Feasibility of Molecular Cytology for the Management of Barrett's Esophagus

描述（由申请人提供）：在过去的40年里，食管腺癌（EAC）的发病率在美国和其他西方国家急剧增加。EAC是一种侵袭性肿瘤，通常在晚期被诊断出来，导致总体生存率很低。然而，如果早期发现，有效的治疗选择包括消融、内镜粘膜切除术和食管切除术，有或没有化疗和放疗。EAC通常发生于慢性胃食管反流病，伴有食管柱状化生，即Barrett食管（BE）。BE是已知的EAC的最大危险因素，因此建议BE患者进入内镜监测项目，以检测不典型增生或早期EAC。然而，使用多次活检的重复内窥镜检查是一种昂贵且侵入性的BE监测模式。我们相信，将食管细胞学与下一代测序相结合，检测驱动发育不良和EAC发展的突变，将为BE监测提供一种新颖、侵入性更小、成本效益更高的方法。在Specific Aim 1中，我们将通过将FDA批准的食管细胞学样本采集设备（EsophaCap(tm)）与一组约100个已知EAC发展驱动基因的靶向新一代测序相结合，来测试这一假设的可行性。该小组将从250个EAC样本的全外显子组序列数据和100个相同肿瘤的RNA-seq数据中确定。这些数据可以通过我们之前发表的工作获得（Dulak等）。《自然遗传学》（2013）和一项由加拿大基因组资助的正在进行的研究。这些数据，以及EsophaCap（tm）在美国的直接使用，使我们在评估分子细胞学测试检测BE患者疾病进展的可行性方面处于独特的地位。具体来说，在Aim 1A中，我们将确定在多个活检样本中发现的EAC驱动突变在匹配的细胞学样本中也可检测到的比例。这将决定我们提出的方法的总体可行性，因为细胞学样本将被正常细胞“污染”，这将稀释突变等位基因的部分。在相关的目的1B中，我们还将通过识别来自同一患者的多个独立活检中的突变来确定BE的突变异质性。这将使我们能够估计检测细胞学样本中所有突变所需的序列深度，也将有助于分析BE的克隆异质性和进化。Specific Aim 2将采用类似的方法来解决关于BE消融的关键问题；消融后形成的新鳞状上皮在基因上是正常的，还是含有可能导致癌症风险的突变？这将通过比较消融前后be和新鳞状上皮的突变来确定。这一数据将有助于确定接受消融治疗的BE患者是否需要随访监测，并将为BE和新鳞状上皮的干细胞起源提供深入了解。

DESCRIPTION (provided by applicant): The incidence of esophageal adenocarcinoma (EAC) has increased dramatically in the US and other Western countries over the past 40 years. EAC is an aggressive tumor that is typically diagnosed at late stage resulting in very poor overall survival. If detected early however, effective treatment options are available including ablation, endoscopic mucosal resection and esophagectomy, with or without chemotherapy and radiation. EAC typically arises in the setting of chronic gastroesophageal reflux disease with the associated development of esophageal columnar metaplasia known as Barrett's Esophagus (BE). BE is the strongest known risk factor for EAC and patients with BE are therefore recommended to enter endoscopic surveillance programs to detect dysplasia or early EAC. However, the use of repeat endoscopies with multiple biopsies is an expensive and invasive model for BE surveillance. We believe that combining esophageal cytology with next-generation sequencing to detect mutations that drive development of dysplasia and EAC will provide a novel, less invasive and more cost-effective approach to BE surveillance. In Specific Aim 1, we will test the feasibility of this hypothesis by combining an FDA approved device for esophageal cytology sample collection (the EsophaCap(tm)) with targeted, next-generation sequencing of a panel of ~100 genes known to be drivers of EAC development. This panel will be identified from whole exome sequence data on 250 EAC samples and RNA-seq data on 100 of the same tumors. This data is available to us through our previously published work (Dulak, et al. Nature Genetics 2013) and an ongoing study funded by Genome Canada. This data, along with access to the EsophaCap(tm) for immediate use in the USA puts us in a unique position to evaluate the feasibility of a molecular cytology test to detect disease progression in patients with BE. Specifically, in Aim 1A we will determine what proportion of EAC driver mutations, identified in multiple biopsy samples, are also detectable in matched cytology samples. This will determine overall feasibility for our proposed approach as the cytology samples will be "contaminated" with normal cells that will dilute the mutant allele fractions. In related aim 1B, we will also determin the mutation heterogeneity in BE by identifying mutations in multiple independent biopsies from the same patients. This will allow us to estimate the sequence depth required to detect all mutations in cytology samples and will also facilitate an analysis of clonal heterogeneity and evolution in BE. Specific Aim 2 will utilize a similar approach to address a critical question regarding ablation of BE; is the neosquamous epithelium that develops following ablation genetically normal or does it harbor mutations that could still incur cancer risk? This will be determined by comparing mutations in BE and neosquamous epithelium pre and post-ablation. This data will help determine the need for follow-up surveillance in BE patients treated with ablative therapy and will provide insight into the origin of stem cells that give rise to BE and neosquamous epithelium.