Page 136 - Haematologica-April 2018

M-M. Ji et al.
scription regulation.2 Two subgroups of PTCL-NOS have
been identified, which are characterized by high expres- sion of either GATA3 or TBX21/T-bet transcription factors and downstream target genes.3 However, actionable bio- markers closely related to the pathogenic mechanism need to be further investigated and may become potential ther- apeutic targets of PTCL-NOS.4,5
Epigenetic alterations play a crucial role in tumor pro- gression.6 Next-generation sequencing technologies have led to the discovery of epigenetic modifier gene mutations in PTCL, such as the DNA methylation genes TET2, TET1 and DNMT3A,7,8 and chromatin remodeler genes ARID1B and ARID2.9,10 Meanwhile, genes of histone methylation, such as KMT2D, KMT2A, KDM6A, SETD2 and EZH2, and those of histone acetylation, including CREBBP and EP300, have also been found in PTCL and other T-lym- phoid malignancies.9,11-13 To further determine their prog- nostic significance and correlation with clinical treatment, here we assessed the mutational pattern of the main epi- genetic modifier genes in patients with PTCL-NOS.
Methods
Patients
A total of 239 patients with previously untreated PTCL-NOS were enrolled in this study. The histological diagnosis was estab- lished according to the World Health Organization (WHO) classi- fication.14 The study was approved by the Shanghai Rui Jin Hospital Review Board with informed consent obtained in accor- dance with the Declaration of Helsinki.
Targeted sequencing
Targeted sequencing was performed on available tumor sam- ples of 125 patients. To determine the mutations of candidate genes, polymerase chain reaction primers were designed by iPLEX Assay Design software (Sequenom, California, USA). Multiplexed libraries of tagged amplicons from patients with PTCL-NOS were generated by the 48×48 Access Array microfluidic platform (Fluidigm, South San Francisco, USA) according to the manufac- turer’s protocol. Deep sequencing was performed with established Illumina protocols on the GAIIx and MiSeq platform (Illumina, California, USA). Matched peripheral blood samples were includ- ed to exclude germline polymorphisms and the mutations were confirmed by Sanger sequencing.
Cell line and reagents
The Jurkat T-leukemia cell line was obtained from the American Type Culture Collection. Cells were grown in RPMI-1640 medi- um, supplemented with 10% heat-inactivated fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37°C. Valproic acid (VPA, V3640) was from Sigma (San Francisco, USA). Suberoylanilide hydroxamic acid (SAHA, S1047) and romidepsin (ROMI, S3020) were from Selleck (Houston, USA). Chidamide, the histone deacetylase (HDAC) inhibitor clinically available in China, was kindly provided by Chipscreen (shenzhen,China).
Lentivirus packaging and transduction
Purified plasmids pGV365-KMT2D (WT), pGV365-KMT2D (V5486M), pGV365-EP300 (WT) and pGV365-EP300 (H1377R) were transfected with package vectors into HEK-293T cells using lipofectamine 2000 (Invitrogen, California, USA; 11668019) according to the manufacturer’s protocol. The supernatant fraction of HEK-293T cell cultures was then condensed to a viral concen- tration of approximately 3×108 transducing units/mL. The lentivi-
ral particles were incubated with Jurkat cells for 72 h. The stably transduced cells were selected by EGFP or mCherry fluorescence protein after transduction.
Statistical analysis
Data were calculated as the mean ± standard deviation from three separate experiments. The Student t-test was applied to compare two normally distributed groups and the Mann-Whitney U test to compare two groups which did not conform to normal distribution. The Bonferroni adjustment was used to perform mul- tiple comparisons. Progression-free survival was calculated from the date when treatment began to the date when the disease pro- gression was recognized or the date of the last follow-up. Overall survival time was measured from the date of diagnosis to the date of death or the last follow-up. Univariate hazard estimates were generated with unadjusted Cox proportional hazards models. Covariates demonstrating statistical significance with P values <0.05 on univariate analysis were included in the multivariate model. All statistical procedures were performed with the SPSS version 20.0 statistical software package or GraphPad Prism 5 soft- ware. P<0.05 was considered statistically significant.
Online supplementary methods
DNA preparation, western blot, immunofluorescence, immunohistochemistry, isobolographic analysis, mRNA-seq library preparation and sequencing analysis, ChIP-seq library preparation and sequencing analysis, the TUNEL assay, murine model and micro-positron emission tomography and computed tomography imaging are described in the Online Supplementary Methods.
Results
Histone modifier genes were frequently mutated in peripheral T-cell lymphoma not otherwise specified
A total of 91 somatic mutations of epigenetic modifier genes were identified in 60 of 125 (48.0%) patients with PTCL-NOS by targeted sequencing (Figure 1A). Most of the somatic mutations were missense mutations (n=72), followed by nonsense (n=10) and frameshift mutations (n=9) (Figure 1B). We observed a preference for C>T/G>A alterations analogous to the somatic single-nucleotide variation spectrum in other cancers (Figure 1C). No corre- lation was found in terms of age and gender. Mutations of histone methylation genes (category I) most frequently occurred in KMT2D (encoding H3K4 methyltransferase, 25/125 patients, 20.0%), followed by those in SETD2 (encoding H3K36 methyltransferase, 6/125 patients, 4.8%), KMT2A (encoding H3K4 methyltransferase, 3/125 patients, 2.4%) and KDM6A (encoding H3K27 demethy- lase, 1/125 patients, 0.8%). No EZH2 mutation was detected. Mutations of histone acetylation genes (category II) were found in EP300 (encoding H3K18 acetyltrans- ferase, 10/125 patients, 8.0%) and in CREBBP (encoding H3K18 acetyltransferase, 5/125 patients, 4.0%). DNA methylation genes TET2, TET1 and DNMT3A (category III), as well as chromatin remodeler genes ARID1B and ARID2 (category IV), were also affected in 12.0%, 3.2%, 3.2%, 4% and 1.6% of the patients, respectively (Figure 1A and Online Supplementary Table S1). In accordance with the conceptual classification of the mutated genes, overlap mutations were seldom present among histone methyla- tion, histone acetylation, DNA methylation or chromatin remodeler genes. In particular, histone methylation gene
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