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BackgroundBRAF V600 mutant circulating cell-free tumor DNA (BRAF V600mut ctDNA) could serve as a specific biomarker in patients with BRAF V600 mutant melanoma. We analyzed the value of BRAF V600mut ctDNA from plasma as a monitoring tool for advanced melanoma patients treated with BRAF/MEK inhibitors.MethodsAllele-specific quantitative PCR analysis for BRAF V600 E/E2/D/K/R/M mutations was performed on DNA extracted from plasma of patients with known BRAF V600 mutant melanoma who were treated with dabrafenib and trametinib.Results245 plasma samples from 36 patients were analyzed. In 16 patients the first plasma sample was obtained before the first dosing of dabrafenib/trametinib. At baseline, BRAF V600mut ctDNA was detected in 75 % of patients (n = 12/16). BRAF V600mut ctDNA decreased rapidly upon initiation of targeted therapy (p < 0.001) and became undetectable in 60 % of patients (n = 7/12) after 6 weeks of treatment. During treatment, disease progression (PD) was diagnosed in 27 of 36 patients. An increase of the BRAF V600mut ctDNA copy number and fraction, identified PD with a sensitivity of 70 % (n = 19/27) and a specificity of 100 %. An increase in the BRAF V600mut ctDNA fraction was detected prior to clinical PD in 44 % of cases (n = 12/27) and simultaneously with PD in 26 % of patients (n = 7/27).ConclusionsQuantitative analysis of BRAF V600mut ctDNA in plasma has unique features as a monitoring tool during treatment with BRAF/MEK inhibitors. Its potential as an early predictor of acquired resistance deserves further evaluation.

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In melanoma, both the induction of immunosuppression by tumor cells and the inflammatory antitumor response can induce an upregulation of counter-regulatory mechanisms such as indoleamine 2,3-dioxygenase (IDO), programmed death-ligand 1 (PD-L1) and CTLA-4 C regulatory T-cells (Tregs) in the tumor microenvironment. Even though these immunosuppressive mediators are targets for immunotherapy, research investigating their expression in the peripheral blood is lacking. We therefore, performed flow cytometry on PBMCs of stage I-IV melanoma patients. IDO expression was detected in plasmacytoid dendritic cells (pDC) and monocytic myeloid-derived suppressor cells (mMDSC), and increased in advanced disease stage (p D 0.027). Tryptophan breakdown confirmed the functional activity of IDO and was linked with increased PD-L1C cytotoxic T-cells (p D 0.009), relative lymphopenia (p D 0.036), and a higher mDC/pDC ratio (p D 0.002). High levels of circulating PD-L1C cytotoxic T-cells were associated with increased CTLA-4 expression by Tregs (p D 0.005) and MDSC levels (p D 0.033). This illustrates that counter-regulatory immune mechanisms in melanoma should be considered as one interrelated signaling network. Moreover, both increased PD-L1C T-cells and CTLA-4 expression in Tregs conferred a negative prognosis, indicating their in vivo relevance. Remarkably, circulating CTLA-4, IDO, and pDC levels were altered according to prior invasion of the sentinel lymph node and IDO expression in the sentinel was associated with more IDOC PBMCs. We conclude that the expression of IDO, PD-L1, and CTLA-4 in the peripheral blood of melanoma patients is strongly interconnected, associated with advanced disease and negative outcome, independent of disease stage. Combination treatments targeting several of these markers are therefore likely to exert a synergistic response.

Endothelial IDO expression is highly consistent in primary, sentinel and metastatic tissues of patients with melanoma, indicating that immune suppression in melanoma is determined very early in the disease course. This supports that IDO expression in melanoma is a marker of antitumour immune response with an independent prognostic value.

The new assessment method for KP, taking into account both history and clinical examination, seems to be a useful and valuable tool for assessing KP in daily practice. Our results support the hypothesis that KP may function as a clinical parameter to assess and predict the clinical profile and course of vitiligo.

