CONTROL-T RESEARCH PROJECT 1

The team

From left: Prof. Ralf Küppers, Prof. Martin-Leo Hansmann, Dr. René Scholtysik, Dr. Friederike Schwartz

Second Funding Period

Accomplishments of the first funding period

RP1 is focused on the pathogenesis of AITL. In the first funding period, it was revealed that somatic mutations in the TET2 tumor suppressor gene are even more frequent in AITL than previously thought. This finding is likely linked to the fact that RP1 used microdissected lymphoma cells for the analysis, whereas prior studies used whole tissue section DNA and thereby might have missed mutations in cases with rare lymphoma cells. Importantly, it was shown in RP1 for the first time that TET2 mutations are frequently also present in a fraction of B cells present in the AITL lymph nodes. This shows that TET2 mutations happen in hematopoietic precursor cells and that such mutated cells also give rise to mature B cells. This finding has important implications for the frequent co-occurrence of B-cell lymphomas in AITL patients, an issue that shall be addressed in the second funding period. After overcoming several technical obstacles for the planned exome and whole genome sequencing analysis of AITL (mainly caused by the lower frequency of lymphoma cells in affected lymph nodes than generally assumed), these sequencing studies are now well under way. RP1 also contributed to a collaborative study with a targeted and exome sequencing approach based on formalin-fixed paraffin-embedded material of numerous AITL and other MTCL, which is currently in the validation phase.

Aims

Aim 1: Evaluation of epigenetic alterations in AITL lymphoma cells

As three of the most frequently mutated genes in AITL, namely TET2, IDH2 and DNMT3A, encode for epigenetic regulators of DNA methylation, it is to be expected that deregulation of epigenetic patterns play a major role in AITL pathogenesis. However, this has not been studied yet. We therefore hypothesise that AITL cells show major alterations in their DNA methylation pattern. We aim to determine deregulated DNA methylation patterns in AITL cells in comparison to normal TFH cells and search for oncogenes and tumor suppressor genes that are affected by deregulated DNA methylation.

Aim 2: Genetic analysis of HSC cells from AITL patients for early somatic mutation events

TET2 mutations occur already in HSC/HPC in AITL patients. However, it is not known whether also other genetic lesions seen in AITL occur at this early developmental stage in AITL patients. We hypothesise that besides TET2 also other genetic lesions occur in HSC/HPC of AITL patients. We therefore aim to clarify whether in addition to TET2 also other mutations happen in HSC/HPC of such patients. To this end, HSC/HPC colonies will be established from AITL patients and targeted as well as exome sequencing will be performed.

Aim 3: Determination of epigenetic and transcriptomic alterations of mutated HSC from AITL patients

From aim 2, we will obtain HSC/HPC colonies with mutations in TET2 and/or other genes, as well as many colonies without such mutations. We hypothesise that such mutated HSC/HPC are altered in their epigenetic pattern, gene expression and functional capabilities. We aim to comprehensively characterize colonies with and without mutations phenotypically, epigenetically and at the transcriptomic level to clarify how the phenotypic features, the DNA methylation pattern, the transcriptome and hence the differentiation potentials of HSC/HPC are altered by the somatic mutations. These studies shall provide insights into the very first step of AITL pathogenesis.

Aim 4: Determination of common and distinct mutation patterns in paired samples of AITL and B-NHL

A unique feature of AITL is the frequent co-occurrence of B-NHL. We hypothesise that early somatic mutations which occurred in HPC or common lymphoid precursors exist in these lymphomas and likely represent a major determinant for this phenomenon. Using paired samples available as paraffin-embedded biopsies, we aim to perform targeted sequencing for candidate genes known from the literature to be frequently mutated in AITL and/or B-NHL to describe the pattern of shared and distinct mutations in the pathogenesis of these lymphomas. For one case with AITL and a diffuse large B-cell lymphoma available as frozen biopsies, whole exome sequencing will be performed to identify shared and distinct mutations at the genome-wide level.

First Funding Period

Abstract

Angioimmunoblastic T cell lymphoma (AITL) is one of the most frequent T cell lymphomas. It has a dismal prognosis, with most patients dying within three years after diagnosis. Only little is known about the pathogenesis of AITL. A peculiar and unique feature of AITL is the frequent development of oligo- or monoclonal B cell lymphoproliferations, sometimes giving rise to overt B cell lymphomas. We aim to identify the genetic lesions and potential oncogenic viruses causing AITL by whole genome next generation sequencing. We will verify the pathogenetic role of mutated genes using mouse models available in the consortium. Using microdissection and single cell PCR, we will clarify whether genetic lesions of AITL are partially also present in the expanded B cell clones or B cell lymphomas, which would explain the high risk of co-development of B and T cell lymphomas in a given patient with AITL. Finally we will analyze whether mutations found in AITL also occur in other lymphoid malignancies. By determining the genetic landscape of AITL we hope to lay the ground for refined diagnostics, prognostication, and novel targeted therapy approaches.

Aims

Aim 1: Identification of genetic lesions in AITL by NGS

We aim to define the landscape of genetic lesions in AITL by whole genome sequencing of AITL cases. Recurrence of lesions will be studied by analysis of a larger cohort of cases for the candidate genes.

Aim 2: Determination whether genetic lesions of AITL tumor T cells are also present in B cell clones

AITL is unique among all lymphoid malignancies with respect to the frequent development of monoclonal B cell proliferations or even frank B cell lymphomas in the background of the T cell lymphoma. This might indicate the presence of genetic lesions already in hematopoietic precursor cells, promoting the occurrence of both T and B cell malignancies from a common pre-malignant ancestor. We aim to clarify whether gene mutations are shared by T and B cell clones in AITL patients and hence occur already in hematopoietic or lymphoid precursor cells.

Aim 3: Evaluation of the pathogenetic role of putative AITL driver mutations in mouse models

We expect to identify numerous novel genetic lesions in AITL. To validate their pathogenetic role, functional studies are needed. We aim to validate the pathogenetic role of several putative driver mutations using mouse models available in the consortium.

Aim 4: Clarification whether mutations found in AITL also occur in other T and B cell lymphomas and leukemias

By analyzing other T and B cell lymphomas and leukemias for mutations in genes found to be mutated in AITL, we aim to determine the specificity of the mutations for AITL and whether they play a pathogenetic role also in other lymphoid malignancies.

Aim 5: Histopathological evaluation of lymphomas

The various mouse lymphoma models generated in other RPs shall be histopathologically evaluated by M.-L. Hansmann.