ALK阳性非小细胞肺癌的免疫组化染色筛查以及EML4-ALK融合亚型异质性的研究

发布时间：2017-12-27 03:32

本文关键词：ALK阳性非小细胞肺癌的免疫组化染色筛查以及EML4-ALK融合亚型异质性的研究　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：过去十年肺癌患者人数快速增长,2015年WHO更新了肺癌分类,强调肿瘤基因改变与组织学诊断同等重要,以驱动基因靶向治疗引领了精准医疗的潮流,必将带来越来越多的具有临床意义的潜在基因的分子检测技术发展。靶向药物治疗间变性淋巴瘤激酶(ALK)基因融合非小细胞肺癌(NSCLC)患者的疗效显著优于传统化疗,准确诊断ALK阳性NSCLC为精准治疗的关键,目前中国分子检测开展尚处于初期阶段,仅少数医院能完成分子诊断,ALK阳性NSCLC检测方法和诊断流程仍需要不断结合临床现状进一步优化。目的:本研究采用多种方法筛选ALK阳性NSCLC,分析其临床病理特征和EML4-ALK融合亚型的异质性,提出一套适合中国国情的诊断流程。方法:(1)收集566例NSCLC石蜡包埋组织,采用Ventana全自动免疫组化染色(Ventana-IHC)、荧光原位杂交(FISH)和序列特异性PCR技术(RT-PCR)方法检测ALK状态,分析Ventana-IHC染色的着色模式,总结ALK阳性NSCLC临床病理特征及治疗预后。(2)采用Ventana-IHC和手工IHC检测60例NSCLC,对比4种不同 ALK抗体D5F3(Ventana)、D5F3(Cell Signaling)、1A4/1H7(OriGene)、5A4(Abcam)联合手工IHC检测ALK染色情况。(3)采用RT-PCR和测序检测EML4-ALK的融合亚型,分析不同亚型的临床病理特征及临床意义。结果:(1)Ventana-IHC 筛查 ALK 阳性 NSCLC 的检出率为 6.7%(38/566),与FISH和PCR结果一致,36例Ventana-IHC异常染色模式经验证为ALK融合阴性。年龄≤60岁患者阳性率(10.6%)高于60岁组(3.5%),有统计差异(P0.05)。无性别差异,不吸烟者多见(78.9%),仅1例合并EGFR基因突变。组织形态上,81.6%为腺癌,其中实体型伴黏液产生多见(47.4%),非腺癌包括3例鳞癌、3例腺鳞癌及1例多形性癌。克唑替尼治疗缓解率为80%。(2)采用手工IHC方法,4 种抗体 D5F3(Ventana)、D5F3(Cell Signaling)、1A4/1H7、5A4 染色敏感性分别为 93.8%、84.4%、93.8%、56.3%,特异性均 100%,与 Ventana-IHC 染色结果一致性为96.7%、91.7%、96.7%、76.7%。手术切除大标本的ALK表达优于活检小标本。(3)存档石蜡包埋组织EML4-ALK融合亚型检出率为52.8%(19/36)。融合亚型V1(52.6%)最多见,其次V3(31.6%,包括1例V3a、1例V3b、4例V3a+V3b混合)和V2(15.8%)。不同融合亚型与患者临床特征(年龄、性别、吸烟、部位)、病理形态(腺癌亚型)以及总生存时间均无统计差异。结论:(1)ALK阳性NSCLC具有独特的临床表现和病理形态,Ventana-IHC可作为检测ALK阳性NSCLC首选方法,可优先检测潜在ALK基因高突变率的肺癌患者。(2)基于已普及且成熟的手工IHC,优选ALK抗体1A4/1H7,初筛ALK染色阳性病例再进一步验证,提出一套简便易行、更适合国内大规模筛查的诊断流程。(3)EM4-ALK的不同融合亚型与临床病理预后无关,尚需扩大样本例数,为EML4-ALK融合NSCLC的分子异质性研究提供实验数据和参考标准。
[Abstract]:The number of lung cancer patients with the rapid growth of the past ten years, 2015 update to WHO lung cancer classification, change the emphasis and organization of tumor gene diagnosis are equally important, to drive the gene targeted therapy leads to precise medical trend, will bring the development of molecular detection technology more and more has the clinical significance of potential gene. Targeted therapy of anaplastic lymphoma kinase (ALK) fusion gene in non-small cell lung cancer (NSCLC) patients with better curative effect than traditional chemotherapy, accurate diagnosis of ALK positive NSCLC as the key to precision treatment, the China molecular detection development is still in its initial stage, only a few hospitals can complete molecular diagnosis, ALK NSCLC positive detection and diagnosis the process still needs to be further optimized according to the clinical status. Objective: to screen ALK positive NSCLC with multiple methods, analyze its clinicopathological characteristics and heterogeneity of EML4-ALK fusion subtypes, and propose a set of diagnostic processes suitable for China's national conditions. Methods: (1) collected 566 cases of NSCLC paraffin embedded tissue by Ventana automated immunohistochemical staining (Ventana-IHC) and fluorescence in situ hybridization (FISH) and sequence specific PCR (RT-PCR) ALK state detection