【作者】 冯晓莉；

【导师】 吕宁；

【作者基本信息】 北京协和医学院 ， 肿瘤学， 2013， 博士

【摘要】 [目的]本研究旨在探讨中国人群的外周T和NK细胞淋巴瘤(PT/NKCL, Peripheral T-cell lymphoma and NK cell lymphoma)临床、病理和分子生物学特点。[方法]收集中国医学科学院肿瘤医院1999年10月至2011年10月间经病理确诊为外周T和NK细胞淋巴瘤的313例患者进行回顾性研究,按照2008年WHO分类标准重新分类。收集临床资料和石蜡包埋组织块。通过组织形态、组织芯片-免疫组化SP方法检测19种蛋白表达、组织芯片-EBER原位杂交以及BIOMED-2标准化TCR重排等进行多方面研究。使用SPSS18.0软件分析结果。两者比较时p<0.05提示有统计学差异。[结果]本组313例患者共有9种类型,根据本研究组检测结果,最常见5种类型依次为结外NK/T细胞淋巴瘤(ENKT, Extranodal NK/T cell lymphoma)(37.1%)、非特殊类型外周T细胞淋巴瘤(PTCL-NOS, Peripheral T-cell lymphoma-not otherwise specified)(31.3%)、血管免疫母细胞T细胞淋巴瘤(AITL, Angioimmunoblastic T-cell lymphoma)(12.8%), ALK阴性的间变性大细胞淋巴瘤(ALCL, ALK-, Anaplastic large cell lymphoma)(7.3%), ALK阳性的间变性大细胞淋巴瘤(ALCL,ALK+, Anaplastic large cell lymphoma)(4.5%)。患者平均年龄46.0岁,男性多见,男女之比为2.10：1。结外发生超过一半(53.7%), ENKT占结外病变的主体,鼻腔最常见。AITL最多发于结内。根据本研究的形态学观察,结果显示PT/NKCL组织形态学变化较大,瘤细胞谱较广。ENKT中凝固性坏死和血管破坏常见,AITL表现间质分支状高内皮静脉增生和残留淋巴组织带状分布,ALCL见偏位的马蹄形核或肾形核细胞等特点。免疫组化检测结果提示所有类型瘤细胞至少表达一种以上的泛T细胞标志物,但在不同类型表达率不同。六种泛T标志物中CD2和CD3在ENKT表达较高,但是在ALCL, ALK+中表达较低；而在ALCL, ALK+中CD8表达最高达64.3%,在AITL中CD4表达最高达80.0%。细胞毒相关标志物TIA-1和GB在ENKT中表达最高分别为88.8%和72.7%,而perforin在ALCL,ALK+中的表达最高为64.3%。常用于B细胞淋巴瘤诊断的标志物CD10在AITL中高表达(67.5%)。候选标志物中nm23和T0P2A在五种常见类型中的表达相似,除了ENKT以外,阳性率都超过60%。VEGF在ENKT表达最低仅为26.7%,而在AITL中表达最高达70.0%。MUM1在ALCL, ALK-和ALCL, ALK+中的表达均较高,分别为60.9%和64.3%。上面提到的各种标志物在不同类型中表达之间的差异性均显示了统计学意义(p<0.05)。EBER原位杂交检测在ENKT阳性率最高,为87.90%。在AITL中,55.0%的病例非肿瘤细胞有EBER表达,基本上在相应的B细胞表达区,散在少量分布。本研究对63个病例进行了BIOMED-2TCR重排检测。22例样本PCR产物使用聚丙烯酰胺凝胶电泳检测,其中PTCL-NOS的TCR重排阳性率最高为90%；ENKT最低为33.3%。55例样本使用荧光毛细管电泳检测,PTCL-NOS阳性率88.9%,也是最高,ENKT最低为36.3%。其中14例用两种方法检测,每个病例的TCR重排阳性/阴性结果完全一致。[结论]本组中国人群PT/NKCL常见类型依次为ENKT、PTCL-NOS、AITL、ALCL,ALK-和ALCL,ALK+。发病部位多见于结外,男性好发,各类型有具体的年龄偏重。常见类型的免疫组化标志物(包括泛T细胞、细胞毒相关、异常表达的B细胞相关和候选标志物)表达有所不同,建议CD2、CD3、CD56、TIA-1和GB可以作为诊断ENKT的免疫组化标志物组合；CD10在诊断AITL中有应用价值；MUM1在鉴别ALCL, ALK-和PLCL-NOS中有一定帮助。另外原位杂交EBER检测在诊断ENKT和AITL有实际意义。细胞毒相关标志物在ALCL, ALK+高表达,提示ALCL, ALK+的发生和细胞毒T细胞有一定相关性；CD10在AITL的高表达,提示AITL的发生和滤泡辅助T细胞有一定相关性；候选标志物nm23、VEGF和T0P2A在PTCL-NOS、AITL和ALCL中的高表达,提示可能和它们的发生有关,也可能成为这些肿瘤靶向治疗的潜在靶点；VEGF在ENKT和AITL中的表达显示了和形态学特点的一致性；MUM1在PT/NKCL中均有异常表达,而且在ALCL中高表达,提示两者之间可能存在一定相关性,同时也可能成为ALCL靶向治疗的潜在靶点。研究方法上,组织微阵列芯片可以有效应用在PT/NKCL的免疫组化及原位杂交研究中；BIOMED-2标准化引物系统分别结合荧光毛细管电泳和聚丙烯酰胺凝胶电泳进行基因克隆性重排检测,两种方法结果相同,均具有临床应用价值,前者能更高效直观地判定TCR重排结果,适合大型实验室应用,后者更经济适合小型实验室应用。更多还原

