Graduation Date

Summer 8-19-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pathology & Microbiology

First Advisor

Wing-Chung (John) Chan

Second Advisor

Rakesh K Singh

Abstract

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of generally aggressive lymphoid malignancies, accounting for 10-15% of all non-Hodgkin lymphomas. Angioimmunoblastic T-cell lymphoma (AITL) represents approximately 20% of all PTCLs and is recognized as a distinct entity. Accurate diagnosis and classification of PTCL remain challenging. With the exception of ALK+ ALCL, patients with PTCL generally have a poor prognosis with standard chemotherapy and even with the availability of many novel drugs, including HDAC inhibitor (romidepsin and belinostat), gemcitabine, and bortezomib. Therefore, deciphering the pathogenesis of this group of diseases is needed to identify novel treatable targets for better therapeutic intervention. The overall goal is to study the genetic and epigenetic alterations in PTCL, particularly AITL, to improve accuracy of diagnosis and identify novel targets for effective therapy.

T-cell clonality of PTCL is routinely evaluated with a PCR-based method using genomic DNA. However, there are limitations with this approach. To determine the utility of RNA-seq for assessing T-cell clonality and TCR repertoire of the neoplastic T-cells in PTCL samples, we assessed TCR transcript including complementarity-determining region 3 (CDR3) sequences. In normal T cells, the CDR3 sequences are extremely diverse, without any clonotype representing more than 2% of the overall TCR population. Dominant clones could be identified by transcriptome sequencing in most cases of AITL (36/40), ALCL (18/22), and PTCL-NOS (11/14) with adequate TCR transcript expression. In monoclonal cases, the dominant clone varied between 10.9% and 98.9% of TCRβ rearrangements. No unique α or β chain usage is observed. RNA-seq is a useful tool for detecting and characterizing clonal rearrangements in PTCL.

We performed targeted resequencing on 92 cases of PTCL and identified frequent mutations affecting TET2, DNMT3A, RHOA, and isocitrate dehydrogenase 2 (IDH2). Whereas IDH2 mutations are largely confined to AITL, mutations of the other three can be found in other types of PTCL although at lower frequencies. We correlated mutational status with gene expression and global DNA methylation changes in AITL. Strikingly, AITL cases with IDH2R172mutations demonstrated a distinct gene expression signature characterized by down-regulation of genes associated with TH1 differentiation (e.g., STAT1 and IFNG) and a striking enrichment of an IL12-induced gene signature. Ectopic expression of IDH2R172K in the Jurkat cell line and CD4+ T cells led to markedly increased levels of 2-hydroxyglutarate, histone-3 lysine methylation, and 5-methylcytosine and a decrease in 5-hydroxymethylcytosine. Correspondingly, clinical samples with IDH2 mutations displayed a prominent increase in H3K27me3 and DNA hypermethylation of gene promoters. Integrative analysis of gene expression and promoter methylation revealed recurrently hypermethylated genes involved in TCR signaling and T cell differentiation that likely contribute to lymphomagenesis in AITL.

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