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

描述（由申请人提供）：过去 40 年来，美国和其他西方国家食管腺癌 (EAC) 的发病率急剧增加。 EAC 是一种侵袭性肿瘤，通常在晚期才被诊断出来，导致总体生存率非常低。然而，如果及早发现，可以选择有效的治疗方案，包括消融、内镜下粘膜切除术和食管切除术，联合或不联合化疗和放疗。 EAC 通常出现在慢性胃食管反流病的情况下，并伴有食管柱状化生（称为巴雷特食管 (BE)）的相关发展。 BE 是已知最强烈的 EAC 危险因素，因此建议 BE 患者参加内窥镜监测计划以检测发育不良或早期 EAC。然而，使用重复内窥镜检查和多次活检是一种昂贵且侵入性的 BE 监测模型。我们相信，将食管细胞学与新一代测序相结合来检测驱动不典型增生和 EAC 发展的突变，将为 BE 监测提供一种新颖的、侵入性较小且更具成本效益的方法。在具体目标 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)) 与对一组约 100 个已知是 EAC 发展驱动因素的基因进行靶向下一代测序相结合，来测试这一假设的可行性。该组将从 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 数据中进行鉴定。我们可以通过我们之前发表的工作（Dulak 等人，Nature Genetics 2013）以及加拿大基因组资助的一项正在进行的研究获得这些数据。这些数据以及在美国立即使用的 EsophaCap(tm) 使我们处于独特的地位，可以评估分子细胞学测试检测 BE 患者疾病进展的可行性。具体来说，在目标 1A 中，我们将确定在多个活检样本中鉴定出的 EAC 驱动突变的比例在匹配的细胞学样本中也可检测到。这将确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”，从而稀释突变等位基因部分。在相关目标 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，并且还将有助于分析 BE 中的克隆异质性和进化。具体目标 2 将采用类似的方法来解决有关 BE 消融的关键问题；消融后形成的新鳞状上皮在遗传上是否正常，或者是否含有仍可能引发癌症风险的突变？这将通过比较消融前后 BE 和新鳞状上皮的突变来确定。这些数据将有助于确定接受消融治疗的 BE 患者是否需要进行随访监测，并将深入了解产生 BE 和新鳞状上皮的干细胞的起源。

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.