本文選題：非小細(xì)胞肺癌　切入點(diǎn)：二代測(cè)序　出處：《中國(guó)人民解放軍醫(yī)學(xué)院》2017年博士論文

【摘要】：研究背景及目的:近年,靶向治療已成為非小細(xì)胞肺癌(non-small lung cancer,NSCLC)重要的治療手段,通過(guò)對(duì)非小細(xì)胞肺癌患者腫瘤組織標(biāo)本進(jìn)行基因檢測(cè),根據(jù)基因檢測(cè)結(jié)果選擇有效的靶向藥物進(jìn)行有針對(duì)性的治療已經(jīng)被普遍接受,然而,要發(fā)現(xiàn)患者的靶向用藥驅(qū)動(dòng)基因就必須進(jìn)行多基因檢測(cè),這樣可以讓患者獲得更多的治療機(jī)會(huì),但是,一代測(cè)序因其復(fù)雜的檢測(cè)過(guò)程過(guò)高的成本要實(shí)現(xiàn)多基因檢測(cè)并不現(xiàn)實(shí)。二代基因檢測(cè)技術(shù)的成熟為多基因檢測(cè)提供了技術(shù)強(qiáng)有力的保障,用外周血中的循環(huán)腫瘤DNA(circulating tumor DNA,ctDNA)進(jìn)行“液體活檢”,樣本相對(duì)于組織活檢容易取得,而且可重復(fù)取樣。ctDNA包含患者的基因突變信息,通過(guò)對(duì)外周血中的ctDNA進(jìn)行重復(fù)檢測(cè)可以監(jiān)測(cè)患者的腫瘤進(jìn)展、用藥療效、耐藥突變等,對(duì)那些腫瘤無(wú)法切除或者穿刺無(wú)法取到的肺癌患者更有意義。用二代測(cè)序技術(shù)進(jìn)行ctDNA液體活檢,已開(kāi)始進(jìn)入臨床應(yīng)用。但目前,組織的多基因檢測(cè)依然是金標(biāo)準(zhǔn),ctDNA多基因檢測(cè)用于臨床的可靠性以及ctDNA能否作為非小細(xì)胞肺癌患者診斷和手術(shù)效果的判斷指標(biāo),有待廣泛的臨床數(shù)據(jù)驗(yàn)證。本研究通過(guò)對(duì)非小細(xì)胞肺癌患者組織和血漿ctDNA檢測(cè),對(duì)兩者的一致性、患者手術(shù)前后的ctDNA突變豐度變化等進(jìn)行分析,來(lái)確證對(duì)血漿ctDNA測(cè)序選擇靶向藥物治療是否可靠、血液ctDNA突變豐度變化是否與腫瘤變化程度相關(guān)、監(jiān)測(cè)血液ctDNA變化判斷腫瘤進(jìn)展與目前監(jiān)測(cè)肺癌標(biāo)志物判斷肺癌患者腫瘤進(jìn)展是否更具有優(yōu)勢(shì)。方法:①收集41例非小細(xì)胞肺癌患者手術(shù)切除的原發(fā)腫瘤組織以及每位患者手術(shù)前、后的外周血,對(duì)上述標(biāo)本進(jìn)行基于Ion Proton二代測(cè)序平臺(tái)的包括BRAF、EGFR、KRAS、TP53、ERBB2、PIK3CA等在內(nèi)的50基因檢測(cè),觀察上述腫瘤驅(qū)動(dòng)基因的突變情況,對(duì)組織和血漿DNA的一致性進(jìn)行比較分析;②取患者手術(shù)前的血清,用化學(xué)發(fā)光免疫分析方法檢測(cè)6種血清標(biāo)志物:CA125,CA19-9,CYFRA21-1,CEA,NSE和SCC,對(duì)比分析血清標(biāo)志物數(shù)值與腫瘤的關(guān)系,并與手術(shù)前ctDNA檢出率進(jìn)行比較。結(jié)果:①外周血ctDNA與原發(fā)腫瘤組織驅(qū)動(dòng)基因突變的一致性為78.1%,敏感性和特異性分別為69.2%和93.3%,陽(yáng)性預(yù)測(cè)值(PPV)為94.7%。②手術(shù)后2天即有91.7%患者血中ctDNA突變的頻率較手術(shù)前明顯下降。③外周血ctDNA比目前臨床應(yīng)用的6種腫瘤生物標(biāo)志物具有更高的陽(yáng)性預(yù)測(cè)值。結(jié)論:靶向測(cè)序檢測(cè)ctDNA可以檢出非小細(xì)胞肺癌患者的突變基因,并反映患者手術(shù)治療的效果,具有一定的可靠性,這種監(jiān)測(cè)外周血中ctDNA變化的方法對(duì)NSCLC患者的臨床管理具有一定的應(yīng)用價(jià)值。
[Abstract]:Background and objective: in recent years, targeted therapy has become an important therapy for non-small lung lung cancer (NSCLC). It has been widely accepted to select effective targeted drugs for targeted therapy based on the results of gene detection. However, to find the driving genes of targeted drugs in patients, we must carry out multi-gene tests. This will give patients more access to treatment, but, Because of the high cost of the complex detection process, it is not realistic for a generation of sequencing to realize multi-gene detection. The maturity of the second-generation gene detection technology provides a strong technical guarantee for multi-gene detection. Using circulating tumor DNA(circulating tumor DNA in peripheral blood to perform "fluid biopsy", samples are easily obtained than tissue biopsies, and repeatable samples contain