method, analysis of color pattern of Ventana-IHC staining, ALK positive NSCLC summarizes the clinical pathological characteristics and prognosis. (2) 60 cases of NSCLC were detected by Ventana-IHC and manual IHC. Compared with 4 different ALK antibodies, D5F3 (Ventana), D5F3 (Cell Signaling), 1A4/1H7 (OriGene), and Signaling (f) combined with manual test to detect the staining condition. (3) RT-PCR and sequencing were used to detect the fusion subtypes of EML4-ALK, and the clinicopathological features and clinical significance of different subtypes were analyzed. Results: (1) the detection rate of ALK positive NSCLC in Ventana-IHC was 6.7% (38/566), which was consistent with FISH and PCR results. 36 cases of Ventana-IHC abnormal staining mode were verified to be ALK fusion negative. The positive rate of 60 (10.6%) patients aged above 60 years old group (3.5%), with statistical difference (P0.05). No asexual difference was found in non smokers (78.9%), and only 1 cases were combined with EGFR gene mutation. In the form of tissue, 81.6% were adenocarcinoma, of which there were more solid mucus (47.4%), non adenocarcinoma including 3 cases of squamous cell carcinoma, 3 cases of adenosscale carcinoma and 1 cases of pleomorphic cancer. The remission rate of kazolinib was 80%. (2) using manual IHC method, the sensitivities of 4 antibodies D5F3 (Ventana), D5F3 (Cell Signaling), 1A4/1H7 and 5A4 were 93.8%, 84.4%, 93.8%, 56.3%, respectively, and the specificity was 100%, which was 96.7%, 91.7%, 96.7% and 76.7% with the results of Ventana-IHC staining. The expression of ALK in large excised specimens is better than that of small biopsy specimens. (3) the detection rate of EML4-ALK fusion subtype in the archived paraffin embedded tissues was 52.8% (19/36). The fusion subtype V1 (52.6%) was the most common, followed by V3 (31.6%, including 1 cases of V3a, 1 cases of V3b, 4 V3a+V3b mixed) and V2 (15.8%). There were no statistical differences between the different fusion subtypes and the patients' clinical features (age, sex, smoking, location), pathological form (adenocarcinoma subtype) and total survival time. Conclusion: (1) ALK positive NSCLC has unique clinical manifestations and pathomorphology. Ventana-IHC can be used as the preferred method to detect ALK positive NSCLC, and it can give priority to detect lung cancer patients with high mutation rate of ALK gene. (2) based on the popularized and mature manual IHC, we optimized the ALK antibody 1A4/1H7 and screened ALK positive cases. Further, we proposed a set of simple and suitable diagnostic process for large-scale screening in China. (3) the different fusion subtypes of EM4-ALK are not related to the prognosis of clinical pathology. It is necessary to expand the number of samples, and provide experimental data and reference standard for the study of molecular heterogeneity of EML4-ALK fusion NSCLC.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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