Methylation Heterogeneity within Human Lung Carcinoids (LCs) Tumors. Methylation Heterogeneity within Human Lung Carcinoids (LCs) Tumors

DNA CpG methylation profiling of LC patients samples were performed to understand genotype to phenotype corrlelations , novel molecular subtypes and cell of origins Lung carcinoids (LCs) are rare and slow growing primary lung neoplasms that are understudied. Here, we performed targeted exome sequencing using a 354-cancer gene panel (n=29), mRNA sequencing (n=30) and DNA methylation assay (n=18) on macro-dissected lung carcinoids. The mutations we identified were enriched for genes involved in covalent histone modification/chromatin remodeling (34.5%) (MEN1, ARID1A, KMT2C and KMT2A were recurrently mutated) as well as DNA repair (17.2%) pathways. Unsupervised clustering and principle component analysis on gene expression and DNA methylation profiles showed 3 robust molecular subtypes (LC1, LC2, LC3) with distinct clinical features. MEN1 gene mutations were found to be enriched and exclusively in the LC2 subtype (p-value<0.001). The LC3 subtype is predominately found at endobronchial lung and earlier age of diagnosis. Immunohistochemical staining of two biomarkers, ASCL1 and S100, is sufficient to stratify the three subtypes. This molecular classification of lung carcinoids into three subtypes may help improve treatment decision and clinical management. Overall design: 18 LCs tumor specimens were obtained from MKSCC and samples were probed for DNA CpG methylation using Illumina Methylation 450K array and processed as per manufacturers instructions

为探究基因型-表型关联、新型分子亚型及细胞起源，本研究对肺癌类癌患者样本开展DNA CpG甲基化谱分析（DNA CpG methylation profiling）。肺癌类癌（Lung carcinoids, LCs）是一类罕见且生长缓慢的原发性肺肿瘤，目前相关研究尚不完善。本研究针对经大块组织解离的肺癌类癌样本，采用354癌症基因面板（354-cancer gene panel）开展靶向外显子组测序（targeted exome sequencing，n=29）、mRNA测序（mRNA sequencing，n=30）及DNA甲基化检测（DNA methylation assay，n=18）。本研究鉴定出的突变显著富集于共价组蛋白修饰/染色质重塑通路（covalent histone modification/chromatin remodeling，占比34.5%，其中MEN1、ARID1A、KMT2C及KMT2A为频发突变基因）与DNA修复通路（DNA repair pathways，占比17.2%）。对基因表达谱与DNA甲基化谱进行无监督聚类（unsupervised clustering）及主成分分析（principal component analysis）后，本研究鉴定出3种具有显著临床特征的稳定分子亚型（LC1、LC2、LC3）。MEN1基因突变显著富集且仅存在于LC2亚型中（p值<0.001）。LC3亚型主要见于支气管内肺癌患者，且确诊年龄更早。通过对ASCL1与S100两种生物标志物（biomarker）进行免疫组化染色（immunohistochemical staining），即可实现这3种亚型的分型。将肺癌类癌划分为3种分子亚型的分类体系，有助于优化治疗决策与临床管理。整体实验设计：本研究从MKSCC获取了18例肺癌类癌肿瘤样本，采用Illumina Methylation 450K芯片对样本进行DNA CpG甲基化检测，并严格按照厂商说明书完成实验流程处理。