中华病理学杂志
中華病理學雜誌
중화병이학잡지
Chinese Journal of Pathology
2014年
8期
541-545
,共5页
神经母细胞瘤%点突变%聚合酶链反应
神經母細胞瘤%點突變%聚閤酶鏈反應
신경모세포류%점돌변%취합매련반응
Neuroblastoma%Point mutation%Polymerase chain reaction
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.
目的 探討兒童神經母細胞瘤(neuroblastoma,NB)ALK蛋白錶達與ALK基因遺傳學及錶觀遺傳學改變的相關性,併分析NB的ALK蛋白錶達異常及ALK基因異常的臨床病理學意義.方法 培養三箇NB細胞株,其中兩株為ALK暘性錶達(SH-SY5Y和SK-N-SH),一株為ALK陰性錶達(SK-N-AS),併收集43例NB石蠟包埋組織標本及相應臨床資料;運用免疫組織化學EnVision法檢測NB細胞株及石蠟包埋組織標本的ALK蛋白錶達;運用熒光原位雜交(FISH)檢測ALK基因的拷貝數;運用PCR技術、Sanger測序技術檢測ALK基因點突變;運用重亞硫痠鹽測序技術(BSP)檢測ALK基因啟動子區CpG島的甲基化狀態.結果 SH-SY5Y和SK-N-SH細胞株的ALK蛋白錶達于胞質,而SK-N-AS無ALK蛋白錶達;43例神經母細胞瘤組織中,ALK蛋白暘性者26例(60.5％,26/43),陰性者17例(39.5％,17/43),ALK蛋白錶達與患者的生存期相關,ALK蛋白暘性錶達者,生存期較短(P =0.020),但ALK蛋白錶達與NB的分化狀態(P=0.503)、髮病部位(P=1.000)及年齡(P =0.063)無相關性.43例NB中,ALK基因擴增者2例(4.6％,2/43),ALK基因穫得者30例(69.7％,30/43),餘者ALK基因正常(25.6％,11/43),ALK基因多拷貝(包括擴增和穫得)與ALK蛋白暘性錶達有明顯相關性(P =0.020),ALK基因多拷貝傾嚮ALK蛋白的暘性錶達;ALK基因多拷貝與NB的分化狀態(P=1.000)、髮病部位(P =0.775)及年齡(P =0.328)無相關性.三箇NB細胞株中均未髮現ALK基因的點突變,43例NB組織中,僅髮現1例(2.3％,1/43)存在ALK基因的點突變[第23號外顯子同義突變A1200A(G4552C)],其ALK蛋白呈陰性錶達,在診斷後2箇月死亡;BSP檢測顯示,三株細胞ALK基因啟動子區CpG島均未甲基化,6例NB患者(其中3例ALK暘性,3例ALK陰性)ALK基因啟動子區CpG島也均未甲基化.結論 大部分NB組織中ALK蛋白錶達暘性,ALK蛋白暘性錶達者提示不良預後.ALK蛋白暘性錶達與ALK基因多拷貝相關,但與ALK基因啟動子區甲基化無明顯相關性.ALK基因多拷貝是NB中比較常見的遺傳學改變.ALK基因點突變率很低,有可能提示不良預後.
목적 탐토인동신경모세포류(neuroblastoma,NB)ALK단백표체여ALK기인유전학급표관유전학개변적상관성,병분석NB적ALK단백표체이상급ALK기인이상적림상병이학의의.방법 배양삼개NB세포주,기중량주위ALK양성표체(SH-SY5Y화SK-N-SH),일주위ALK음성표체(SK-N-AS),병수집43례NB석사포매조직표본급상응림상자료;운용면역조직화학EnVision법검측NB세포주급석사포매조직표본적ALK단백표체;운용형광원위잡교(FISH)검측ALK기인적고패수;운용PCR기술、Sanger측서기술검측ALK기인점돌변;운용중아류산염측서기술(BSP)검측ALK기인계동자구CpG도적갑기화상태.결과 SH-SY5Y화SK-N-SH세포주적ALK단백표체우포질,이SK-N-AS무ALK단백표체;43례신경모세포류조직중,ALK단백양성자26례(60.5％,26/43),음성자17례(39.5％,17/43),ALK단백표체여환자적생존기상관,ALK단백양성표체자,생존기교단(P =0.020),단ALK단백표체여NB적분화상태(P=0.503)、발병부위(P=1.000)급년령(P =0.063)무상관성.43례NB중,ALK기인확증자2례(4.6％,2/43),ALK기인획득자30례(69.7％,30/43),여자ALK기인정상(25.6％,11/43),ALK기인다고패(포괄확증화획득)여ALK단백양성표체유명현상관성(P =0.020),ALK기인다고패경향ALK단백적양성표체;ALK기인다고패여NB적분화상태(P=1.000)、발병부위(P =0.775)급년령(P =0.328)무상관성.삼개NB세포주중균미발현ALK기인적점돌변,43례NB조직중,부발현1례(2.3％,1/43)존재ALK기인적점돌변[제23호외현자동의돌변A1200A(G4552C)],기ALK단백정음성표체,재진단후2개월사망;BSP검측현시,삼주세포ALK기인계동자구CpG도균미갑기화,6례NB환자(기중3례ALK양성,3례ALK음성)ALK기인계동자구CpG도야균미갑기화.결론 대부분NB조직중ALK단백표체양성,ALK단백양성표체자제시불량예후.ALK단백양성표체여ALK기인다고패상관,단여ALK기인계동자구갑기화무명현상관성.ALK기인다고패시NB중비교상견적유전학개변.ALK기인점돌변솔흔저,유가능제시불량예후.
Objective 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.
