Independent Loss of Methylthioadenosine Phosphorylase (MTAP) in Primary Cutaneous T-Cell Lymphoma  Wesley J. Woollard, Nithyha P. Kalaivani, Christine L. Jones, Catherine Roper, Lam Tung, Jae Jin Lee, Bjorn R. Thomas, Isabella Tosi, Silvia Ferreira, Carl Z. Beyers, Robert C.T. McKenzie, Rosie M. Butler, Anna Lorenc, Sean J. Whittaker, Tracey J. Mitchell  Journal of Investigative Dermatology  Volume 136, Issue 6, Pages 1238-1246 (June 2016) DOI: 10.1016/j.jid.2016.01.028 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Gene CN analysis for MTAP and CDKN2A in MF and SS. (a) MTAP and (b) CDKN2A CN was determined by Q-PCR in 84 HCs, 52 MF IB-IIA, 73 MF IIB-IV, and 101 SS III-IV samples (64 PBMC samples and 39 skin samples). Horizontal lines represent 3 standard deviations from the HC mean. Schematic representations of (c) MTAP and (d) CDKN2A showing positions of Q-PCR probe sets 1 and 2. Gene CN of (e) MTAP and (f) CDKN2A determined by probe sets 1 (y-axis) and 2 (x-axis). Red lines represent 3 standard deviations from the HC mean for the corresponding probe set. Probe set 2 shows 75.0% and 58.3% concordance with probe set 1 for MTAP and CDKN2A, respectively. CN, copy number; MF, mycosis fungoides; MTAP, methylthioadenosine phosphorylase; SS, Sézary syndrome. Journal of Investigative Dermatology 2016 136, 1238-1246DOI: (10.1016/j.jid.2016.01.028) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Comparison of methods to call gene copy number in SS. From left to right: (a) Normalized exome depth across each exon plotted as a heat map, see the Materials and Methods section for the description of normalized depth calculation. Exons are arranged telomeric to centromeric along the x-axis and SS samples that were assayed by Q-PCR are plotted on the y-axis. (b) Normal or loss (ivory/blue, respectively) as called by the “ExomeDepth” package. (c) Normal or loss as assayed by Q-PCR probe set 1, MTAP-P1 = exon 8, CDKN2A-P2 = exon 3. (d) Normal or loss as assayed by Q-PCR probe set 2, gray represents samples not assayed by probe set 2 MTAP-P2 = intron 4, CDKN2A-P1 = intron 1. MTAP, methylthioadenosine phosphorylase; Q-PCR, quantitative reverse transcriptase in real time; SS, Sézary syndrome. Journal of Investigative Dermatology 2016 136, 1238-1246DOI: (10.1016/j.jid.2016.01.028) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Expression of CDKN2A (p16) and MTAP in CD4+ lymphocytes of patients with SS. Relative expression of (a) MTAP and (b) CDKN2A (p16) compared with cyclophilin (PPIA) in CD4+ lymphocytes of HCs and 20 patients. The y-axis shows standard deviations from the HC mean ΔΔCT. Points represent Z-scores and red lines represent 3 standard deviations of the HC ΔΔCT. Samples from tumors where copy number changes have been identified are highlighted with red dots. Samples with no identified copy number change are represented by blue dots. HC, healthy control; MTAP, methylthioadenosine phosphorylase; SS, Sézary syndrome. Journal of Investigative Dermatology 2016 136, 1238-1246DOI: (10.1016/j.jid.2016.01.028) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Functional evidence linking MTAP promoter hypermethylation and expression. (a) Demethylation restores MTAP RNA expression in MyLa cells. Lanes (1) SeAx and (2) MyLa cells treated with DMSO alone. Lanes 3–8 cell lines treated with 5 μM 5′-azadeoxycytidine (3) SeAx (4) MyLa, 24 hours; (5) SeAx (6) MyLa, 48 hours; (7) SeAx (8) MyLa, 72 hours. Lane (9) water control. RNA was analyzed by RT-PCR for (A) cyclophilin expression as an endogenous control and (B) MTAP expression. (b) Intracellular flow cytometry confirms demethylation of MyLa cells induces MTAP protein expression. SeAx and MyLa cells treated as described and MTAP protein expression determined by flow cytometry, 10,000 events were acquired per sample. Data expressed as delta mean fluorescent intensity (ΔMFI) calculated using Flowjo software. MTAP, methylthioadenosine phosphorylase; RT-PCR, reverse transcriptase-PCR. Journal of Investigative Dermatology 2016 136, 1238-1246DOI: (10.1016/j.jid.2016.01.028) Copyright © 2016 The Authors Terms and Conditions