated with increased toxicity risk. Tooth extraction prior to RT was associated with increased osteoradionecrosis risk (HR = 6.4, p = .006). Conclusion: We reported on outcomes in the largest patient series to date treated with a hypofractionated boost for OPSCC. Efficacy was good with survival rates comparable to conventionally fractionated (chemo)radiotherapy. Grade ≥3 toxicity profiles showed high rates of soft tissue necrosis and osteoradionecrosis. Strategies to mitigate severe toxicity risks are under investigation to improve the tolerability of the SBRT boost.

Specimen‐driven intraoperative assessment of the resection margins provides immediate feedback if an additional excision is needed. However, relocation of an inadequate margin in the wound bed has shown to be difficult. The objective of this study is to assess a reliable method for accurate relocation of inadequate tumor resection margins in the wound bed after intraoperative assessment of the specimen.