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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.

MicroRNAs are commonly aberrantly expressed in many cancers. Very little is known of their role in T-cell lymphoma, however. We therefore elucidated the complete miRNome of purified T cells from 21 patients diagnosed with Sézary Syndrome (SzS), a rare aggressive primary cutaneous T-cell (CD4(+)) lymphoma. Unsupervised cluster analysis of microarray data revealed that the microRNA expression profile was distinct from CD4(+) T-cell controls and B-cell lymphomas. The majority (104 of 114) of SzS-associated microRNAs (P < .05) were down-regulated and their expression pattern was largely consistent with previously reported genomic copy number abnormalities and were found to be highly enriched (P < .001) for aberrantly expressed target genes. Levels of miR-223 distinguished SzS samples (n = 32) from healthy controls (n = 19) and patients with mycosis fungoides (n = 11) in more than 90% of samples. Furthermore, we demonstrate that the down-regulation of intronically encoded miR-342 plays a role in the pathogenesis of SzS by inhibiting apoptosis, and describe a novel mechanism of regulation for this microRNA via binding of miR-199a* to its host gene. We also provide the first in vivo evidence for down-regulation of the miR-17-92 cluster in malignancy and demonstrate that ectopic miR-17-5p expression increases apoptosis and decreases cell proliferation in SzS cells.

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.