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Phospholipase C Gamma 1 (PLCG1) is frequently mutated in primary cutaneous T-cell lymphoma (CTCL). This study functionally interrogated nine PLCG1 mutations (p.R48W, p.S312L, p.D342N, p.S345F, p.S520F, p.R1158H, p.E1163K, p.D1165H, and the in-frame indel p.VYEEDM1161V) identified in Sézary Syndrome, the leukemic variant of CTCL. The mutations were demonstrated in diagnostic samples and persisted in multiple tumor compartments over time, except in patients who achieved a complete clinical remission. In basal conditions, the majority of the mutations confer PLCγ1 gain-of-function activity through increased inositol phosphate production and the downstream activation of NFκB, AP-1, and NFAT transcriptional activity. Phosphorylation of the p.Y783 residue is essential for the proximal activity of wild-type PLCγ1, but we provide evidence that activating mutations do not require p.Y783 phosphorylation to stimulate downstream NFκB, NFAT, and AP-1 transcriptional activity. Finally, the gain-of-function effects associated with the p.VYEEDM1161V indel suggest that the C2 domain may have a role in regulating PLCγ1 activity. These data provide compelling evidence to support the development of therapeutic strategies targeting mutant PLCγ1., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

FK228 (romidepsin) and suberoylanilide hydroxamic acid (vorinostat) are histone deacetylase inhibitors (HDACi) approved by the US Food and Drug Administration for cutaneous T-cell lymphoma (CTCL), including the leukemic subtype Sézary syndrome. This study investigates RAD23B and STAT3 gene perturbations in a large cohort of primary Sézary cells and the effect of FK228 treatment on tyrosine phosphorylation of STAT3 (pYSTAT3) and RAD23B expression. We report RAD23B copy number variation in 10% (12/119, P ≤ 0.01) of SS patients, associated with reduced mRNA expression (P = 0.04). RAD23B knockdown in a CTCL cell line led to a reduction in FK228-induced apoptosis. Histone deacetylase inhibitor treatment significantly reduced pYSTAT3 in primary Sézary cells and was partially mediated by RAD23B. A distinct pattern of RAD23B-pYSTAT3 co-expression in primary Sézary cells was detected. Critically, Sézary cells harboring the common STAT3 Y640F variant were less sensitive to FK228-induced apoptosis and exogenous expression of STAT3 Y640F, and D661Y conferred partial resistance to STAT3 transcriptional inhibition by FK228 (P ≤ 0.0024). These findings suggest that RAD23B and STAT3 gene perturbations could reduce sensitivity to histone deacetylase inhibitors in SS patients., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

Methylthioadenosine phosphorylase (MTAP) and the tumor suppressor genes CDKN2A-CDKN2B are frequently deleted in malignancies. The specific role of MTAP in cutaneous T-cell lymphoma subgroups, mycosis fungoides (MF) and Sézary syndrome (SS), is unknown. In 213 skin samples from patients with MF/SS, MTAP copy number loss (34%) was more frequent than CDKN2A (12%) in all cutaneous T-cell lymphoma stages using quantitative reverse transcription PCR. Importantly, in early stage MF, MTAP loss occurred independently of CDKN2A loss in 37% of samples. In peripheral blood mononuclear cells from patients with SS, codeletion with CDKN2A occurred in 18% of samples but loss of MTAP alone was uncommon. In CD4(+) cells from SS, reduced MTAP mRNA expression correlated with MTAP copy number loss (P < 0.01) but reduced MTAP expression was also detected in the absence of copy number loss. Deep sequencing of MTAP/CDKN2A-CDKN2B loci in 77 peripheral blood mononuclear cell DNA samples from patients with SS did not show any nonsynonymous mutations, but read-depth analysis suggested focal deletions consistent with MTAP and CDKN2A copy number loss detected with quantitative reverse transcription PCR. In a cutaneous T-cell lymphoma cell line, promoter hypermethylation was shown to downregulate MTAP expression and may represent a mechanism of MTAP inactivation. In conclusion, our findings suggest that there may be selection in early stages of MF for MTAP deletion within the cutaneous tumor microenvironment., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

