Melanoma is a skin cancer which treatment requires early diagnosis and large surgical removal. The histopathological diagnosis of a melanocytic tumour is sometimes difficult between a benign nevus and a malignant melanoma. We built an immunomarker-based score to differentiate nevi from melanomas. Two independent sets of 308 (first set) and 62 (validation set) formalin-fixed and paraffin embedded tumour samples were studied using p16-Ki-67 and HMB45-MelanA dual-staining immunohistochemistry. In the first set of tumours, high Ki-67 index, low to null p16 immunohistochemistry and absence of HMB45 immunohistochemistry gradient were more frequent in melanomas (156 primary tumours and 78 metastases) than in nevi (74 tumours). Nevertheless, none of these single parameters was able to differentiate all primary melanomas from all nevi. We built a scoring system based on the addition of semi-quantitative scorings of Ki-67 (0: 20 %) and p16 (0:>50 % stained cells; 1:11–50 %; 2:1–10 %; 3:0 %) and HMB45 staining (0: gradient present; 1: doubtful/inconclusive gradient; 2: gradient absent). A p16-Ki-67-HMB45 total score from 0 to 9 permitted to classify nevi (score <4) and primary melanomas (score ≥4) with a sensitivity of 97.4 % and a specificity of 97.3 % in the first set of tumours. Sensibility and specificity of 100 % were obtained in a second set (validation set) of 62 tumours (46 melanomas and 16 nevi). The total scoring also allowed analyzing 11 difficult or initially misdiagnosed tumours in our files. We propose a valuable triple p16-Ki-67-HMB45 immunohistochemistry scoring system to help pathologists in the differential diagnosis of melanomas and nevi.

The determination of NRAS and BRAF mutation status is a major requirement in the treatment of patients with metastatic melanoma. Mutation specific antibodies against NRASQ61R and BRAFV600E proteins could offer additional data on tumor heterogeneity. The specificity and sensitivity of NRASQ61R immunohistochemistry have recently been reported excellent. We aimed to determine the utility of immunohistochemistry using SP174 anti-NRASQ61R and VE1 anti-BRAFV600E antibodies in the theranostic mutation screening of melanomas. 142 formalin-fixed paraffin-embedded melanoma samples from 79 patients were analyzed using pyrosequencing and immunohistochemistry. 23 and 26 patients were concluded to have a NRAS-mutated or a BRAF-mutated melanoma respectively. The 23 NRAS Q61R and 23 BRAF V600E-mutant samples with pyrosequencing were all positive in immunohistochemistry with SP174 antibody and VE1 antibody respectively, without any false negative. Proportions and intensities of staining were varied. Other NRAS Q61L, NRAS Q61K, BRAF V600K and BRAF V600R mutants were negative in immunohistochemistry. 6 single cases were immunostained but identified as wild-type using pyrosequencing (1 with SP174 and 5 with VE1). 4/38 patients with multiple samples presented molecular discordant data. Technical limitations are discussed to explain those discrepancies. Anyway we could not rule out real tumor heterogeneity. In our study, we showed that combining immunohistochemistry analysis targeting NRASQ61R and BRAFV600E proteins with molecular analysis was a reliable theranostic tool to face challenging samples of melanoma.

The therapeutic management of patients with endoscopic resection of colorectal cancer invading the submucosa (i.e. pT1 CRC) depends on the balance between the risk of cancer relapse and the risk of surgery-related morbidity and mortality. The aim of our study was to report on the histopathological risk factors predicting lymph node metastases and recurrences in an exhaustive case series comprising every pT1 CRC (of adenocarcinoma subtype only) diagnosed in Finistère (France) during 5-years. For 312 patients with at least 46 months follow-up included in the digestive cancers registry database, histopathological factors required for risk stratification in pT1 CRC were reviewed. Patients were treated by endoscopic resection only (51 cases), surgery only (138 cases), endoscopic resection followed by surgery (102 cases) or transanal resection (21 cases). Lymph node metastases were diagnosed in 19 patients whereas 15 patients had an extra-nodal recurrence (7 local recurrences only, 4 distant metastases only and 4 combining local and distant recurrences). Four patients with distant metastases died of their cancer. Poor tumor differentiation, vascular invasion and high grade tumor budding on HES slides were notably identified as strong risk-factors of lymph node metastases but the prediction of extra-nodal recurrences (local, distant and sometimes fatal) was less obvious, albeit it was more frequent in patients treated by transanal resection than with other treatment strategies. Beyond good performances in predicting lymph node metastases and guiding therapeutic decision in patients with pT1 CRC, our study points that extra-nodal recurrence of cancer is more difficult to predict and requires further investigations.

Evaluating lymphocytic infiltration of minor salivary gland biopsy in primary Sjögren’s syndrome is challenging. We developed and evaluated a digital method for quantifying B and T lymphocytes in whole minor salivary gland biopsy slides. Minor salivary gland biopsies were immunostained with anti-CD20/anti-CD3 antibodies using red/brown chromogens. Slides were digitised and spliced into mosaics of smaller JPEG format images in which red and brown pixels were counted. ImageJ Cell counter was used for validation. Agreement between the digital and manual methods was evaluated using Bland-Altman plots and the interclass correlation coefficient. External validation relied on the Chisholm-Mason, Tarpley, and focus-score methods. Of 62 minor salivary gland biopsy slides, 61.3 % had a Chisholm-Mason grade ≥ III or a focus score ≥1. The number of pixels correlated well with manual cell counts (r = 0.95 for red pixels vs. B cell count and r = 0.91 for brown pixels vs. T cell count). Interclass correlation coefficients between digital and manual counts were excellent (0.92 for B/T cells). B-cell proportion showed a significant positive correlation with the focus score (Spearman’s coefficient 0.463, p < 0.0001). Median B-cell proportion was lower in minor salivary gland biopsies with Chisholm grades I–II (2.5 % (0.2–13.9)) than III–IV (30.0 % (15.5–45.2)) and increased with Tarpley’s class (1, 2.2 % (0.2–6.6); 2, 27.2 % (13.0–38.9); and 3–4, 48.5 % (29.4–56.4); p < 0.001 for all comparisons). Minor salivary gland biopsy B-cell proportion was also significantly correlated with several markers of clinical and biological activity of the disease, especially with markers of systemic B-cell hyperactivation. The digital procedure proved accurate compared to the reference standard, producing reliable results for whole tissue sections. ClinicalTrials.gov [ NCT00740948 ]. Registered 22 August 2008.