【Abstract】 ObjectiveThis study aimed to explore the clinicopathological and molecular biological characteristics of peripheral T and NK cell lymphoma (PT/NKCL) in Chinese population.MethodsThree hundred and thirteen (313) cases of PT/NKCLs diagnosed during the period from1999to2011in a pathology department of one center, Cancer Hospital of Chinese Academy of Medical Sciences, were retrospectively reclassified according to WHO scheme (2008). The clinical data and paraffin-embedded tissue blocks were collected. Tissue microarray-immunohistochemical detection of protein expression, tissue microarray-in situ hybridization as well as PCR BIOMED-2TCR rearrangement was applied. The results were analyzed by SPSS18.0software.Differences were considered statistically significant when2-sided p value was less than0.05.ResultsThis group of313patients were classified into9types, the top5types were extranodal NK/T cell lymphoma (ENKT)(37.1%), peripheral T cell lymphoma-not otherwise specified (PTCL-NOS)(31.3%), angioimmunoblastic T-cell lymphoma (AITL)(12.8%),anaplastic large cell lymphoma, ALK-(ALCL,ALK-)(7.3%),anaplastic large cell lymphoma, ALK+(ALCL, ALK+)(4.5%)The average age of cases was46.0years old. The ratio of male to female was2.10:1. Predominance of extranodal involvement was noted (53.7%). ENKT was the most common extranodal type, while AITL showed mainly nodal diseases.Morphologically, the cytological spectrum was extremely broad. Coagulation necrosis and vascular damage of ENKT, branching high endothelial venule and the ragged distribution of residual lymphocytes of AITL, as well as horse-shoe or kidney-shape nucleus cells of ALCL were common features respectively for each type.Immunohistochemically, at least one pan T marker expressed in tumor cells of all types. But the expression rates were variant in different types. Both CD3and CD2overexpressed in ENKT, while they expressed lower in ALCL, ALK+. The highest expression of CD8occurred in ALCL, ALK+(64.3%). The highest expression of CD4was in AITL (80.0%).Cytotoxic related markers TIA-1and GB were both highest expressions in ENKT, while perforin was highest expression in ALCL. ALK+. CD10often used in B cell lymphoma diagnosis overexpressed in AITL. Mentioned candidate markers, the expressions of nm23and TOP2A were similar in the most common five types. Both of them overexpressed more than60.0%in the common types except ENKT. The VEGF expression was lowest in ENKT (26.7%), while was highest in AITL (70.0%).MUM1expressed in all top five types, and the highest expression was64.3%in ALCL, ALK+followed by60.9%in ALCL, ALK-Generally, the different expressions of above mentioned markers showed statistically significant in different types (p<0.05).The positive rate of EBER in ENKT was up to87.90%.22of40(55.0%) cases of AITL expressed EBER positive singles in non-tumor cells, coexpressing in scattered distribution B cells.TCR rearrangements in63cases were checked by PCR BIOMED-2. DNA productions of22samples were tested with polyacrylamide gel electrophoresis, and55were tested by fluorescence detection with capillary electrophoresis. The highest positive rate of TCR rarrangement in PTCL-NOS was90%with the former method and88.9%with the latter one respectively. The lowest expression was33.3%and36.3%in ENKT using two different methods for PCR product detection.14patients with both methods for DNA detection, the results of TCR rearrangement positive/negative were completely consistent each other.ConclusionThe most common five types of PT/NKCL in Chinese population were ENKT, PTCL-NOS, AITL, ALCL, ALK-and ALCL, ALK+. Each type had a specific age bias. The main occurence in extranodal and male patients were clinical features.Immunohistochemical markers (including Pan T cell, cytotoxic, B cell associated and candidate markers) expressed variant in different types. The group of markers containing CD2, CD3, CD56, TIA-1and GB were recommended to diagnose ENKT. CD10was valuable for AITL. MUM1was useful to make a differential diagnosis between ALCL, ALK-and PTCL-NOS. In addition, EBER routine detection can be applied in the diagnosis of ENKT and AITL.The high expression of cytotoxic associated markers in ALCL, ALK+suggested that ALCL, ALK+may occur from cytotoxic T cells. The overexpression of CD10in AITL may suggest the relationship between AITL and follicular helper T cells. The overexpressions of candidate markers nm23, VEGF and T0P2A in PTCL-NOS, AITL and ALCL might provide some relationships among them. These three markers may be as targets for therapy in the future. The expression characteristics of VEGF indicated the consistence with morphological features in ENKT and AITL. In this research, abnormal expression of MUM1were in PT/NKCL. Furthermore, the overexpression of MUM1was observed in ALCL. So MUM1may play some roles in the development and may become the potencial marker of targeted therapy in ALCL.Tissue microarray can be efficiently used in the PT/NKCL immunohistochemical and in situ hybridization study. Standardization of BIOMED-2primer system respectively combined with fluorescence capillary electrophoresis and polyacrylamide gel electrophoresis for ckecking TCR rearrangement showed the consistent results. So both methods can be used in routine work. The former, which was more efficient and visual, was suitable for large group.The latter, which was more economical, can be used in small lab.更多还原