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Differentiation between Sézary syndrome and erythrodermic inflammatory dermatoses can be challenging, and a number of studies have attempted to identify characteristic immunophenotypic changes and molecular biomarkers in Sézary cells that could be useful as additional diagnostic criteria. In this European multicenter study, the sensitivity and specificity of these immunophenotypic and recently proposed but unconfirmed molecular biomarkers in Sézary syndrome were investigated. Peripheral blood CD4(+) T cells from 59 patients with Sézary syndrome and 19 patients with erythrodermic inflammatory dermatoses were analyzed for cell surface proteins by flow cytometry and for copy number alterations and differential gene expression using custom-made quantitative PCR plates. Experiments were performed in duplicate in two independent centers using standard operating procedures with almost identical results. Sézary cells showed MYC gain (40%) and MNT loss (66%); up-regulation of DNM3 (75%), TWIST1 (69%), EPHA4 (66%), and PLS3 (66%); and down-regulation of STAT4 (91%). Loss of CD26 (≥80% CD4(+) T cells) and/or CD7 (≥40% CD4(+) T cells) and combination of altered expression of STAT4, TWIST1, and DNM3 or PLS3 could distinguish, respectively, 83% and 98% of patients with Sézary syndrome from patients with erythrodermic inflammatory dermatoses with 100% specificity. These additional diagnostic panels will be useful adjuncts in the differential diagnosis of Sézary syndrome versus erythrodermic inflammatory dermatoses., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Cutaneous T-cell lymphoma (CTCL) tumor cells lack the ability of activated T cells to undergo TCR/CD3-mediated activation-induced cell death (AICD). In this issue, the study reported by Wu et al. demonstrates that c-CBL (Casitas B-lineage Lymphoma proto-oncogene) is overexpressed in CTCL. When CTCL cells lose c-CBL, AICD is enhanced. Furthermore, combination therapy with methotrexate (a known demethylating agent for the CD95 gene) in combination with the loss of c-CBL increases CTCL cell death. Therefore, inhibition of c-CBL could represent a method of sensitizing lymphoma cells to enhance AICD. Armed with their novel data, the investigators envision combination therapies that target c-CBL to reactivate AICD in the malignant T cells whenever responsiveness to TCR/CD3 signaling is retained.

Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non-mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRAS(G13D) mutation; one Sézary syndrome and one CD30(+) CTCL harbored a NRAS(Q61K) amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRAS(Q61K) mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRAS(Q61K) mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors.

Cutaneous T-cell lymphoma (CTCL) has been suggested by in vitro experiments to represent a malignant CD4+ T-cell proliferation with a regulatory T-cell (Treg) phenotype (CD4+CD25+FOXP3+). We investigated percentages of FOXP3+ and CD25+ cells in the blood of 15 Sézary, 14 mycosis fungoides (MF), and 10 psoriasis (Pso) patients and 20 normal healthy donors (NHDs). We found similar numbers of FOXP3+ cells in MF (10.4% of blood CD4+ cells) and Pso (11.1%) patients and NHDs (9.8%). In 8 of 15 (53%) Sézary patients, significantly reduced percentages of FOXP3+ cells were seen in blood (2.9%) and skin (10.4%). Interestingly, 6 of 15 (40%) Sézary patients showed significantly increased percentages of FOXP3+ cells (39.7% (blood), 20.3% (skin)); however, these cells did not express CD25. In these latter patients, clone-specific TCR-Vbeta-chain antibodies were used to demonstrate that these FOXP3+CD25- cells were monoclonal CTCL tumor cells. FOXP3+CD25- CTCL tumor cells showed a highly demethylated status of the foxp3 gene locus similar to Treg cells, and they were functionally able to suppress IL-2 mRNA induction in TCR-stimulated conventional T cells. Thus, FOXP3+CD25- CTCL tumor cells with functional features of Treg cells define a subgroup of Sézary patients who might carry a different prognosis and might require differential treatment.

The aim of this study was to analyze the influence of cholinergic and anticholinergic drugs on epidermal physiology using organotypic cocultures (OTCs). Blocking of all acetylcholine receptors (AChRs) by combined treatment with mecamylamine and atropine or treatment with strychnine (blocking alpha9nAChR) for 7-14 days resulted in a complete inhibition of epidermal differentiation and proliferation. Blockage of nicotinic (n)AChR with mecamylamine led to a less pronounced delay in epidermal differentiation and proliferation than blockage of muscarinic (m)AChR with atropine, evidenced by reduced epithelial thickness and expression of terminal differentiation markers like cytokeratin 2e or filaggrin. In OTCs treated with atropine, mecamylamine, or strychnine, we could demonstrate intracellular lipid accumulation in the lower epidermal layers, indicating a severely disturbed epidermal barrier. In addition, we observed prominent acantholysis in the basal and lower suprabasal layers in mecamylamine-, atropine-, and strychnine-treated cultures, accompanied by a decreased expression of cell adhesion proteins. This globally reduced cell adhesion led to cell death via intrinsic activation of apoptosis. In contrast, stimulation of nAChR and mAChR with cholinergic drugs resulted in a significantly thickened epithelium, accompanied by an improved epithelial maturation. In summary, we show that epidermal AChR are crucially involved in the regulation of epidermal homeostasis.