【摘要】：背景近年來,針對程序性死亡因子1(PD-1)及其配體1(PD-L1)的免疫治療在非小細(xì)胞肺癌中獲得了突飛猛進(jìn)的進(jìn)展。盡管免疫治療療效顯著,但僅僅只有一小部分患者可以從中獲益,如何選擇有效的生物標(biāo)志物篩選出潛在的獲益人群是目前面臨的主要問題。值得欣喜的是,近年來研究發(fā)現(xiàn)腫瘤突變負(fù)荷、DNA錯配修復(fù)缺陷、腫瘤浸潤淋巴細(xì)胞數(shù)量及腫瘤/免疫細(xì)胞PD-L1表達(dá)水平可以有效預(yù)測PD-1抑制劑的療效。并且,這些因素之間也存在著彼此相互影響的關(guān)系。這些發(fā)現(xiàn)給我們提出來另一個問題:是否存在其他生物標(biāo)志物可以同時影響上述兩個或多個預(yù)測因素從而可以達(dá)到更強的預(yù)測價值?方法我們通過利用公共數(shù)據(jù)庫(TCGA、GEO及broad)作為探索集,并用我中心二代測序數(shù)據(jù)作為驗證集,從基因組、轉(zhuǎn)錄組及蛋白組進(jìn)行系統(tǒng)分析。臨床免疫治療療效與基因突變的相關(guān)性分別從既往臨床試驗結(jié)果(MSKCC)和我中心(GLCI)免疫治療數(shù)據(jù)進(jìn)行分析。免疫組化及sanger測序用于相關(guān)分子檢測�；蚋患�(GSEA)對相關(guān)信號通路進(jìn)行分析。結(jié)果本研究首先通過運用TCGA及GEO數(shù)據(jù)庫相關(guān)信息對肺腺癌常見基因突變(TP53/KRAS/EGFR/STK11)與腫瘤免疫微環(huán)境中免疫檢查點蛋白PD-L1及腫瘤浸潤淋巴細(xì)胞進(jìn)行相關(guān)性分析。我們發(fā)現(xiàn)了 TP53突變及TP53/KRAS雙突變促進(jìn)肺腺癌組織PD-L1表達(dá)、腫瘤淋巴細(xì)胞浸潤及效應(yīng)T細(xì)胞-IFN-γ信號通路的激活。初步確立了上述突變對腫瘤免疫微環(huán)境的影響。進(jìn)而我們對腫瘤突變負(fù)荷及突變譜進(jìn)行分析,通過從公共數(shù)據(jù)庫的探索集(TGCA和Broad)到我中心二代測序的驗證集(GLCI)初步證明了 TP53及KRAS突變與肺腺癌腫瘤突變負(fù)荷增加及堿基顛換發(fā)生頻率增加相關(guān)。同時利用基因富集分析發(fā)現(xiàn)TP53及KRAS突變通過干擾腫瘤細(xì)胞細(xì)胞周期調(diào)控、DNA復(fù)制及DNA損傷修復(fù)等過程從而增加腫瘤基因突變發(fā)生風(fēng)險。鑒于上述分子機制上的發(fā)現(xiàn)而提出了 TP53及KRAS突變可能是肺腺癌免疫治療的預(yù)測標(biāo)志物的假設(shè)。為了證明上述理論推斷,我們首先通過利用公共數(shù)據(jù)庫中既往已發(fā)表的免疫治療臨床試驗患者信息(MSKCC),初步證明了 TP53及KRAS突變尤其是TP53/KRAS雙突變患者其免疫治療療效顯著優(yōu)于野生型患者。進(jìn)而我們又分析了真實世界中(GLCI)免疫治療患者的臨床治療信息,結(jié)果同樣提示TP53及KRAS突變患者具有更好的免疫治療效果。結(jié)論我們的研究首次探明了 TP53及KRAS突變對肺腺癌PD-L1表達(dá)、腫瘤淋巴細(xì)胞浸潤及腫瘤突變負(fù)荷的影響。并進(jìn)一步從臨床免疫治療中初步確立了TP53及KRAS突變,尤其是TP53/KRAS雙突變對肺腺癌PD-1單抗免疫治療敏感性增加的預(yù)測價值。上述發(fā)現(xiàn)對未來篩選免疫治療有效生物標(biāo)志物提供了新的思路。
[Abstract]:Background Immunotherapy of programmed death factor 1 (PD-1) and its ligand 1 (PD-L1) has made rapid progress in non-small cell lung cancer (NSCLC) in recent years. Although the efficacy of immunotherapy is remarkable, only a small number of patients can benefit from it. How to select effective biomarkers to select potential beneficiaries is the main problem. It is gratifying to note that recent studies have found that the defect of mismatch repair of tumor mutation load, the number of tumor infiltrating lymphocytes and the expression of PD-L1 in tumor / immune cells can effectively predict the efficacy of PD-1 inhibitors. Moreover, these factors also have a mutual influence on each other. These findings raise another question: is there any other biomarker that can affect two or more of these predictive factors at the same time to achieve greater predictive value? Methods by using the common database (TCGA,GEO and broad) as the exploration set and the second generation sequencing data of our center as the validation set, we systematically analyzed the genome, transcriptome and proteome. The relationship between the efficacy of clinical immunotherapy and gene mutation was analyzed from the results of previous clinical trials (MSKCC) and (GLCI) immunotherapy