摘要目的 探讨儿童神经母细胞瘤(neuroblastoma,NB)ALK蛋白表达与ALK基因遗传学及表观遗传学改变的相关性,并分析NB的ALK蛋白表达异常及ALK基因异常的临床病理学意义.方法 培养三个NB细胞株,其中两株为ALK阳性表达(SH-SY5Y和SK-N-SH),一株为ALK阴性表达(SK-N-AS),并收集43例NB石蜡包埋组织标本及相应临床资料;运用免疫组织化学EnVision法检测NB细胞株及石蜡包埋组织标本的ALK蛋白表达;运用荧光原位杂交(FISH)检测ALK基因的拷贝数;运用PCR技术、Sanger测序技术检测ALK基因点突变;运用重亚硫酸盐测序技术(BSP)检测ALK基因启动子区CpG岛的甲基化状态.结果 SH-SY5Y和SK-N-SH细胞株的ALK蛋白表达于胞质,而SK-N-AS无ALK蛋白表达;43例神经母细胞瘤组织中,ALK蛋白阳性者26例(60.5％,26/43),阴性者17例(39.5％,17/43),ALK蛋白表达与患者的生存期相关,ALK蛋白阳性表达者,生存期较短(P =0.020),但ALK蛋白表达与NB的分化状态(P=0.503)、发病部位(P=1.000)及年龄(P =0.063)无相关性.43例NB中,ALK基因扩增者2例(4.6％,2/43),ALK基因获得者30例(69.7％,30/43),余者ALK基因正常(25.6％,11/43),ALK基因多拷贝(包括扩增和获得)与ALK蛋白阳性表达有明显相关性(P =0.020),ALK基因多拷贝倾向ALK蛋白的阳性表达;ALK基因多拷贝与NB的分化状态(P=1.000)、发病部位(P =0.775)及年龄(P =0.328)无相关性.三个NB细胞株中均未发现ALK基因的点突变,43例NB组织中,仅发现1例(2.3％,1/43)存在ALK基因的点突变[第23号外显子同义突变A1200A(G4552C)],其ALK蛋白呈阴性表达,在诊断后2个月死亡;BSP检测显示,三株细胞ALK基因启动子区CpG岛均未甲基化,6例NB患者(其中3例ALK阳性,3例ALK阴性)ALK基因启动子区CpG岛也均未甲基化.结论 大部分NB组织中ALK蛋白表达阳性,ALK蛋白阳性表达者提示不良预后.ALK蛋白阳性表达与ALK基因多拷贝相关,但与ALK基因启动子区甲基化无明显相关性.ALK基因多拷贝是NB中比较常见的遗传学改变.ALK基因点突变率很低,有可能提示不良预后.

abstractsObjective To correlate the abnormal expression of anapastic lymphoma kinase (ALK) protein with the genetic and epigenetic changes of ALK,and to analyze its clinical application in pediatric neuroblastoma.Methods Three neuroblastoma (NB) cell lines (two ALK positive:SH-SY5Y and SK-N-SH,one ALK negative:SK-N-AS) and 43 paraffin-embedded NB tissues were included in the study.In both cell lines and clinical cases,immunohistochemistry was used to detect ALK protein expression; PCR and Sanger sequencing were used to detect ALK point mutation; fluorescence in situ hybridization (FISH)was used to detect ALK abnormality and bisulfite sequencing PCR (BSP) was used to detect methylation of CpG island in the promoter area of ALK.Results The cell lines SH-SY5Y and SK-N-SH were positive for ALK expression (cytoplasm),while the SK-N-AS was negative; among the 43 cases of NB,26 (60.5％,26/43) were positive for ALK protein (membrane and cytoplasm),and the rest were negative.Survival analysis showed ALK protein expression was related to survival time,with ALK positive cases having shorter survival time than ALK negative cases (P =0.020).But ALK protein expression had no association with tumor differentiation (P =0.503),tumor sites (P =1.000) and age of patients (P =0.063).FISH showed ALK amplification in two cases (4.6％,2/43),ALK gain was found in 30 cases (69.7％,30/43),and the remaining cases had normal ALK copy (25.6％,11/43).The presence of extra copies (amplification and gain) of ALK was associated with ALK positive protein expression (P =0.020),but there was no association with tumor differentiation (P =1.000),tumor sites (P =0.775) and age of patients (P =0.328).No point mutation was found in all three cell lines.Of the 43 NB cases,only one case (2.3％,1/43) showed point mutation in exon 23,and was a synonymous mutation [A1200A (G4552C)].The case was ALK negative,but the patient died two months after diagnosis.BSP analysis showed that CpG island in ALK promoter region were all unmethylated in three cell lines and 6 NB cases (including 3 ALK positive,3 ALK negative).Conclusions ALK protein is expressed in most NB,and the expression indicates poor outcome.ALK expression is associated with extra copies of ALK,but there is no association with the methylation status of CpG island of ALK; the presence of extra copies of ALK is the most common genetic aberration in NB.Point mutation of ALK is rare,and may predict poor prognosis in pediatric NB.