成熟T／NK细胞淋巴瘤中鼠类肉瘤滤过性病毒致癌同源体B1基因突变和表达的检测及意义

Detection and significance of BRAF gene in mature T/NK cell lymphoma

目的研究成熟T／NK细胞淋巴瘤组织以及细胞系中鼠类肉瘤滤过性病毒致癌同源体B1（BRAF）基因突变和表达情况，探讨BRAF基因的表达与成熟T／NK细胞淋巴瘤临床病理特征和临床疗效的关系。方法采用直接测序法检测Jurkat细胞、Hut-78细胞、YTS细胞、正常外周血淋巴细胞、58例不同类型成熟T／NK细胞淋巴瘤组织和29例反应性增生淋巴结组织中BRAF基因第15号外显子的1799位点和第11号外显子的463、465和468位点突变情况。采用实时荧光定量PCR和Westernblot法检测Jurkat细胞、Hut-78细胞、YTS细胞和正常外周血淋巴细胞中BRAFmRNA和蛋白的表达情况。采用免疫组化方法检测成熟T／NK细胞淋巴瘤组织和反应性增生淋巴结组织中BRAF蛋白的表达，分析BRAF基因的表达与非特指型外周T细胞淋巴瘤患者临床病理特征和疗效的关系。结果成熟T／NK细胞淋巴瘤组织和细胞系中BRAF基因均未出现第15号外显子的1799位点和第11号外显子的463、465和468位点突变。正常淋巴细胞、YTs细胞、Hut-78细胞和Jurkat细胞中BRAFmRNA的相对表达量分别为1．000、5．207±0．013、8．412±0．615和36．720±1．797，Jurkat细胞中BRAFmRNA的相对表达量明显高于正常淋巴细胞、Hut-78细胞和YTS细胞（P〈0．01）。正常淋巴细胞、YTS细胞、Hut-78细胞和Jurkat细胞中BRAF蛋白的相对表达量分别为0．051±0．003、0．102±0．013、0．113±0．017和0．304±0．010，Jurkat细胞中BRAF蛋白的相对表达量高于正常淋巴细胞、Hut-78细胞和YTS细胞（P〈0．05）。58例不同亚型成熟T／NK细胞淋巴瘤组织中，仅有6例（10．3％）呈BRAF蛋白阳性表达，且全部为非特指型外周T细胞淋巴瘤。BRAF蛋白表达与非特指型外周T细胞淋巴瘤患者的性别、年龄、B症状、临床分期、血清乳酸脱氢酶水平均无关（均P〉0．05）。BRAF蛋白在治疗有效组中的阳性率（8．3％）明显低于治疗无效组（55．6％，P=0．046）。结论BRAF基因的表达有可能成为外周T细胞淋巴瘤恶性生物学特性的标志物和临床治疗靶点。

Objective we aimed to investigate the mutation and expression of BRAF gene in mature T/NK cell lymphoma tissues and cell lines, explore the correlation between gene alterations and elinicopathological features and clinical outcomes of mature T/NK cell lymphoma. Methods Firstly, we detected common mutant sites of BRAF （locus 1 799 mutation in exon 15 and loci 463, 465 and 468 mutation in exon 11 ） in lymphoma Jurkat, Hut-78 and YTS cell lines, normal peripheral blood lymphocytes, different types of mature T/NK cell lymphoma and reactive hyperplasia lymph nodes by direct sequencing. Then we measured the expression of BRAF in Jurkat, Hut-78, YTS ceils and normal peripheral blood lymphocytes by real time-PCR and Western-blot detection. We also used immunohistochemistry （IHC） to detect the expression of BRAF in mature T/NK cell lymphoma tissues and reactive hyperplasia lymph nodes,and to analyze the correlation between the expression of BRAF and clinocopathological features and clinical outcomes. Results We did not find common BRAF mutation in mature T/NK cell lymphoma tissues and cell lines, and the relatively expression of BRAF gene mRNA in normal peripheral blood lymphocytes, YTS, Hut-78 and Jurkat cells were 1.000, 5.207±0.013,8.412±0.615 and 36.720± 1.797, respectively, and protein expressions were 0.051 ±0.003,0.102±0.013,0.113 ± 0.017 and 0.304±0.010, respectively, and the expression of BRAF in peripheral T cell lymphoma Jurkat cells was significantly higher than that of Hut-78, YTS cells and normal lymphocytes （P〈0.05）. Only 6 of 58 peripheral T cell lymphomas （10.3%） had positive BRAF expression, and were the subgroups of peripheral T cell lymphoma-unspecified type. The statistical data did not show any correlation between positive expression of BRAF and gender, age, clinical stage, location, lactate dehydrogenase in the 21 cases of peripheral T cell lymphoma-unspecified type （P〈 0.05）, but the positive rate of BRAF in the effective treatment group （8.3%） was significantly lower than that of the invalid group （55.6%, P〈 0.05 ）. Conclusion The expression of BRAF gene may become a marker of malignant biological characteristics and clinical therapeutic target of peripheral T cell lymphoma.