data of our center. Immunohistochemistry and sanger sequencing were used for the detection of related molecules. The related signal pathways were analyzed by gene enrichment method (GSEA). Results in this study, the correlation between common gene mutation (TP53/KRAS/EGFR/STK11) and immunological checkpoint protein (PD-L1) and tumor infiltrating lymphocytes in tumor immune microenvironment was analyzed by using TCGA and GEO database. We found that TP53 mutation and TP53/KRAS double mutation promoted the expression of PD-L1, tumor lymphocyte infiltration and activation of effector T cell -IFN- 緯 signaling pathway in lung adenocarcinoma. The effect of the above mutation on tumor immune microenvironment was preliminarily established. And then we analyze the mutation load and the mutation spectrum of the tumor. From the exploration set (TGCA and Broad) of the common database to the verification set (GLCI) of the second generation sequencing of our center, it was preliminarily proved that the mutations of TP53 and KRAS were correlated with the increase of mutation load and the frequency of base transversion in lung adenocarcinoma. At the same time, gene enrichment analysis showed that TP53 and KRAS mutations increased the risk of tumor gene mutation by interfering with the process of tumor cell cycle regulation, such as DNA replication and DNA damage repair. In view of the above discovery of molecular mechanism, it is suggested that TP53 and KRAS mutations may be predictive markers of immunotherapy for lung adenocarcinoma. In order to prove the above theoretical inference, We first demonstrated that TP53 and KRAS mutations, especially in patients with double TP53/KRAS mutations, were more effective than wild-type patients by using the previously published information of patients with immunotherapy clinical trials in the public database, and the results showed that TP53 and KRAS mutations, especially in patients with double TP53/KRAS mutations, were significantly better than those in wild type patients. Furthermore, we analyzed the clinical information of (GLCI) immunotherapy patients in the real world. The results also indicated that TP53 and KRAS mutation patients had better immunotherapy effect. Conclusion our study demonstrated for the first time the effects of TP53 and KRAS mutations on PD-L1 expression, tumor lymphocyte infiltration and tumor mutation load in lung adenocarcinoma. Furthermore, the predictive value of TP53 and KRAS mutations, especially TP53/KRAS double mutations, for the increased sensitivity of PD-1 monoclonal antibody to immunotherapy of lung adenocarcinoma was preliminarily established from clinical immunotherapy. These findings provide a new idea for screening effective biomarkers for immunotherapy in the future.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R734.2

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