T-plastin (PLS3) is an actin-bundling protein normally expressed in epithelial cells but absent in cells of hematopoietic origin. Aberrant PLS3 expression has been demonstrated in lymphocytes from Sézary syndrome (SS) patients and has been proposed as a biomarker for SS; however, the mechanism underlying dysregulation of PLS3 has not been determined. In this study, PLS3 mRNA expression was demonstrated in 21/35 (60%) SS patients and in 3/8 (38%) mycosis fungoides patients, all of whom had clonal blood involvement. No evidence for PLS3 mutations within coding or promoter regions was found, but significant hypomethylation of CpG dinucleotides 95-99 within the PLS3 CpG island was observed and this was restricted to the PLS3+ population. A polyclonal antibody specific to PLS3 was raised to examine coexpression of PLS3 with a panel of T-cell differentiation markers. All PLS3+ cells were CD3+CD4+ and CD26-, suggesting that loss of CD26 is consistently associated with gain of PLS3, whereas all other markers were distributed heterogeneously. However, a patient-specific TCR copy number assay also demonstrated heterogeneity in PLS3 expression in tumor cell populations. Importantly, our findings demonstrate PLS3 expression in the majority of SS patients and provide insight into the molecular regulation of PLS3 expression in CTCL.

Sézary Syndrome (SS) is an aggressive leukemic variant of primary cutaneous T-cell lymphoma characterized by the presence of tumor or Sézary cells that generally display a mature memory T-cell immunophenotype. Sézary cells proliferate poorly and therefore their accumulation may be due to defective T-cell homeostasis involving resistance to apoptosis. In this study, we analyzed Fas expression in CD4+ lymphocytes at the mRNA and protein levels in a large cohort of SS patients as compared with healthy controls. Fas mRNA expression was dysregulated in 34/47 patients, with significant under- and overexpression of Fas mRNA detected in 21 and 13 patients respectively (P<0.01). Examination of cell-surface Fas expression showed correlation with the observed downregulation of mRNA in CD4+ T cells. Mutational analysis demonstrated that functional FAS gene mutations are rare. Moreover, 16 SS patients who showed significant under-expression of Fas mRNA also showed significant positional hypermethylation within the FAS CpG island, which was not present in healthy controls or SS patients determined to have normal or overexpression of Fas mRNA. These data demonstrate that dysregulation of Fas expression is a common feature of SS, and provide a rationale for targeted therapies to restore the extrinsic Fas-dependent apoptotic pathway in this malignancy.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL, LT) is the most aggressive type of primary cutaneous B-cell lymphoma. In a recent study on 12 patients it was found that inactivation of CDKN2A by either deletion of 9p21.3 or promoter hypermethylation is correlated with a worse prognosis. In the present EORTC multicenter study, skin biopsies of 64 PCLBCL, LT patients were analyzed by multiplex ligation-dependent probe amplification to validate these previous results and to fine-map the losses in this region. Although no minimal common region of loss could be identified, most homozygous loss was observed in the CDKN2A gene (43 of 64; 67%) encoding p16 and p14ARF. Promoter hypermethylation of p16 and p14ARF was found in six and zero cases, respectively. Survival was markedly different between patients with versus without aberrations in the CDKN2A gene (5-year disease-specific survival 43 versus 70%; P=0.06). In conclusion, our results confirm that deletion of chromosome 9p21.3 is found in a considerable proportion of PCLBCL, LT patients and that inactivation of the CDKN2A gene is associated with an unfavorable prognosis. In most patients the deletion involves a large area of at least several kilobase pairs instead of a small minimal common region.

Mycosis fungoides and its leukemic variant, Sezary syndrome, are the most common primary cutaneous T-cell lymphomas (CTCLs). In an ex vivo study, we investigated the percentage, phenotype, and suppressive function of CD4+CD25+ regulatory T cells (Tregs) from peripheral blood of CTCL patients. The percentage of Tregs did not differ significantly between patients and controls. Functional assays demonstrated a dichotomy in Treg function: in four out of 10 patients CD4+CD25+ T cells were incapable of suppressing autologous CD4+CD25- T-cell proliferation, whereas suppressive function was intact in the other six patients. Suppressive activity of Tregs inversely correlated with the peripheral blood tumor burden. T-plastin gene expression, used as a Sezary cell marker, confirmed that Sezary cells were heterogeneous for CD25 expression. Mixed lymphocyte reactions demonstrated that CD4+CD25- T cells from patients who lacked functional Tregs were susceptible to suppression by Tregs from healthy controls, and had not become suppressive themselves. Furthermore, we found reduced expression of Foxp3 in the CD4+CD25+ Tregs of these patients relative to the other six CTCL patients and controls. Our findings thus indicate a dysfunction of peripheral Tregs in certain CTCL patients, which correlates with tumor burden.

