Cellular proliferation is correlated with the progression of melanoma. Accordingly, the proliferation index of H&E-stained thin melanomas was recently included in the staging system of the American Joint Committee on Cancer. Yet, the immunohistochemical markers of proliferation phosphohistone H3 and Ki67 may improve such indices. To accurately quantify these markers, they should be combined with a melanocytic marker, for example, MART1 in an immunohistochemical double stain; also enabling automated quantification by image analysis. The aim of the study was to compare the prognostic impact of phosphohistone H3/MART1, Ki67/MART1, and H&E stains in primary cutaneous melanoma, and to determine the difference between indices established in hot spots and the global tumor areas. The study included 153 consecutive stage I/II melanomapatients. The follow-up time was 8-14 years for event-free melanoma. Recurrent disease occurred in 43 patients; 37 died of melanoma. Both events occurred in only three thin melanomas. Their paraffin-embedded tissue was stained for phosphohistone H3/MART1, Ki67/MART1, and with H&E. And proliferation indices were established in 1-mm 2 hot spots and in the global tumor areas. In multivariate Cox analyses, only hot spot indices of phosphohistone H3/MART1 and Ki67/MART1 were independent prognostic markers. Phosphohistone H3/MART1 tended to be better than Ki67/MART1 with adjusted hazard ratios of 3.66 (95% CI, 1.40-9.55; P ¼ 0.008) for progression-free survival and 3.42 (95% CI, 1.29-9.04; P ¼ 0.013) for melanoma-specific death. In all stains, prognostic performance was substantially improved by using hot spots instead of the global tumor areas. In conclusion, phosphohistone H3/MART1 and Ki67/MART1 were superior to H&E stains, and hot spots superior to the global tumor areas. Given the potential for automated analysis, these double stains seem to be robust alternatives to conventional mitotic detection by H&E in stage I/II melanomas in general. This was particularly true for thick melanomas whereas no specific analyses for thin melanomas only could be performed.

Patients diagnosed with BRAF V600E mutated cutaneous melanoma show response to treatment with the BRAF inhibitor Vemurafenib. Different methods for BRAF mutation detection exist; however, only the Cobas 4800 BRAF V600 Mutation Test has been approved by the US Food and Drug Administration for patient selection. The results from this test depend on the percentage of tumor cells in the samples, which clinically may be estimated with substantial variation. We have evaluated five different methods: the Cobas test, Sanger sequencing, pyrosequencing, TaqMan-based allele-specific PCR, and Competitive Amplification of Differentially Melting Amplicons (CADMA), for detection of BRAF c.1799T>A (V600E) mutations in 28 formalin-fixed paraffin-embedded (FFPE) cutaneous melanoma samples. We show that the frequency of the BRAF V600E mutation is influenced by the analytical sensitivity of the applied method. However, a 100% consensus was observed among all five methods when the tumor tissue fraction was more than 10% of all tissue or more than 50% of cell-dense tissue. When using Sanger sequencing, pyrosequencing, or the Cobas test, it may be advisable to perform macrodissection before mutation testing if the tumor cell fraction is low. CADMA and TaqMan may not require macrodissections for a reliable test. Therefore, the use of more sensitive methods may have a future in testing for BRAF mutations in clinical settings.

Cadherin switch and reduced/absent PTEN expression are associated in melanoma, and both factors may play important roles in the progression of melanoma.

The receptor tyrosine kinase KIT and its ligand, stem cell factor (SCF), are essential for the proliferation and survival of normal melanocytes. In melanomas arising on mucosal, acral, and chronically sun-damaged skin, activating KIT mutations have been identified as oncogenic drivers and potent therapeutic targets. Through an initial whole-genome screen for aberrant promoter methylation in melanoma, we identified the KIT promoter as a target for hypermethylation in 43/110 melanoma cell lines, and in 3/12 primary and 11/29 metastatic cutaneous melanomas. Methylation density at the KIT promoter correlated inversely with promoter activity in vitro and in vivo, and the expression of KIT was restored after treatment with the demethylating agent 5-aza-2'-deoxycytidine. Hypermethylation of KIT showed no direct or inverse correlations with well-documented melanoma drivers. Growth of melanoma cells in the presence of SCF led to reduced KIT expression and increased methylation density at the KIT promoter, suggesting that SCF may exert a selection pressure for the loss of KIT. The frequent loss of KIT in cutaneous melanoma by promoter hypermethylation suggests that distinct KIT signaling pathways have opposing roles in the pathogenesis of melanoma subtypes.

Distinction between benign and malignant melanocytic lesions may be difficult by today's methods, even for highly skilled dermatopathologists, emphasizing the need for improved diagnostic tools. We have studied the discriminative abilities of immunohistochemical (IHC) double stains using the IHC markers Ki67 combined with MART1, and HMB45 combined with MITF. Paraffin-embedded tissue sections from 50 melanomas and 78 benign nevi were stained using a simple simultaneous IHC double staining technique. Both simple semiquantitative estimates of the immunopositivity in the deepest third of the lesions and full-scale quantitative measurements of the Ki67 and HMB45 indices were performed, and scores for melanomas and nevi were compared. The differences between melanomas and nevi were significant (P < 0.0001) using either analysis or stain. The misclassification rates for melanomas and nevi were generally lower for Ki67/MART1 stains than for HMB45/MITF stains. In the simple semiquantitative Ki67/MART1 analysis, the misclassification rates were 6% (2%-17%) for melanomas and 12% (6%-21%) for nevi. In full-scale quantitative analysis the corresponding rates were 4% (1%-14%) and 8% (4%-16%), and by combining Ki67 and HMB45 indices, the misclassification rates were 0% (0%-7%) for melanomas and 13% (7%-22%) for nevi. We conclude that both semiscale and fullscale quantitative analyses of Ki67/MART1 stains are valuable diagnostic tools to distinguish melanomas and nevi with a large degree of certainty. The HMB45/MITF stains may serve as adjuncts to predict malignancy and the diagnostic potential of combining the HMB45 and Ki67 indices are promising. The IHC double stains may potentially reduce misinterpretations of melanomas in histopathology.