本文關(guān)鍵詞：ALK陽(yáng)性非小細(xì)胞肺癌的免疫組化染色篩查以及EML4-ALK融合亞型異質(zhì)性的研究　出處：《南方醫(yī)科大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 非小細(xì)胞肺癌 間變性淋巴瘤激酶 免疫組化 抗體 篩查 融合亞型

【摘要】：過(guò)去十年肺癌患者人數(shù)快速增長(zhǎng),2015年WHO更新了肺癌分類(lèi),強(qiáng)調(diào)腫瘤基因改變與組織學(xué)診斷同等重要,以驅(qū)動(dòng)基因靶向治療引領(lǐng)了精準(zhǔn)醫(yī)療的潮流,必將帶來(lái)越來(lái)越多的具有臨床意義的潛在基因的分子檢測(cè)技術(shù)發(fā)展。靶向藥物治療間變性淋巴瘤激酶(ALK)基因融合非小細(xì)胞肺癌(NSCLC)患者的療效顯著優(yōu)于傳統(tǒng)化療,準(zhǔn)確診斷ALK陽(yáng)性NSCLC為精準(zhǔn)治療的關(guān)鍵,目前中國(guó)分子檢測(cè)開(kāi)展尚處于初期階段,僅少數(shù)醫(yī)院能完成分子診斷,ALK陽(yáng)性NSCLC檢測(cè)方法和診斷流程仍需要不斷結(jié)合臨床現(xiàn)狀進(jìn)一步優(yōu)化。目的:本研究采用多種方法篩選ALK陽(yáng)性NSCLC,分析其臨床病理特征和EML4-ALK融合亞型的異質(zhì)性,提出一套適合中國(guó)國(guó)情的診斷流程。方法:(1)收集566例NSCLC石蠟包埋組織,采用Ventana全自動(dòng)免疫組化染色(Ventana-IHC)、熒光原位雜交(FISH)和序列特異性PCR技術(shù)(RT-PCR)方法檢測(cè)ALK狀態(tài),分析Ventana-IHC染色的著色模式,總結(jié)ALK陽(yáng)性NSCLC臨床病理特征及治療預(yù)后。(2)采用Ventana-IHC和手工IHC檢測(cè)60例NSCLC,對(duì)比4種不同 ALK抗體D5F3(Ventana)、D5F3(Cell Signaling)、1A4/1H7(OriGene)、5A4(Abcam)聯(lián)合手工IHC檢測(cè)ALK染色情況。(3)采用RT-PCR和測(cè)序檢測(cè)EML4-ALK的融合亞型,分析不同亞型的臨床病理特征及臨床意義。結(jié)果:(1)Ventana-IHC 篩查 ALK 陽(yáng)性 NSCLC 的檢出率為 6.7%(38/566),與FISH和PCR結(jié)果一致,36例Ventana-IHC異常染色模式經(jīng)驗(yàn)證為ALK融合陰性。年齡≤60歲患者陽(yáng)性率(10.6%)高于60歲組(3.5%),有統(tǒng)計(jì)差異(P0.05)。無(wú)性別差異,不吸煙者多見(jiàn)(78.9%),僅1例合并EGFR基因突變。組織形態(tài)上,81.6%為腺癌,其中實(shí)體型伴黏液產(chǎn)生多見(jiàn)(47.4%),非腺癌包括3例鱗癌、3例腺鱗癌及1例多形性癌�？诉蛱婺嶂委熅徑饴蕿�80%。(2)采用手工IHC方法,4 種抗體 D5F3(Ventana)、D5F3(Cell Signaling)、1A4/1H7、5A4 染色敏感性分別為 93.8%、84.4%、93.8%、56.3%,特異性均 100%,與 Ventana-IHC 染色結(jié)果一致性為96.7%、91.7%、96.7%、76.7%。手術(shù)切除大標(biāo)本的ALK表達(dá)優(yōu)于活檢小標(biāo)本。(3)存檔石蠟包埋組織EML4-ALK融合亞型檢出率為52.8%(19/36)。融合亞型V1(52.6%)最多見(jiàn),其次V3(31.6%,包括1例V3a、1例V3b、4例V3a+V3b混合)和V2(15.8%)。不同融合亞型與患者臨床特征(年齡、性別、吸煙、部位)、病理形態(tài)(腺癌亞型)以及總生存時(shí)間均無(wú)統(tǒng)計(jì)差異。結(jié)論:(1)ALK陽(yáng)性NSCLC具有獨(dú)特的臨床表現(xiàn)和病理形態(tài),Ventana-IHC可作為檢測(cè)ALK陽(yáng)性NSCLC首選方法,可優(yōu)先檢測(cè)潛在ALK基因高突變率的肺癌患者。(2)基于已普及且成熟的手工IHC,優(yōu)選ALK抗體1A4/1H7,初篩ALK染色陽(yáng)性病例再進(jìn)一步驗(yàn)證,提出一套簡(jiǎn)便易行、更適合國(guó)內(nèi)大規(guī)模篩查的診斷流程。(3)EM4-ALK的不同融合亞型與臨床病理預(yù)后無(wú)關(guān),尚需擴(kuò)大樣本例數(shù),為EML4-ALK融合NSCLC的分子異質(zhì)性研究提供實(shí)驗(yàn)數(shù)據(jù)和參考標(biāo)準(zhǔn)。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R734.2

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