Molecular characterization of T-cell receptor junctional region sequences in cutaneous T-cell lymphoma had not been previously reported. We have examined in detail the features of the T-cell receptor beta (TCRB) gene rearrangements in 20 individuals with well-defined stages of cutaneous T-cell lymphoma (CTCL) comprising 10 cases with early-stage mycosis fungoides (MF) and 10 cases with late-stage MF or Sezary syndrome. Using BIOMED-2 PCR primers, we detected a high frequency of clonally rearranged TCR gamma and TCRB genes (17/20 and 15/20 cases, respectively). We carried out sequencing analysis of each complete clonal variable (V)beta-diversity (D)beta-joining(J)beta fingerprint generated by PCR amplification, and determined the primary structure of the Vbeta-Dbeta-Jbeta junctional regions. We observed considerable diversity in the T-cell receptor Vbeta gene usage and complementarity-determining region 3 loops. Although we found that TCRB gene usage in CTCL and normal individuals share common features, our analysis also revealed preferential usage of Jbeta1 genes in all cases with advanced stages of disease.

Fifty-one mycosis fungoides samples were analyzed for microsatellite instability (MSI) using the panel of markers recommended for hereditary nonpolyposis colorectal cancer kindred and a panel we designed for cutaneous T cell lymphoma in order to compare detection rates and determine if MSI is a genome-wide phenomenon. Samples demonstrating MSI were analyzed for abnormalities of the hMLH1 gene including loss of heterozygosity, mutations, and promoter hypermethylation. MSI was detected in 16% using the hereditary nonpolyposis colorectal cancer panel and 22% with the cutaneous T cell lymphoma panel. Overall, 27% demonstrated MSI and 73% had a stable phenotype. hMLH1 gene studies did not detect loss of heterozygosity or reveal any mutations. Promoter hypermethylation was detected in nine of 14 patients with MSI, however (64%). In addition hMLH1 and hMSH2 protein expression was studied using immunohistochemical techniques. Five of nine patients with MSI and hMLH1 promoter methylation showed abnormal hMLH1 protein expression with normal hMSH2 gene expression. All other patients tested demonstrated normal hMLH1 and hMSH2 protein expression. MSI was found to be more prevalent in tumor stage mycosis fungoides (47%) than early stage disease (20%) and was associated with an older age of onset of mycosis fungoides. MSI may be a consequence of hMLH1 promoter hypermethylation in mycosis fungoides patients and may prevent transcription in a subset of patients. This suggests that the development of a mutator phenotype may contribute to disease progression in mycosis fungoides.

It is now recognized that dendritic cells (DC) isolated from mouse spleen play an important role in activating T lymphocytes. These DC, which show many similarities to veiled cells found in the paracortical regions of spleen and lymph node, may be closely related to the epidermal Langerhans cells. It is known that DC are extremely effective allostimulators. We have also found that although DC lack demonstrable phagocytic ability, they are extremely potent at presenting soluble polypeptide antigens to primed T cells. Since T lymphocytes comprise several distinct subsets (particularly cytotoxic, helper, and suppressor) in our most recent studies we have asked whether DC are able to trigger all these different subsets of T cells. We examined the ability of different spleen cell types coupled with the hapten NP to induce antigen-specific T suppressors for a delayed type hypersensitivity (DTH) response. It was found that T suppressors were generated only when hapten was conjugated to a spleen-derived antigen-presenting cell. Further analysis revealed that macrophages but not DC were able to induce defined sets of suppressor cells in vivo (although DC were able to trigger a very powerful DTH response). We also examined the ability of DC to activate T cells which are required to cooperate with B cells in the production of antibody. Even though DC were able to trigger T lymphocytes to produce lymphokines, these activated T cells did not act as helper cells in a standard hapten-carrier system. Possible mechanisms for this dichotomy of DC function are discussed.