陈海泉教授
经3年多临床攻关，复旦大学附属肿瘤医院陈海泉教授领衔的肺癌转化性研究课题组，发明了一种能够快速而准确检测出携带“ALK融合基因”的肺癌分子诊断技术，相关论文已发表在国际著名肿瘤学期刊《临床癌症研究》上。国际权威学术杂志《癌症研究》将该成果誉为“突破性进展”，并专门刊文报道。
肺癌分为小细胞肺癌和非小细胞肺癌，“ALK融合基因”是癌基因，存在于3%～7%的非小细胞肺癌中，以该癌基因为靶点的分子靶向药物Crizotinib可显著提高肺癌患者的生存率。但如何快速而准确地诊断出携带“ALK融合基因”的肺癌，一直是困扰临床医生和研究者的世界性难题。目前，国际上“ALK融合基因”检测技术复杂，至少需2天时间完成，每例价格1500美元。
陈海泉在一次研究中意外发现，当“ALK融合基因”发生断裂后，人体内的“激酶域”表达会显著增高，而“非激酶域”则不表达或低表达。根据这一特点，陈海泉教授课题组创造性地发明了一种“实时定量的ALK融合基因检测”新技术，通过检测“ALK融合基因”断裂点前后ALK基因的表达水平差异，快速而准确地诊断出ALK融合基因。经验证，应用该新技术对950例非小细胞肺癌标本的检测结果发现了40例携带“ALK融合基因”的阳性标本，敏感性、特异性均达到100%。此外，该技术还具有高通量、低成本等优势，可在90分钟内完成48例样本的检测，而每例样本的检测成本不超过30元人民币。同时，该技术所需的仪器设备极为普通、简单，易于在各级医疗科研单位中普及推广。
据悉，为表彰陈海泉的杰出贡献，美国胸科医师学院已决定，在即将举行的2012年年会上，授予其“阿尔弗雷德・索弗研究奖”。
The Use of Quantitative Real-Time Reverse Transcriptase PCR for 5′ and 3′ Portions of ALK Transcripts to Detect ALK Rearrangements in Lung Cancers
Rui Wang, Yunjian Pan, Chenguang Li, Haichuan Hu, Yang Zhang, Hang Li, Xiaoyang Luo, Jie Zhang, Zhaoyuan Fang, Yuan Li, Lei Shen, Hongbin Ji, David Garfield, Yihua Sun, and Haiquan Chen
Purpose: Approximately 3% to 7% of non–small cell lung cancers (NSCLC) harbor an ALK fusion gene, thus defining a tumor group that may be responsive to targeted therapy. The breakpoint in ALK consistently occurs at exon 20 and EML4 or other fusion partners, thus driving a strong expression of ALK kinase domain and resulting in an unbalanced expression in 5′ and 3′ portions of ALK transcripts. We have developed a rapid and accurate method by simultaneously detecting the expression in 5′ and 3′ portions of ALK mRNA.
Experimental Design: Quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to examine expression levels of the 5′ and 3′ portions of ALK transcripts in177 NSCLCs, in which EGFR, KRAS, HER2, and BRAF mutations were absent. If unbalanced ALK mRNA expression was seen, ALK rearrangement was assumed to exist. ALK FISH was used to confirm the accuracy of qRT-PCR. RT-PCR and 5′ RACE coupling sequencing identified the fusion variants.
Results: Real-time RT-PCR showed excellent sensitivity and specificity (100% and 100%, respectively) for detection of ALK rearrangements in resected specimens. In addition, six novel ALK fusion variants were identified, including one KIF5B-ALK (E17;A20) and five EML4-ALK variants (E6a;A19, E6a/b ins 18;A20, E17b ins 39;A20, E10a/b, E13;A20, and E17 ins 65;A20).
Conclusions: Real-time RT-PCR is a rapid and accurate method for diagnosing ALK-rearranged lung cancers. Coupling of 5′ RACE to this method should further facilitate rapid identification of novel ALK fusion genes. Clin Cancer Res; 18(17); 4725–32. ©2012 AACR.
