文章所在期刊：Clinical Cancer Research

介紹

PD-1/PDL-1在臨床治療NSCLC（non-small cell carcinoma）病人中表現(xiàn)了空前的潛力固灵。然而只有少數(shù)人對當(dāng)前的免疫治療有反應(yīng)，驅(qū)動病人產(chǎn)生藥物敏感/抵擋性的機(jī)制并不是完全清楚坪仇。因而鑒定一些生物標(biāo)志物以提升病人的反應(yīng)率非常重要莉擒，當(dāng)前研究已經(jīng)闡述了tumor mutational load, DNA mismatch repair deficiency, the intensity of CD8+ cell infiltrates, intratumoral PD-L1 expression是抗PD-1/PD-L1治療反應(yīng)的生物標(biāo)記物抠蚣。鑒于這些因素存在關(guān)聯(lián)作用并在個體中經(jīng)常一起被發(fā)現(xiàn)连躏，因而有沒有其他的因素可以同時刺激（調(diào)控）上面提到的這些因素呢？如果存在這種因素坎缭，那么它肯定比上述提到的4種標(biāo)志物更有價值（它反應(yīng)了更為本質(zhì)的內(nèi)部驅(qū)動）白群。這就是這篇文章研究的核心尚胞。

一些研究已經(jīng)表明了LUAD中TP53和STK11突變非常普遍，而且經(jīng)常伴隨KRAS的突變川抡》妫基于這個結(jié)果须尚，作者假設(shè)激活一些特定的腫瘤發(fā)生的通路會對基因表達(dá)（改變）有廣譜的效應(yīng)，那么可以想象到基因組上的突變會對腫瘤微環(huán)境造成重要的影響侍咱。而且一些研究也發(fā)現(xiàn)了TP53或KRAS突變的NSCLC相對于野生型會表達(dá)更高水平的PD-L1蛋白耐床；TP53功能的喪失降低了基因組的不穩(wěn)定性，與DNA損失修復(fù)相關(guān)楔脯，也表現(xiàn)了TP53突變的腫瘤有更高的mutational burden撩轰。

以上介紹都說明了TP53/KRAS與之前提到的四個生物標(biāo)志物有密切聯(lián)系，所以作者推測TP53/KRAS就是他在問題中想要尋找的“另一種因素”昧廷，對它們的分析和理解可以區(qū)分病人亞型（更有針對性）以提升免疫治療的效果堪嫂。

研究目的

臨床研究已經(jīng)表明了用靶向PD-1/PD-L1通路在治療NSCLC中有光明的前景，但是我們并未能完全理解木柬、區(qū)分對免疫治療有反應(yīng)的亞型病人的因素皆串。

實驗設(shè)計

整合基因組、轉(zhuǎn)錄組眉枕、蛋白質(zhì)組和臨床等多個維度的LUAD公共數(shù)據(jù)庫（探索數(shù)據(jù)集）和內(nèi)部數(shù)據(jù)庫（驗證數(shù)據(jù)集）恶复、免疫治療病人的數(shù)據(jù)進(jìn)行分析∷偬簦基因富集分析（GSEA）用來測定特定病人子群的潛在相關(guān)基因表達(dá)signature谤牡。

方法

Clinical Cohorts

• TCGA: 462 patients with mRNA expression profiling and gene mutation data.
• GSE72094: 442 patients with detailed mRNA expression data and EGFR/KRAS/TP53/STK11 sanger sequencing analysis.
• Broad cohort: 183 lung adenocarcinomas and matched normal tissues with detail information about mutation load and mutation spectrum.
• A total of 85 lung adenocarcinomas from the Guangdong Lung Cancer Institute (GLCI), Guangdong General Hospital (GCH) were underwent whole genome sequencing (WGS).

Immunotherapeutic patients

Clinical and mutation data for 34 NSCLC (29 ADC) patients were retrieved from cbioPortal (http://www.cbioportal.org/study.do?cancer_study_id=luad_mskcc_2015). Another group consisted of 20 NSCLC (15 ADC) patients were collected prospectively in the Guangdong Lung Cancer Institute from August, 2015 to August, 2016. Tumor specimens
142 were obtained for Sanger sequencing and IHC analysis.

mRNA Expression Profiling and Reverse Phase Protein Array (RPPA) analysis

Gene expression data for the GSE72094 lung adenocarcinomas have been deposited in the GEO repository (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE72094). Proteomic analysis was based on Reverse Phase Protein Array (RPPA) form TCGA database. The RPPA methodology and data analysis pipeline have been previously described (ref 21). For TCGA, level 3 data were downloaded directly from the TCGA portal and utilized in subsequent analyses.

Mutation Data Analysis

For the discovery set, somatic mutation data (level 2) of the 462 lung adenocarcinomas were retrieved from the TCGA data portal (https://tcga-data.nci.nih.gov/tcga/findArchives.htm). To assess the mutational load, the number of mutated genes carrying at least one nonsynonymous mutation in the coding region was computed for each tumor. Somatic mutation data of 183 lung adenocarcinomas in Broad cohort was retrieved from cbioPortal (http://www.cbioportal.org/study.do?cancer_study_id=luad_broad). Somatic substitutions and covered bases within their trinucleotide sequence context were analyzed to characterize the mutation spectrum of 183 lung adenocarcinoma. Mutation spectrum for each sample was calculated as the percentage of each of six possible single nucleotide changes (AT>CG AT>GC, AT>TA, GC>AT, GC>CG, GC>TA) among all single nucleotide substitutions. The most frequent mutation signatures were C→T transitions and C→A transversions.

For the validation set (GLCI), we conducted whole-exome sequencing of DNA from tumors and matched normal blood from 85 lung adenocarcinoma patients. Enriched exome libraries were sequenced on the HiSeq 2000 platform (Illumina) to >100X coverage. Alignment, base-quality score recalibration and duplicate-read removal were performed, germline variants were excluded, mutations annotated and indels evaluated as previously described(ref 4, 5, 24). Mutations between clinical groups were compared using the Mann-Whitney test. 用的非參檢驗，確實突變數(shù)不適合參數(shù)檢驗姥宝。

Gene Expression Data Analysis (GSEA) and Pathway Analysis

For Gene Set Enrichment Analysis (GSEA)(ref 25), the javaGSEA Desktop Application was downloaded from http://software.broadinstitute.org/gsea/index.jsp. GSEA was used to associate the gene signature with the TP53 or KRAS mutation status (TP53-mut vs. TP53-wt; KRAS-mut vs. KRAS-wt). The genes identified to be on the leading edge of the enrichment profile were subject to pathway analysis. Fold change values were exported for all genes and analyzed with version 2.2.0 of GSEA, using the GseaPreranked module. The normalized enrichment score (NES) is the primary statistic for examining gene set enrichment results The nominal P value estimates the statistical significance of the enrichment score. A gene set with nominal P ≤ 0.05 was considered to be significantly enriched in genes.

免疫組化翅萤、Sanger測序跳過。

Statistical Analyses

Statistical analyses were conducted using GraphPad Prism (version 7.01, La Jolla, CA) and SPSS version 22.0 (SPSS, Inc., Chicago, IL). Scatter dot plot and Box and whisker plots indicate median and 95% confidence interal (CI). Statistical tests were used to analyze the clinical and genomic data, including the Mann-Whitney U, Chi-square, Fisher’s exact and Kruskal-Wallis. Kaplan-Meier curves analysis of progression free survival (PFS) were compared using the log-rank test. All reported P values are two-tailed, and for all analyses, P≤0.05 is considered statistically significant, unless otherwise specified.