Underexpression of PTPRK mRNA is due to promoter hypermethylation and monoallelic deletions in NKTCL. (A) Gel images showing the PTPRK mRNA expression levels detected by semiquantitative RT-PCR in 5 NKTCL cell lines after treatment with 5-aza-dC for 3 or 6 days. Pharmacologic demethylation with 5-aza-dC restored PTPRK mRNA expression in all 4 non–PTPRK-expressing NKTCL cell lines. The mRNA expression in each sample was normalized to β-actin expression. (B) Immunofluorescence image of a non–PTPRK-expressing NKYS cell line showing the re-expression of PTPRK protein after treatment with the 5-aza-dC demethylating agent; an antibody to the N-terminus of PTPRK was used (arrows indicate green fluorescent cellular membranous and cytoplasmic staining). Original magnification ×1000. (C) (Top) Gel image revealing the methylation status of the PTPRK promoter, as determined by MSP in 2 preparations of normal NK cells and 5 NKTCL cell lines. The PTPRK promoter was unmethylated in PTPRK-expressing normal NK cells and NK malignant cell lines, whereas PTPRK was methylated in non–PTPRK-expressing NKTCL cell lines. (Bottom) Detailed methylation analysis of the CpG sites in the PTPRK promoter was performed via BGS in normal NK cells, untreated NKTCL cells, and 5-aza-dC–treated malignant cells. BGS confirmed the PTPRK promoter methylation status determined by MSP. The amount that each circle is filled represents the percentage of methylated cytosines detected via BGS from 8 to 10 sequenced colonies. (D) The PTPRK promoter was frequently methylated in NKTCL primary tumors. (Top) The composite gel images show the PTPRK promoter methylation status as determined by MSP in 27 NKTCL primary tumors. BGS confirmed the PTPRK promoter methylation status determined by MSP (bottom). The CpG sites of each representative case are presented in the row as individual circles. (E) Gel image of PTPRK gene allelic loss analysis in 6 representative cases of primary NKTCL tumors as determined by semiquantitative multiplex PCR (30 cycles) using the β-globin gene as normal allele control. Samples with a PTPRK/β-globin allelic ratio of < 0.75, as determined by densitometry, were considered to have a monoallelic PTPRK gene deletion (case #14, case #15, and case #16). (F) Methylation of the PTPRK promoter significantly correlated with inferior OS in NKTCL patients treated with the SMILE protocol. Kaplan-Meier curves indicating DFS (left panel) and OS (right panel) distributions according to methylation status of the PTPRK promoter in NKTCL patients. Both Kaplan-Meier curves were compared using the log-rank, Breslow, and Tarone-Ware tests, and the P values are presented alongside the survival plots. The results revealed that methylation of the PTPRK promoter was significantly correlated with inferior DFS and OS in NKTCL patients treated with the SMILE regimen. The treatment outcome data were available for the 17 NKTCL patients treated with the SMILE regimen who were included in this study. M, methylated; MW, molecular weight markers; U, unmethylated; UMD, universal methylated DNA; UUD, universal unmethylated DNA.