Molecular Profiling of MALT lymphoma at different anatomical sites

博士项目战略主题：生物科学促进对健康的综合认识粘膜相关淋巴组织(MALT)淋巴瘤是边缘带B细胞淋巴瘤最常见的亚型，是第三大最常见的B细胞淋巴瘤。它发生在胃、甲状腺、肺、眼附件和小肠等不同的解剖部位。仅对少数MALT淋巴瘤亚型进行了外显子组测序(WES)。这种突变图谱与靶向小组测序一起确定了大量不同的基因变化，这些变化在不同的解剖部位以不同的频率发生。其中一个遗传学发现是程序性死亡配体1(PD-L1或CD274)，它优先出现在甲状腺麦芽淋巴瘤中，其中很大一部分突变是有害的。在我为期3个月的轮换项目中，为了进一步表征这些PD-L1突变在甲状腺麦芽中的作用，我试图使用MLPA分析来调查PD-L1是否也是甲状腺麦芽中染色体缺失的靶标，以评估PD-L1潜在的拷贝数变异。我调查了35个甲状腺麦芽病例，发现了甲状腺麦芽中PD-L1和JAK2的频繁缺失。这些发现将使我能够将样本中发现的缺失与现有的下一代测序(NGS)数据以及患者的临床数据相关联。为了进一步研究PD-L1的作用，建立PD-L1和PD-1的表达水平以及这两种蛋白和不同免疫细胞在甲状腺MALT淋巴瘤肿瘤微环境中相互作用的水平是至关重要的。这可以通过对带有和不带有PD-L1缺失或有害突变的甲状腺MALT切片的B细胞和T细胞标记物以及PD-1和PD-L1的免疫荧光染色来进行，这为我们进一步了解MALT淋巴瘤的肿瘤微环境和PD-L1失活的潜在意义提供了进一步的了解。杜氏实验室最近发表了一项研究，该小组已经成功地对相对大量的MALT淋巴瘤病例进行了NGS，尽管由于当时样本有限，他们只能对13例甲状腺MALT样本进行WES。目前，杜氏实验室有76份甲状腺MALT样本已经使用93基因组进行了测序，尽管基因板可以提供大量信息，但由于与WES相反的基因组面积较小，它们只能涵盖疾病突变概况的一部分。因此，为了进一步阐明MALT淋巴瘤的不同基因突变，我将首先对已经使用靶向测序小组进行分析的甲状腺MALT淋巴瘤进行WES，重点关注没有PD-L1突变的甲状腺MALT淋巴瘤，以进一步描述甲状腺MALT淋巴瘤的遗传格局。此外，通过对WES结果的分析，可以创建一个基因小组，用于分析MALT淋巴瘤的不同解剖位置，重点放在对甲状腺MALT样本进行的WES中发现的复发和潜在致病突变上。

PhD project strategic theme: Bioscience for an integrated understanding of healthMucosa-associated lymphoid tissue (MALT) lymphoma is the most common subtype of Marginal Zone B cell lymphoma, and the third most frequent B cell lymphoma. It arises in diverse anatomic sites including the stomach, thyroid, lung, ocular adnexa and small intestine.Whole exome sequencing (WES) has been carried out only on a small number of MALT lymphoma subtypes. This mutation profiling together with targeted panel sequencing has identified a large number of distinct genetic changes which occur at variable frequency at different anatomic sites. One of these genetic findings is Programmed death-ligand 1 (PD-L1 or CD274) which preferentially occurs in thyroid MALT lymphoma, with a large percentage of the mutations being deleterious.To further characterise the role of these PD-L1 mutations in thyroid MALT in my 3-month rotation project I sought to investigate whether PD-L1 is also targeted by chromosomal deletions in thyroid MALT using an MLPA assay to assess the potential copy number variation of PD-L1. I investigated 35 thyroid MALT cases and identified frequent deletions of PD-L1 and JAK2 in thyroid MALT. These findings will allow me to correlate the deletions identified in the samples with already existing next generation sequencing (NGS) data as well as clinical data from the patients.To further investigate the role of PD-L1 it would be vital to establish the expression levels of PD-L1 and PD-1, and the level at which the two proteins and the different immune cells present interact in the tumour micro-environment of thyroid MALT lymphoma. This can be undertaken through immunofluorescent staining of B cell and T cell markers as well as PD-1 and PD-L1 of thyroid MALT sections with and without identified PD-L1 one deletions or deleterious mutations, providing us with further insight into the tumour microenvironment of MALT lymphoma and the potential significance of the PD-L1 inactivation.The Du laboratory has recently published a study where the group have successfully undertaken NGS on a relatively large number of MALT lymphoma cases, although due to the limited samples they had at the time they could only undertake WES of 13 samples of thyroid MALT.Currently the Du laboratory has 76 thyroid MALT samples which have been sequenced using a 93-gene panel, although gene panels can provide a vast amount of information they will only cover a part of the mutational profile of the disease due to the reduced area of the genome being analysed in antithesis to WES. Thus, to further elucidate the different genetic mutations in MALT lymphoma, I will firstly undertake WES of thyroid MALT lymphomas which have already been analysed using a targeted sequencing panel, focusing on thyroid MALT lymphomas which do not have PD-L1 mutations, in order to further characterise the genetic landscape of thyroid MALT lymphoma. Moreover, upon analysis of the WES results, a gene panel which can be used to analyse different anatomic sites of MALT lymphoma can be created focusing on recurrent and potentially pathogenic mutations identified in the WES undertaken on the thyroid MALT samples.