Pathogenesis of angioimmunoblastic T-cell lymphoma

血管免疫母细胞性T细胞淋巴瘤是最常见的T细胞淋巴瘤之一。它的预后很差，大多数患者在诊断后三年内死亡。AITL的一个独特特征是寡聚或单克隆B细胞增殖的频繁发展，有时引起明显的B细胞淋巴瘤。有迹象表明，AITL中TET 2基因的突变已经发生在造血干细胞或前体细胞（HSC/HPC）中。在第一个资助期内，我们建立了一种方法，从显微切割的PD 1阳性AITL肿瘤细胞中进行全外显子组测序，并从流式分选的AITL细胞中进行全基因组测序。目前正在对第一批案件进行评估。我们还对来自福尔马林固定活检的AITL病例进行了靶向和外显子组测序分析。通过对12例AITL病例的受累淋巴结的T和B细胞进行显微解剖和PCR分析，我们发现在大多数样品中，一部分B细胞也携带存在于AITL克隆中的TET 2或IDH 2突变。这证实了这些突变通常发生在HSC/HPC中，并表明这些突变的祖细胞也产生TET 2突变的B细胞。这可能是AITL患者发生B细胞淋巴瘤的危险因素之一。在第二个资助期内，我们的目标是完成AITL的外显子组和全基因组测序研究，以确定导致AITL的进一步遗传病变和潜在致癌病毒。我们将使用一个可用的小鼠模型验证所选突变基因的致病作用（目的1）。由于AITL中的几种复发性突变影响表观遗传调节因子（TET 2、IDH 2、DNMT 3A），我们的目标是表征AITL中DNA甲基化的改变作为该疾病的进一步致病原理（目的2）。我们的目标是从AITL患者的HSC/HPC中建立细胞系，并对这些细胞系进行靶向和外显子组测序，以揭示在AITL发展的早期阶段已经发生的遗传病变的模式（目的3）。我们还旨在使用这样的细胞系来确定TET 2突变对HSC/HPC的DNA甲基化和基因表达模式的功能后果（目的4）。最后，我们计划使用AITL病例的靶向深度测序，同时或随后B-NHL，通过揭示共享和不同遗传病变的模式来深入了解此类双重淋巴瘤的发病机制（目的5）。通过确定AITL、其前体细胞和相关B-NHL的遗传和表观遗传景观，我们的目标是全面表征小鼠模型中AITL的多步转化过程以及来自患者的不同细胞群体。

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. A unique feature of AITL is the frequent development of oligo- or monoclonal B-cell proliferations, sometimes giving rise to overt B-cell lymphomas. There is indication that mutations in the TET2 gene in AITL already occur in hematopoietic stem or precursor cells (HSC/HPC). In the first funding period, we established a method to perform whole exome sequencing from microdissected PD1-positive AITL tumor cells, and also performed whole genome sequencing from flow-sorted AITL cells. The evaluation of the first cases is currently under way. We also contributed to a targeted and exome sequencing analysis of AITL cases from formalin-fixed biopsies. By microdissection and PCR analysis of T and B cells from involved lymph nodes of 12 AITL cases we revealed that in most samples also a fraction of B cells carries the TET2 or IDH2 mutations present in the AITL clone. This confirms that these mutations typically occur in HSC/HPC, and shows that these mutated progenitor cells give also rise to TET2-mutated B cells. This might be one of the risk factors for the development of B-cell lymphomas in AITL patients. A detailed single cell IgV gene and TET2 mutation analysis of four selected AITL uncovered that the TET2-mutated B cells were mostly polyclonal B cells.In the second funding period, we aim to finish the exome and whole genome sequencing study of AITL to identify further genetic lesions and potential oncogenic viruses causing AITL. We will verify the pathogenetic role of selected mutated genes using an available mouse model (aim 1). As several recurrent mutations in AITL affect epigenetic regulators (TET2, IDH2, DNMT3A), we aim to characterize alterations of DNA methylation in AITL as a further pathogenetic principle in this disease (aim 2). We aim to establish cell lines from HSC/HPC of AITL patients and perform targeted and exome sequencing of these lines to reveal the pattern of genetic lesions already occurring at this early stage of AITL development (aim 3). We also aim to use such lines to determine the functional consequence of TET2 mutations on the DNA methylation and gene expression pattern of the HSC/HPC (aim 4). Finally, we plan to use targeted deep sequencing of AITL cases with concurrent or subsequent B-NHL to gain insight into the pathogenesis of such dual lymphomas by revealing the pattern of shared and distinct genetic lesions (aim 5). By determining the genetic and epigenetic landscape of AITL, their precursor cells and related B-NHL we aim to comprehensively characterize the multi-step transformation process in AITL in a mouse model as well as different cell populations from patients.