Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis).
Using microarrays, we show that he most induced (miR-326, miR-663b, and miR-711) and repressed (miR-203 and miR-205) miRNAs distinguish CTCL from benign skin diseases with > 90% accuracy in a training set of 90 samples and a test set of 58 blinded samples. These miRNAs also distinguish malignant and benign lesions in an independent set of 50 patients with PTL and skin inflammation and in experimental human xenograft mouse models of psoriasis and CTCL. Quantitative (q)RT-PCR analysis of 103 patients with CTCL and benign skin disorders validates differential expression of 4 of the 5 miRNAs and confirms previous reports on miR-155 in CTCL. A qRT-PCR–based classifier consisting of miR-155, miR-203, and miR-205 distinguishes CTCL from benign disorders with high specificity and sensitivity, and with a classification accuracy of 95%, indicating that miRNAs have a high diagnostic potential in CTCL.
Expression profiles in training set for highly significant miRNAs.
We analyzed microarray measurements of 688 miRNAs in the training set of 90 samples with t tests to discover differences in expression between samples of subjects with CTCL and those of BDN subjects. The 27 miRNAs that displayed highly significant (Bonferroni corrected P < .001) and strong differences (at least 50% change) are presented in the heat map. Samples are arranged in columns, miRNAs in rows, and both are hierarchically clustered using Euclidean distance with average linkage of nodes. Red-to-yellow shades indicate increased relative expression; blue shades indicate reduced expression; green indicates median expression. The top 5 most significantly induced or repressed miRNAs are shown in bold.
qRT-PCR–based classification of samples from patients with CTCL and benign skin disease.
(A) A Cp based sample score (S) were calculated for each sample. Patients are ordered by increasing values of this score. The solid line shows the cutoff between patients with CTCL (green) and patients with benign skin disease (orange). The dotted lines show the cutoffs for the low confidence region.
(B) Classification performance using the cutoffs defined in panel A. P values were calculated using Fisher exact test.
(C) ROC curve ROC showing the sensitivity and specificity for various cutoff values on the sample score of the samples.
(D) Relative expression of the 3 miRNAs used in the classification in samples from patients with CTCL and benign skin disease. Error bars indicate +/- 1 SD.
Ulrik Ralfkiaer, Peter H. Hagedorn, Nannie Bangsgaard, Marianne B. Løvendorf, Charlotte B. Ahler, Lars Svensson, Katharina L. Kopp, Marie T. Vennegaard, Britt Lauenborg, John R. Zibert, Thorbjørn Krejsgaard, Charlotte M. Bonefeld, Rolf Søkilde, Lise M. Gjerdrum, Tord Labuda, Anne-Merete Mathiesen, Kirsten Grønbæk, Mariusz A. Wasik, Malgorzata Sokolowska-Wojdylo, Catherine Queille-Roussel, Robert Gniadecki, Elisabeth Ralfkiaer, Carsten Geisler, Thomas Litman, Anders Woetmann, Christian Glue, Mads A. Røpke, Lone Skov and Niels Odum
In cutaneous T cell lymphomas (CTCL), miR-21 is aberrantly expressed in skin and peripheral blood and displays anti-apoptotic properties in malignant T cells. It is, however, unclear exactly which cells express miR-21 and what mechanisms regulate miR-21. Here, we demonstrate miR-21 expression in situ in both malignant and reactive lymphocytes as well as stromal cells. qRT-PCR analysis of 47 patients with mycosis fungoides (MF) and Sezary Syndrome (SS) con rmed an increased miR-21 expression that correlated with progressive disease. In cultured malignant T cells miR-21 expression was inhibited by Tofacitinib (CP-690550), a clinical-grade JAK3 inhibitor. Chromatin immunoprecipitation (ChIP) analysis showed direct binding of STAT5 to the miR-21 promoter.
Cytokine starvation ex vivo triggered a decrease in miR-21 expression, whereas IL-2 induced an increased miR-21 expression in primary SS T cells and cultured cytokine-dependent SS cells (SeAx). siRNA-mediated depletion of STAT5 inhibited constitutive- and IL-2- induced miR-21 expression in cytokine- independent and dependent T cell lines, respectively. IL-15 and IL-2 were more potent than IL-21 in inducing miR-21 expression in the cytokine-dependent T cells. In conclusion, we provide rst evidence that miR-21 is expressed in situ in CTCL skin lesions, induced by IL-2 and IL-15 cytokines, and is regulated by STAT5 in malignant T cells. Thus, our data provide novel evidence for a pathological role of IL-2Rg cytokines in promoting expression of the oncogenic miR-21 in CTCL.
Lise M. Lindahl, Simon Fredholm, Claudine Joseph, Boye Schnack Nielsen, Lars Jønson, Andreas Willerslev-Olsen, Maria Gluud, Edda Blümel, David L. Petersen, Nina Sibbesen, Tengpeng Hu, Claudia Nastasi, Thorbjørn Krejsgaard, Ditte Jæhger, Jenny L. Persson, Nigel Mongan, Mariusz A. Wasik, Ivan V. Litvinov, Denis Sasseville, Sergei B. Koralov, Charlotte M. Bonefeld, Carsten Geisler, Anders Woetmann, Elisabeth Ralfkiaer, Lars Iversen, Niels Odum
Intense miR-21 ISH signal is seen in the dermal in ltrate, and weaker miR-21 staining over epidermis, including the Pautrier ́s microabcesses, (A, C, E), whereas only background staining appeared for the scrambled control (B, D, F, H). Neoplastic lymphocytes (large atypical cells, example indicated by large arrow in (G) and stromal cells, likely including macrophages, showed a cytoplasmatic staining for miR-21 (G). Similar ndings were observed in other patient samples where neoplastic T-cells and stromal cells were stained for miR-21 (N = 10).
Nina A. Sibbesen, Katharina L. Kopp, Ivan V. Litvinov, Lars Jønson, Andreas Willerslev-Olsen, Simon Fredholm, David L. Petersen, Claudia Nastasi, Thorbjørn Krejsgaard, Lise M. Lindahl, Robert Gniadecki, Nigel P. Mongan, Denis Sasseville, Mariusz A. Wasik, Lars Iversen, Charlotte M. Bonefeld, Carsten Geisler, Anders Woetmann and Niels Odum
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