Bronchial premalignant lesions have distinct molecular subtypes associated with future histologic progression

Exposure to cigarette smoke creates a field of injury throughout the entire respiratory tract inducing genomic alterations that lead to an “at-risk” airway where and lung cancers develop. Lung squamous cell carcinoma (SCC) arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions(PMLs). The presence of high-grade persistent or progressive PMLs is a marker of increased lung cancer risk both at the lesion site elsewhere in the lung. Effective tools to identify and treat premalignant lesions at highest risk of progression to invasive carcinoma are lacking. We profiled via RNA-Seq airway brushing and biopsies (divided into two cohorts: discovery and validation) obtained from high-risk smokers undergoing lung cancer screening via serial auto-fluorescence bronchoscopy procedures. We identified four distinct molecular subtypes (Proliferative, Inflammatory, Secretory, and Normal) in the bronchial biopsies that correspond to a spectrum of biological and morphological alterations. The Proliferative subtype was enriched with dysplastic PMLs from current smokers that exhibit up-regulation of KRT5 and KI67 as well as metabolic and cell cycle pathways, and down-regulation of cilium-associated processes and FOXJ1 expression. Molecular subtype classification in the validation cohort biopsies replicated these significant clinical and biological associations. Airway brushes from normal fluorescing areas of the large airway classified as the Proliferative subtype specifically predicts the presence of biopsies of this same subtype. Within the Proliferative subtype, genes associated with interferon signaling and antigen processing and presentation were observed to be down-regulated among dysplastic biopsies that persistent or progress in the future. Innate and adaptive immune cells were computationally predicted to be depleted in these biopsies and this was confirmed via immunofluorescence staining of adjacent biopsies. These findings provide a proof of concept that molecular biomarkers of endobronchial biopsies can enhance histopathological grading and that immunoprevention strategies are an important future direction in intercepting the progression of PMLs to lung cancer. Endobronchial biopsies and brushings were obtained from high-risk subjects undergoing lung cancer screening at approximately 1-year intervals by white light and auto-fluorescence bronchoscopy and computed tomography at the Roswell Park Cancer Institute in Buffalo, NY. Subjects were selected that had biopsies collected in repeat locations via serial bronchoscopies. mRNA sequencing was performed on a discovery cohort (DC) of samples comprising of endobronchial biopsies and brushes collected between 2010 and 2012 (n=30 subjects, n=197 biopsies, and n=91 brushings). mRNA sequencing was subsequently performed on a validation cohort (VC) of samples comprising of endobronchial biopsies and brushes collected between 2010 and 2015 (n=20 subjects, n=111 biopsies, and n=49 brushings). The submitter declares that the raw data will be made available in dbGaP (https://www.ncbi.nlm.nih.gov/gap) due to patient privacy concerns.

香烟烟雾暴露会在整个呼吸道造成损伤区域，诱导基因组改变，进而形成“风险易感”气道，最终引发肺癌。肺鳞状细胞癌（Lung squamous cell carcinoma, SCC）起源于支气管气道的上皮层，其发生通常伴随癌前病变（premalignant lesions, PMLs）的形成。高级别持续性或进展性癌前病变的存在，不仅提示病变局部的肺癌风险升高，也提示肺部其他部位的肺癌风险上升。目前尚缺乏能够识别并干预最易进展为浸润性癌的癌前病变的有效手段。我们通过RNA测序（RNA-Seq）对经系列自体荧光支气管镜检查进行肺癌筛查的高危吸烟者的气道刷检样本与活检样本进行了转录组分析，所有样本分为两个队列：发现队列与验证队列。我们在支气管活检样本中鉴定出四种独特的分子亚型：增殖型（Proliferative）、炎症型（Inflammatory）、分泌型（Secretory）与正常型（Normal），这些亚型对应一系列生物学与形态学改变。增殖型亚型富集了当前吸烟者的异型增生性癌前病变，这类病变表现出KRT5与KI67的上调，同时代谢通路与细胞周期通路激活，而纤毛相关生物学过程及FOXJ1表达则出现下调。验证队列活检样本的分子亚型分类验证了上述显著的临床与生物学关联。取自大气道正常荧光区域的气道刷检样本若被归类为增殖型亚型，则可特异性预测该部位活检样本亦属于该亚型。在增殖型亚型中，未来会持续存在或进展的异型增生活检样本中，与干扰素信号通路、抗原加工呈递相关的基因均出现下调。通过计算预测，这类活检样本中的固有免疫与适应性免疫细胞均出现耗竭，这一结果通过相邻活检样本的免疫荧光染色得到了验证。本研究结果证实了一个概念：支气管内镜活检样本的分子生物标志物可辅助提升组织病理学分级的准确性，而免疫预防策略将成为阻断癌前病变进展为肺癌的重要未来研究方向。本研究的支气管内镜活检与气道刷检样本取自纽约州布法罗市罗斯威尔帕克癌症研究所（Roswell Park Cancer Institute）的高危受试者，这些受试者接受了约每年1次的白光与自体荧光支气管镜检查及计算机断层扫描（computed tomography, CT）肺癌筛查。研究对象均为经系列支气管镜检查在同一部位重复获取活检样本的受试者。研究团队对发现队列（discovery cohort, DC）的样本进行了mRNA测序，该队列样本采集于2010至2012年间，包含30名受试者的197份活检样本与91份气道刷检样本。后续又对验证队列（validation cohort, VC）的样本进行了mRNA测序，该队列样本采集于2010至2015年间，包含20名受试者的111份活检样本与49份气道刷检样本。本数据集提交者声明，鉴于受试者隐私保护相关问题，原始数据将上传至dbGaP数据库（https://www.ncbi.nlm.nih.gov/gap）。