MicroRNAs (miRNAs) are post-transcriptional modulators of gene expression which play important roles in tumorigenesis and cancer metastasis. Since they are often highly deregulated in various types of cancer, miRNAs may be effective treatment targets. miRNA profiling studies of melanoma have led to the identification of several tumor suppressor miRNAs. One of these include miR-145, although functional data proving its specific function are limited. Therefore, in this study, we examined the expression levels of miR-145 in three melanoma cell lines (BLM, FM3P and WM793). Additional gain-of-function experiments revealed that miR-145 exerts an anti-proliferative effect in the primary, non-invasive melanoma cell line, WM793, whereas cell migration and the invasive potential of metastatic melanoma cells was suppressed following transfection with miR-145 mimics. In order to investigate the mechanisms by which miR-145 exerts its invasion suppressor function, we examined the expression level of target genes [fascin homolog 1 (FSCN1), myosin‑Va (MYO5A and SOX9] and that of an indirect target (RAB27A) following the overexpression of miR-145. The results showed that SOX9, MYO5A and RAB27A were not involved in the biological effects caused by miR-145 mimics. Surprisingly, we discovered that miR-145 in melanoma, in contrast to many other tumor types, does not necessarily act via the target, FSCN1, since the downregulation of FSCN1 did not inhibit cell proliferation or migration but, on the contrary, increased cell invasion in two out of the three melanoma cell lines examined. Our in vitro data is in accordance with previously reported in vivo data describing the low expression of FSCN1 in malignant melanomas when compared to dysplastic nevi, suggesting that the expression of FSCN1 decreases as the formation and progression stage of melanoma advances. In conclusion, our data provide evidence that miR-145 is an invasion suppressor in metastatic melanoma cells. Despite the fact that it remains unclear which genes or pathways are regulated by miR-145 in melanoma, miR-145 may serve as a useful therapeutic agent in melanoma when re-expressed in situ.

IMPORTANCE Skin cancer is the most frequent cancer type. It remains unknown if and how screening programs can be organized in a cost-effective manner. OBJECTIVE To compare the 2 screening strategies of systematic total-body examination (TBE) and lesion-directed screening (LDS), with a focus on the participation rate, detection rate, anxiety, and cost. DESIGN, SETTING, AND PARTICIPANTS Population-based cross-sectional screenings by a team of 6 dermatologists were organized in 2 sociodemographically similar regions. The TBE was organized in a community of 9325 inhabitants 18 years and older (Wichelen, East Flanders, Belgium) during a 5-day screening (March 14-18, 2014). The LDS was organized in a sociodemographically comparable community (Nevele, East Flanders, Belgium) of 9484 adult inhabitants during a 4-day screening (April 22 and 25-27, 2014). The first population received a personal invitation for a standard TBE. In the second population, individuals were invited for an LDS if they had a lesion meeting 1 or more of the following criteria: ABCD rule (A, asymmetry; B, borders; C, colors; and D, differential structures), ugly duckling sign, new lesion lasting longer than 4 weeks, or red nonhealing lesions. MAIN OUTCOMES AND MEASURES In total, 1982 individuals were screened, and 47 skin cancers (2.4%) were histologically confirmed, including 9 melanomas (0.5%), 37 basal cell carcinomas (1.9%), and 1 squamous cell carcinoma or Bowen disease (0.1%). RESULTS The positive predictive value for all suspicious lesions was 56.6% (47 of 83). The participation rate was 17.9% (1668 of 9325) in the TBE group vs 3.3% (314 of 9484) in the LDS group (P < .01). The skin cancer detection rate per 100 participants did not differ significantly between the 2 groups, with rates of 2.3% (39 of 1668) in the TBE group vs 3.2% (8 of 248) in the LDS group (P = .40). The operational effectiveness per 100 invitees was 0.4% (39 of 9325) in the TBE group vs 0.1% (8 of 9484) in the LDS group (P < .01). In addition, LDS was 5.6 times less time consuming than TBE. Participants in the LDS group had significantly higher baseline anxiety levels compared with participants in the TBE group (3.7 vs 3.3 points on a visual analog scale, P < .01). In screenees without a suspicious lesion, anxiety levels significantly dropped after screening. CONCLUSIONS AND RELEVANCE Total-body examination yielded a higher absolute number of skin cancers. Lesion-directed screening had a similar detection rate of 3.2% (8 of 248) but was 5.6 times less time consuming. When performed by dermatologists, LDS is an acceptable alternative screening method in health care systems with limited budgets or long waiting lists.