Pathogenesis of angioimmunoblastic T-cell lymphoma

血管免疫母细胞T细胞淋巴瘤(AITL)是最常见的T细胞淋巴瘤之一。它的预后很糟糕，大多数患者在确诊后三年内死亡。AITL的一个独特特征是经常出现寡聚或单克隆性B细胞增殖，有时会导致显性B细胞淋巴瘤。有迹象表明，AITL中的TET2基因突变已经出现在造血干细胞或前体细胞(HSC/HPC)中。在第一个资助期，我们建立了一种方法，从显微解剖的PD1阳性的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。我们还对福尔马林固定活检中的AITL病例进行了有针对性的外显子组测序分析。通过对12例AITL受累淋巴结中T和B细胞的显微解剖和聚合酶链式反应分析，我们发现在大多数样本中，也有一部分B细胞携带AITL克隆中存在的TET2或IDH2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会产生TET2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。对4个AITL进行了详细的单细胞IGV基因和TET2突变分析，发现TET2突变的B细胞主要是多克隆B细胞。在第二个资助期，我们的目标是完成AITL的外显子组和全基因组测序研究，以确定进一步的遗传损伤和潜在的致癌病毒。我们将使用可用的小鼠模型来验证所选突变基因的致病作用(目标1)。由于AITL中的几个反复发生的突变影响表观遗传调节因子(TET2，IDH2，DNMT3A)，我们的目标是表征AITL中DNA甲基化的变化，作为本病的进一步发病原理(目标2)。我们的目标是从AITL患者的HSC/HPC建立细胞系，并对这些细胞系进行有针对性的和外显子组测序，以揭示AITL发育的早期阶段已经发生的遗传损伤的模式(AIM 3)。我们还打算利用这些株系来确定TET2突变对HSC/HPC的DNA甲基化和基因表达模式的功能后果(目标4)。最后，我们计划对并发或继发B-NHL的AITL病例进行有针对性的深度测序，通过揭示共同和不同的遗传损害的模式来深入了解这种双重淋巴瘤的发病机制(目标5)。通过确定AITL、它们的前体细胞和相关的B-NHL的遗传和表观遗传格局，我们的目的是在小鼠模型和患者的不同细胞群中全面描述AITL的多步骤转化过程。

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.