CAJ | 학술논문

目的研究中国胶质瘤人群中异柠檬酸脱氢酶(IDH)1和IDH2的基因突变情况,及其与p53和表皮生长因子受体(EGFR)的关系,为胶质瘤的诊断、鉴别诊断提供依据,探讨其在肿瘤发生和评估患者预后中的价值.方法对234例胶质瘤石蜡包埋标本及同时收集到的30例血液标本进行检测,提取DNA用PCR-Sanger测序来检测IDH1和IDH2的基因突变情况,同时应用免疫组织化学EnVision二步法检测IDH1 R132H、p53和EGFR的蛋白表达情况.在IDH突变的少突胶质细胞肿瘤中,采用免疫荧光双重标记IDH1R132H和胶质纤维酸性蛋白(GFAP),对IDH1R132H的蛋白表达进行细胞定位.结果 (1) 234例胶质瘤的石蜡标本,基因测序仅检测出IDH1杂合性突变,且均为p:Arg132His(c:G395A)型,未检测出IDH2突变.突变率为31.6％ (74/234),其中少突星形细胞瘤(9/13)、间变型少突星形细胞瘤(7/11)、少突胶质细胞瘤(18/26,69.2％)和间变型少突胶质细胞瘤(8/10)中IDH1突变比例高于弥漫型星形细胞瘤(17/47,36.2％)、间变型星形细胞瘤(5/18)和胶质母细胞瘤(14.5％,10/69)；星形细胞肿瘤中,随着肿瘤级别升高,其突变率呈线性下降(P=0.007)；胶质母细胞瘤中,继发性胶质母细胞瘤突变比例高于原发性胶质母细胞瘤[5/14比5/55(9.1％),P =0.036]；而血液标本均未见突变；(2)抗体IDH1R132H表达于细胞质,能特异地识别肿瘤细胞,且免疫组织化学染色与基因测序结果高度一致(P=0.001)；(3)p53蛋白在IDH突变的弥漫型星形细胞瘤、间变型星形细胞瘤及继发性胶质母细胞瘤中表达率高,与IDH突变呈高度正相关(P值分别为0.007、0.026、0.038)；(4) EGFR蛋白表达与IDH突变无相关性.结论 IDH突变是高发于胶质瘤早期阶段的体细胞杂合性突变,可首选免疫组织化学标记抗体IDH1R132H快速检测；IDH突变与p53和EGFR联合应用和分析有助于胶质瘤的诊断和鉴别诊断.
목적 연구중국효질류인군중이저몽산탈경매(IDH)1화IDH2적기인돌변정황,급기여p53화표피생장인자수체(EGFR)적관계,위효질류적진단、감별진단제공의거,탐토기재종류발생화평고환자예후중적개치.방법 대234례효질류석사포매표본급동시수집도적30례혈액표본진행검측,제취DNA용PCR-Sanger측서래검측IDH1화IDH2적기인돌변정황,동시응용면역조직화학EnVision이보법검측IDH1 R132H、p53화EGFR적단백표체정황.재IDH돌변적소돌효질세포종류중,채용면역형광쌍중표기IDH1R132H화효질섬유산성단백(GFAP),대IDH1R132H적단백표체진행세포정위.결과 (1) 234례효질류적석사표본,기인측서부검측출IDH1잡합성돌변,차균위p:Arg132His(c:G395A)형,미검측출IDH2돌변.돌변솔위31.6％ (74/234),기중소돌성형세포류(9/13)、간변형소돌성형세포류(7/11)、소돌효질세포류(18/26,69.2％)화간변형소돌효질세포류(8/10)중IDH1돌변비례고우미만형성형세포류(17/47,36.2％)、간변형성형세포류(5/18)화효질모세포류(14.5％,10/69)；성형세포종류중,수착종류급별승고,기돌변솔정선성하강(P=0.007)；효질모세포류중,계발성효질모세포류돌변비례고우원발성효질모세포류[5/14비5/55(9.1％),P =0.036]；이혈액표본균미견돌변；(2)항체IDH1R132H표체우세포질,능특이지식별종류세포,차면역조직화학염색여기인측서결과고도일치(P=0.001)；(3)p53단백재IDH돌변적미만형성형세포류、간변형성형세포류급계발성효질모세포류중표체솔고,여IDH돌변정고도정상관(P치분별위0.007、0.026、0.038)；(4) EGFR단백표체여IDH돌변무상관성.결론 IDH돌변시고발우효질류조기계단적체세포잡합성돌변,가수선면역조직화학표기항체IDH1R132H쾌속검측；IDH돌변여p53화EGFR연합응용화분석유조우효질류적진단화감별진단.
Objective To investigate mutation status of isocitrate dehydrogenase (IDH) 1 and IDH2 genes in Chinese patients with gliomas in correlation with clinicopathological characteristics.Methods Formalin-fixed and paraffin-embedded (FFPE) tissue samples of 234 gliomas were collected including the matched blood samples in 30 patients.DNA was extracted,followed by PCR-Sanger sequencing to detect IDH1 and IDH2 gene mutations.Immunohistochemistry was performed using mutation-specific antibody recognizing IDH1R132H mutation.Immunostains for p53 and epidermal growth factor receptor (EGFR) were also performed.Oligodendroglial tumors with IDH mutation were double stained with IDH1R132H and GFAP by immunofluorescence to investigate the location of IDH1 R132H expression.Results (1) By IDH1 heterozygous somatic mutation analysis,Arg132His (c:G395A) was found in 31.6％ (74 of 234) of the cases.IDH mutations were more frequent in oligoastrocytomas (9/13),anaplastic oligoastrocytomas (7/11),oligodendrogliomas (18/26,69.2％),anaplastic oligodendrogliomas (8/10),and less frequent in diffuse astrocytomas (17/47,36.2％),anaplastic astrocytomas (5/18),and glioblastomas (10/69,14.5％).The mutation rate inversely correlated with the tumor grade in a linear fashion in astrocytic tumors (P =0.007).Primary glioblastomas were characterized by a lower frequency of mutations than secondary glioblastomas (5/55 vs.5/14,P =0.036); IDH mutation was not detected in pilocytic astrocytoma and ependymoma.No IDH2 mutation was identified in this study cohort.(2) Immunohistochemistry of IDH1R132H demonstrated a strong cytoplasmic staining in 80 cases,which was highly correlated with IDH mutation status (P =0.001).IDH1R132H was highly specific to tumor cells.(3) p53 immunostain was significantly correlated the IDH mutation in diffuse astrocytoma,anaplastic astrocytoma and secondary glioblastomas (P =0.007,0.026,0.038 respectively).(4) No correlation between EGFR and IDH mutation was found.Conclusions High prevalence of IDH heterozygous somatic mutation occurs in the earlier stage of gliomas,which can be detected by mutation-specific antibody IDH1R132H.Furthermore,evaluation of p53 and EGFR expression combined with IDH mutation analysis may significantly aid in the diagnosis and differential diagnoses of gliomas in Chinese patients.