mutational information about the patient's genes. Repeated detection of ctDNA in peripheral blood can monitor tumor progression, drug efficacy, drug resistance mutation, and so on. It makes more sense for lung cancer patients whose tumors are unresectable or can't be punctured. CtDNA fluid biopsies using second-generation sequencing techniques have begun to be used clinically. Tissue polygenic detection is still the gold standard ctDNA polygene test for clinical reliability and whether ctDNA can be used as a diagnostic and surgical outcome in patients with non-small cell lung cancer (NSCLC). In this study, the consistency of ctDNA in tissues and plasma of patients with non-small cell lung cancer (NSCLC) and the changes of ctDNA mutation abundance before and after operation were analyzed. To confirm the reliability of selective drug therapy for plasma ctDNA sequencing, and whether the change in blood ctDNA mutation abundance is related to the degree of tumor change. Monitoring the changes of ctDNA in blood to judge the progress of tumor was superior to monitoring the markers of lung cancer. Methods 41 patients with non-small cell lung cancer were collected from 41 patients with non-small cell lung cancer and each patient was treated before surgery. In the peripheral blood of the patients, 50 genes, including BRAFFN EGFRX, TP53, ERBB2PIK3CA and so on, were detected on the basis of Ion Proton second generation sequencing platform, and the mutation of the tumor driving genes was observed. The consistency between tissue and plasma DNA was compared and analyzed. The serum samples were collected from patients before operation. Six serum markers, CYFRA21-9, CYFRA21-1 and SCC, were detected by chemiluminescence immunoassay, and the relationship between serum markers and tumor was compared and analyzed. The detection rate of ctDNA was compared with that before operation. Results the consistency between the mutation of ctDNA and the driving gene of primary tumor tissue was 78.1, the sensitivity and specificity were 69.2% and 93.33.The positive predictive value was 94.72.The positive predictive value was 91.7% 2 days after operation. The frequency of ctDNA mutation in human blood was significantly lower than that before operation. 3. 3 ctDNA in peripheral blood was significantly lower than that of 6 tumor biomarkers in clinical use. Conclusion: the detection of ctDNA by targeted sequencing can detect non-small cell lung cancer. The patient's mutant gene, The method of monitoring the changes of ctDNA in peripheral blood is valuable for clinical management of patients with NSCLC.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【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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