MicroRNA Expression Profile in Mycosis Fungoides – Preliminary Findings

Abstract

Dear Editor, Mycosis Fungoides (MF) is the commonest cutaneous lymphoma, comprising about 12% of all T-cell lymphomas in India.[1] MF presents as patches, plaques or tumors. While the diagnosis of tumor stage MF is easy, lesions of patch and plaque stages pose problems in diagnosis.[2] There is a clinicopathologic overlap with several inflammatory conditions. Currently, there is no single diagnostic standard for early MF. It rests on a combination of clinical findings, histopathology, immunohistochemistry and T-cell receptor clonality testing.[3] miRNAs(miR) have great potential as biomarkers as they are robust, tissue-specific and can be obtained from both blood and tissues. Their value as diagnostic markers have been shown in several cancer types but is still nascent in MF.[4] We attempted to explore the role of miRs in patch and plaque stage MF in Indian patients. We selected 24 MF samples whose diagnosis was established based on clinical features, histopathology and follow up. We chose a set of 7miRs namely miR 203, miR205, miR223, miR155, miR 93, miR 92a, miR 21 along with two controls RNU48 and RUN44. The first three miRs have a tumor suppressor function, while the rest serve as OncomiRs. RNU44, 48 served as controls. Primer sequences designed using Primer3Plus software and then validated on Ensemble & NCBI Blast. After deparaffinisation and overnight incubation with proteinase K, RNA was extracted using RNAwiz and estimated by Ribogreen fluorimetry, the yield ranged from 5.06 to 49.6 ng. Real time PCR was done on the 26 samples on LightCycler 480 (Roche). Pre-incubation and initial denaturation of the template was at - 95°C for 10 min and amplification was done for 45 cycles at 95°C for 15 sec and 60°C for 1 min and Ct values were obtained. There were 24 samples from 21 patients. Their age ranged from 24-83 years and were predominantly men (14:7). There were 8 patch stage MF, 9 plaque stage and 7 were recurrent MF. There was no significant difference in the expression of any of the 7 miRs between the patch and plaque stages (p-values ranged from 0.423-0.963). There was no difference between patch/plaque stages and recurrent lesions. (p-values ranged from 0.481-0.963). We compared the average Ct values of the 3 tumor suppressor miRs versus the 4 OncomiRs, to look for any difference between patch, plaque and recurrent lesions. There was no significant difference in these either. (p-value ranged from 0.877 to 1.484) A few studies have focussed on the differential expression of miRs in distinguishing MF from inflammatory diseases. van Kester et al studied 19 tumor stage MF and compared their miR expression patterns with 12 benign skin conditions.[5] They found that 49 miR were differentially expressed in the two groups and miR 93, which has an anti-apoptotic function, showed the highest discrimination.[5] Ralfkiaer et al showed that early MF displays a miR profile distinct from advanced T-cell lymphomas.[6] They were also able to demonstrate different profiles in patients with progressive vs. non-progressive disease. McGirt et al demonstrated that miR223, which targets TOX gene, needed for T-cell development is overexpressed in early MF and diminishes with progression to advanced disease.[7] There is only one study which has compared miR profiles in patch vs. plaque stages of MF. Sorensen et al found 11 miRNAs were overexpressed in plaques as compared to patches, most significantly miR-142, miR-21 and miR-22.[8] We did not find any difference in miR (including miR21) expression between patch and plaque stages or in patients who had recurrences. Variation in results could be due to the methodology used (whole tissue vs. microarray), quantity and quality of RNA and density of the infiltrate.[8] Nevertheless, it seems that although clinically distinct, patch and plaque stage MF are more similar biologically. We will expand the scope to compare miR expression profiles of MF with various inflammatory skin diseases in the next part of our ongoing study. To summarise, miR expression does not appear to be useful in distinguishing patch vs. plaque stage MF. Our findings need to be validated in larger samples in Indian patients. Financial support and sponsorship Rajiv Gandhi University of Health Sciences, Bangalore for funding the project Conflicts of interest There are no conflicts of interest.