弥漫性大 B 细胞淋巴瘤中 PRDM 1基因甲基化状态与预后的相关性

摘要

Objective To investigate PRDM1 gene methylation status , immune classification and their prognostic significance in diffuse large B cell lymphoma (DLBCL).Methods Immunohistochemical (IHC) staining for CD20, CD10, bcl-2, bcl-6, PRDM1/Blimp-1 and MUM1 was carried out in 100 cases of DLBCL specimens and 20 reactive lymphoid proliferation samples . All patients were classified into germinal center B cell-like ( GCB) and activated B cell-like ( ABC) subtype according to Hans′algorithmin. PRDM1 gene methylation was detected by methylation-specific PCR ( MSP ) and its relationship with clinicopathologic parameters was analyzed .OCI-Ly1 ( GCB type) and OCI-Ly3 ( ABC type) cell lines were transfected by Small interfering RNA ( siRNA ) with cationic lipid reagent transfection mediated , and the PRDM1/Blimp-1 expression in before and after transfected cell lines were detected with reverse transcription -PCR and Western blot methods .The relationship between PRDM 1 gene methylation , clinicopathologic parameter and survival was analyzed using one-way analysis of variance .Results One hundred patients were classified into 73 (73%) cases of GCB subtypes and 27 (27%) cases of ABC.PRDM1/Blimp-1 was expressed in 21 DLBCL and highly expressed in 20 reactive lymphoid proliferation .PRDM1 gene methylation was detected in 23%(23/100) of DLBCL, while no methylation was detected in all 20 reactive lymphoid proliferation.The difference of the PRDM1 methylation status between DLBCL and the control samples was statistically significant (P=0.004).However, there was no significant correlation between the PRDM 1 gene methylation and clinicopathologic parameters ( P >0.05 ) .Reverse transcription-PCR and Western blot showed that PRDM1 gene expression was reduced in siRNA-induced group compared with blank control group and negative control group .One-way analysis of variance revealed that aged ≥60 years, performance status score above 3, and the presence of general symptoms were associated with significantly lower overall survival rate.Conclusions PRDM1 gene silencing with aberrant CpG methylation is probably one of the critical events in the oncogenesis of DLBCL .This may have important implications as a candidate marker for diagnosis and targeted gene therapy .Meanwhile in vitro siRNA transfected OCI-Ly1 and OCI-Ly3 cell lines confirm that PRDM1 gene is a suppressor gene in DLBCL and may represent a novel therapeutic target .%目的：探讨弥漫性大B细胞淋巴瘤（ DLBCL）组织中PRDM1基因启动子甲基化状态与免疫分型及预后的相关性，初步揭示该基因甲基化在DLBCL发生发展中的作用机制。方法选取初治DLBCL石蜡包埋组织100例，反应性增生淋巴结20例作为对照。免疫组织化学EnVision法检测CD20、CD10、bcl－6、bcl－2、PRDM1／Blimp－1、MUM1蛋白表达，根据Hans分型法分型。甲基化特异性聚合酶链反应（ MSP）法检测肿瘤组及对照组中PRDM1基因启动子甲基化状态并分析其与临床特征之间的关系。应用阳离子脂质体试剂转染法介导的小干扰RNA转染OCI－Ly1（ GCB型）和OCI－Ly3（ ABC型）细胞系，逆转录PCR和Western blot方法检测siRNA转染前后细胞系中PRDM1／Blimp－1的表达情况。单因素生存分析（ Kaplan－Meier）法分析PRDM1基因启动子甲基化及各临床特征与生存期之间的关系。结果100例DLBCL 患者中， GCB 型27例（27％）， ABC 型73例（73％）。 PRDM1／Blimp－1在DLBCL组阳性表达21例（21％），反应性增生淋巴结中广泛阳性（100％）。 PRDM1基因启动子甲基化在肿瘤组共有23例（23％），而对照组中均为非甲基化，两者之间差异有统计学意义（P＝0．004）。 PRDM1基因启动子甲基化在不同年龄段、性别、Ann Arbor分期、血清乳酸脱氢酶水平、国际预后指数、免疫分型等临床病理参数之间无明显相关性。沉默表达siRNA转染细胞系，逆转录PCR和Western blot结果显示PRDM1／Blipm－1在转染组表达较空白对照组及阴性对照组下降。单因素生存分析显示在年龄≥60岁、功能状态（ PS ）评分3～4分、有B症状组DLBCL患者生存率明显较低。结论在DLBCL患者中存在PRDM1基因启动子区CpG岛甲基化，由其所致的PRDM1基因表达沉默或减少可能是DLBCL发生的一个重要因素。体外沉默siRNA转染OCI－Ly1和OCI－Ly3细胞系，证实PRDM1基因在DLBCL中起到抑癌基因作用，PRDM1基因甲基化有望作为一个候选分子诊断标志物及可能的治疗靶点。