IDH2 R172 Mutations Define a Unique Subgroup of Patients in Angioimmunoblastic T-Cell Lymphoma

Abstract

Background: Angioimmunoblastic T-cell lymphoma (AITL) is a common subtype of peripheral T-cell lymphoma (PTCL) with distinct pathological features and poor prognosis. Currently used chemotherapy is mostly unsuccessful with a 3-year overall survival of less than 30%. We and others have identified frequent mutations affecting IDH2 at arginine-172 (R172),TET2, DNMT3A and RHOA in AITL. The biochemical and functional consequences of IDH2R172 mutations in T cells have not been demonstrated. In this study, we performed targeted re-sequencing of epigenetic regulators, including IDH2, TET2 and DNMT3A in molecularly defined PTCL cases and analyzed the biochemical changes associated with IDH2R172 mutations as well as alterations in gene expression profiling (GEP), DNA methylation and histone modification that may improve our understanding of the pathogenetic mechanisms in AITL. Methods: We performed targeted re-sequencing of epigenetic regulators IDH2, TET2 and DNMT3A in AITL (n = 39) and PTCL subtypes (n = 53) with corresponding GEP. Due to lack of appropriate cell lines derived from AITL, we chose to use Jurkat T cells, a T-ALL cell line frequently used for T-cell functional studies and normal CD4+T cell to study the biochemical consequences of IDH2R172 mutations in T-cells. Liquid chromatography- tandem mass spectrometry was utilized for the detection of intracellular level of the 2-hydroxyglutarate and levels of 5-methycytosine and 5-hydroxymethycytosine in genomic DNA. Alterations of histone lysine tri-methylation were assessed by immunohistochemistry in AITL specimens and western blotting in vitro. Results: TET2 mutations appear to be the founder mutations in AITL with 82.1% (32/39) mutated cases and present as the major clone in the majority of mutant cases (75%; 24/32). TET2 mutations were also observed at a lower frequency in PTCL-NOS molecular subgroups (TBX21 (46%; 10/18); GATA3 (41%; 5/12)) and ALK negative-ALCL (33.3%, 4/12). The mutations in DNMT3A were observed at similar frequency in AITL (38.5%, 15/39) and PTCL-NOS subgroups [TBX21 (33%; 6/18); GATA3 (25%; 3/12)). However, IDH2R172 mutations were found predominantly in AITL (33.3%, 13/39), but rarely in PTCL-NOS subgroups (6.7%, 1/15 in PTCL-NOS). Remarkably, IDH2R172 mutant cases formed a unique cluster in unsupervised hierarchical clustering, and IDH2R172mutation defined a unique subset within AITL with a distinct gene expression signature. We observed that ectopic expression of IDH2R172K in the Jurkat T cell line led to a markedly increased level of intracellular 2-HG and up-regulation of the repressive histone methylation mark H3K27me3. Furthermore, a significant increase in 5-methylcytosine and corresponding decrease in 5-hydroxymethycytosine in genomic DNA was also observed in IDH2R172K transduced Jurkat and primary CD4+ T cells. Consistent with these findings, significant increase in aglobal DNA hypermethylation in proximal promoter regions and a global increase of the repressive histone mark H3K27me3 was observed in AITL harboring IDH2R172 mutants. Integrative analysis of GEP and promoter methylation identified several recurrently hypermethylated genes including negative regulators of the NF-kB pathway. Conclusion: IDH2R172 mutations define a unique subgroup of patients in angioimmunoblastic T-cell lymphoma with a distinct gene expression profile. IDH2R172 mutations are associated with global promoter hypermethylation in genomic DNA and trimethylation of H3K27 in AITL specimens. The current findings suggest that abnormal methylation associated with IDH2R172 mutations contribute to lymphomagenesis in AITL. Disclosures Fu: Nanostring: The author is a potential inventor on a patent application using Nanostring technology for the Lymph2Cx assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Greiner:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties.

Angioimmunoblastic T‐cell lymphoma contains multiple clonal T‐cell populations derived from a common TET2 mutant progenitor cell

“…In the 2016 WHO lymphoma classification, a broad category of nodal lymphoma of follicular helper cell origin, including AITL, PTCL-TFH, and follicular T-cell lymphoma, was introduced due to their overlapping morphological, immunophenotypic, genetic, and clinical presentations, while also bearing certain distinct features. Several studies show that RHOA and IDH2 mutations are strongly associated with the TFH immunophenotype and more extensive characteristics typical of AITL such as proliferation of follicular dendritic cells, high endothelial vessels, and presence of clear cells, with IDH2 mutation defining a unique subgroup of AITL [12,[45][46][47]. In support of these observations, we found RHOA mutation only in AITL and PTCL-TFH but not in PTCL-NOS, and IDH2 mutation exclusively in AITL in the present study.…”

“…Although exome and targeted sequencing have identified a wide spectrum of genetic changes in AITL, and also demonstrated a remarkable similarity in the mutation profile between AITL and peripheral T-cell lymphoma (PTCL) with a TFH cell phenotype, suggesting their close relationship [5][6][7][8][9][10][11][12]. In addition, these studies revealed distinct classes of genetic changes that occur at different stages of AITL development.…”

Approach to nodal-based T-cell lymphomas

“…[38][39][40] IDH2 mutations modify IDH2 enzymatic activity resulting in the production of an oncometabolite (2 hydroxyglutarate) ultimately altering DNA and histone methylation. 39,41 AITL with IDH2 mutations have a characteristic morphology with prominent medium to large clear cells, and are characterised by a strong TFH phenotype, especially strong CD10 and CXCL13 expression. 42 Genomic imbalances are frequent as well; gains of chromosomes 5 and 21 are frequent, especially in IDH2-mutated cases, and copy number losses in genes regulating the PI3K-AKT-mTOR pathway are enriched in IDH2-wild type cases.…”

GATA‐3 in T‐cell lymphoproliferative disorders

“…AITL cases harboring concurrent TET2 and IDH2 mutations have distinct gene expression and epigenetic profiles, including upregulation of T FH -associated genes and promoter and histone hypermethylation. 22 Loss-of-function mutations in the DNA methyltransferase DNMT3A are also observed, and usually occur with TET2 mutations, in approximately one-third of AITL cases.…”

“…DNMT3A and RHOA mutations are similarly observed, whereas IDH2 mutations are rare in PTCL, NOS. 22 Whether TET2 and DNMT3A mutations are also present within nonmalignant cells within the tumor microenvironment, as observed in AITL, and the mechanism by which these mutations may foster lymphomagenesis in a non-cell-autonomous manner in that context remains an open question. In contrast to the mutational landscape in these epigenetic regulators, significant differences in clinically relevant chromosomal copy number alternations have been observed between GATA-3 and T-bet PTCL.…”