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.

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.

Clinical diagnosis of early melanoma (Breslow thickness less than 0.8 mm) is crucial to disease-free survival. However, it is subjective and can be exceedingly difficult, leading to missed melanomas, or unnecessary excision of benign pigmented skin lesions. An objective technique is needed to improve the diagnosis of early melanoma. We have developed a method to improve diagnosis of (thin) melanoma, based on Raman spectroscopy. In an ex vivo study in a tertiary referral (pigmented lesions) centre, high-wavenumber Raman spectra were collected from 174 freshly excised melanocytic lesions suspicious for melanoma. Measurements were performed on multiple locations within the lesions. A diagnostic model was developed and validated on an independent data set of 96 lesions. Approximately 60% of the melanomas included in this study were melanomas in situ. The invasive melanomas had an average Breslow thickness of 0.89 mm. The diagnostic model correctly classified all melanomas (including in situ) with a specificity of 43.8%, and showed a potential improvement of the number needed to treat from 6.0 to 2.7, at a sensitivity of 100%. This work signifies an important step towards accurate and objective clinical diagnosis of melanoma and in particular melanoma with Breslow thickness <0.8 mm.

Differentiated vulvar intraepithelial neoplasia (dVIN) is the precursor lesion of HPV-negative vulvar squamous cell carcinoma (VSCC). The histopathological diagnosis of dVIN can be challenging, as it often resembles vulvar non-neoplastic epithelial disorders (NNED), especially lichen sclerosus (LS). We aimed to establish the most specific and reproducible histological features of dVIN and assessed cytokeratin 13 (CK13) and cytokeratin 17 (CK17) immunohistochemistry as a diagnostic aid. Consecutive cases of dVIN (n = 180) and LS (n = 105) from the period 2010 to 2013 were reviewed using a checklist of histological features. Each feature was recorded as ‘present’ or ‘absent’ and statistical comparison (dVIN vs LS) was made. Interobserver agreement between two pairs of pathologists was assessed for a subset of cases of dVIN (n = 31) and LS and other NNED (n = 23). Immunohistochemistry with CK13, CK17, MIB1 and p53 was performed on dVIN, LS, and other NNED cases. Macronucleoli, features of disturbed maturation and angulated nuclei were significantly more common in dVIN than LS (p < 0.001). We found ‘substantial agreement’ for the diagnosis of dVIN (κ = 0.71). Macronucleoli and deep keratinisation had the highest agreement. In dVIN, the mean percentage of cells staining with CK13 was 15 and with CK17, this was 74. For LS, the mean percentage of cells staining with CK13 was 31, and with CK17, this was 41. By plotting receiver operating characteristic curves (ROC), an area under the curve (AUC) of 0.52 was obtained for CK13, and an AUC of 0.87 was obtained for CK17. The most helpful histological features for diagnosing dVIN were macronucleoli, features of disturbed maturation, and angulated nuclei. Increased CK17 expression may have promise for supporting dVIN diagnosis.