BACKGROUND Sentinel lymph node (SLN) biopsy remains crucial for melanoma staging. The European Organisation for Research and Treatment of Cancer Melanoma Group recommends performing immunohistochemical stainings for reproducible identification of melanoma metastases. S100 protein (pS100) is a commonly used melanocytic antigen because of its high sensitivity in spite of relatively low specificity. SRY-related HMG-box 10 protein (SOX10) is a transcription factor characterising neural crest-derived cells. It is uniformly expressed mostly in the nuclei of melanocytes, neural, and myoepithelial cells. Pathologists sometimes prefer SOX10 as a melanoma marker, but it has not yet been investigated on a large-scale to confirm that it is reliable and recommendable for routine SLN evaluation. METHODS Four hundred one treatment-naïve lymph node (LN) metastatic melanomas were included in high-density tissue microarrays and were assessed for the presence of SOX10 and pS100 by immunohistochemistry. The slides were digitalised, shared and evaluated by a panel of experienced melanoma pathologists. RESULTS The vast majority of melanomas were double-positive for pS100 and SOX10 (93.2%); a small percentage of the cases (3.9%) were double-negative melanomas. Discordance between the two markers was observed: 1.9% pS100(-)/SOX10(+) and 0.75% pS100(+)/SOX10(-). SOX10 was not expressed by immune cell types in the LN, resulting in a less controversial interpretation of the staining. CONCLUSIONS SOX10 is as equally specific as pS100 for the detection of melanoma metastases in LNs. The interpretation of SOX10 staining is highly reproducible among different centres and different pathologists because of the absence of staining of immune cells.

OBJECTIVES Depth of invasion (DOI) is the most important predictor for lymph node metastasis (LNM) in early stage (T1-T2) oral cancer. The aim of this study is to validate the cut-off value of 4 mm on which the decision to perform an Elective Neck Dissection (END) is made. MATERIALS AND METHODS We performed a retrospective study in patients with pathologically proven early stage oral cavity squamous cell carcinoma (OCSCC) without clinical or radiological signs of LNM, who were treated between 2013 and 2018. An END was performed when DOI was ≥ 4 mm and a watchful waiting protocol was applied in patients with DOI < 4 mm. RESULTS Three hundred patients were included. END was performed in 77% of patients with DOI ≥ 4 mm, of which 36% had occult LNM (pN+). Patients in the watchful waiting group (48%) developed a regional recurrence in 5.2% for DOI < 4 mm and 24.1% for DOI ≥ 4 mm. For DOI ≥ 4 mm, regional recurrence free survival was higher for patients who were treated with END compared to watchful waiting (p = 0.002). A Receiver-Operator-Curve -analysis showed that a DOI cut-off value of 4.0 mm was the optimal threshold for the prediction of occult LNM (95.1% sensitivity, 52.9% specificity). CONCLUSION A DOI of ≥ 4 mm is an accurate cut-off value for performing an END in early stage OCSCC. END results in higher survival rates and lower regional recurrence rates in patients with DOI ≥ 4 mm.

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.

Using tissue microarrays it was shown that membranous C-terminal MET immunoreactivity and ectodomain (ECD) shedding are associated with poor prognosis in oral cancer. Seen the potential diagnostic value, extrapolation of these results to whole tissue sections was investigated. Since MET orchestrates epithelial to mesenchymal transition (EMT), results were benchmarked to loss of E-cadherin; a readout for EMT known to be associated with poor prognosis. C-terminal MET, N-terminal MET, and E-cadherin immunoreactivities were examined on formalin-fixed paraffin-embedded parallel sections of 203 oral cancers using antibody clones D1C2, A2H2-3 and NCH-38. Inter- and intra-antibody relations were examined using a novel scoring system, nonparametric distribution and median tests. Survival analyses were used to examine the prognostic value of the observed immunoreactivities. Assessment of the three clones revealed MET protein status (no, decoy, transmembranous C-terminal positive), ECD shedding and EMT. For C-terminal MET positive cancers, D1C2 immunoreactivity is independently associated with poor overall survival (OS; HR=2.40; 95% CI, 1.25 to 4.61 and P=0.008) and disease free survival (DFS; HR=1.83; 95% CI 1.07 - 3.14; P=0.027). For both survival measures, this is also the case for ECD shedding (43.4% with HR=2.30; 95% CI, 1.38 to 3.83 and P=0.001 versus HR=1.87; 95% CI 1.19 - 2.92; P=0.006) and loss of E-cadherin (55.3% with HR=2.21; 95% CI, 1.30 to 3.77 and P=0.004 versus HR=1.90; 95% CI 1.20 - 3.01; P=0.007). The developed scoring system accounts for MET protein status, ECD shedding, EMT, and is prognostically informative. These findings may contribute to development of companion diagnostics for MET-based targeted therapy.

The precursor lesion of vulvar squamous cell carcinoma (VSCC), namely vulvar intraepithelial neoplasia (VIN), is classified as: human papillomavirus (HPV)-related high grade squamous intraepithelial lesion (HSIL), and HPV-independent differentiated VIN (dVIN). Traditionally, histology and immunohistochemistry (IHC) have been the basis of diagnosis and classification of VIN. HSIL shows conspicuous histological atypia, and positivity on p16-IHC, whereas dVIN shows less obvious histological atypia, and overexpression or null-pattern on p53-IHC. For both types of VIN, other diagnostic immunohistochemical markers have also been evaluated. Molecular characterization of VIN has been attempted in few recent studies, and novel genotypic subtypes of HPV-independent VSCC and VIN have been identified. This systematic review appraises the VSCC precursors identified so far, focusing on histology and biomarkers (immunohistochemical and molecular). To gain further insights into the carcinogenesis and to identify additional potential biomarkers, gene expression omnibus (GEO) datasets on VSCC were analyzed; the results are presented.

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.