Identification of Cell Surface Targets Based on Gene Amplification/Overexpression

在过去的几十年里，食管远端和胃食管交界处腺癌的发病率以超过350%的惊人速度增加（1）。这是任何癌症类型中观察到的发病率增加最多的一种。当这些癌症被早期发现时，患者在手术切除后具有相对良好的结果。这种疾病的快速血管和淋巴扩散以及对放疗和化疗的不良临床反应导致大多数患者的预后不良。尽管采用了多模式治疗，食管癌的5年生存率仍然很低，仅为5-15%（2，3）。这突出了开发新方法用于癌前病变（如Barrett化生）患者早期癌症检测的重要性，并改进新的治疗方式以提高这种致命疾病患者的生存率。 肠（巴雷特）化生的存在，其中正常鳞状粘膜是 被柱状上皮替代，与食管腺癌相关（2-6）。这种疾病是 在美国和其他西方社会，肥胖人口迅速增加。在 在有酸反流症状的患者中，初次内镜检查时发现Barrett粘膜约占10-12%（5，6）。据估计，Barrett化生患者发生腺癌的风险是普通人群的30 - 40倍（3，4）。非异型增生巴雷特化生进展为低度异型增生、高度异型增生，最终发展为浸润性腺癌，证明了这种疾病的恶性潜力。出于这个原因，已知患有巴雷特的患者需要定期进行内窥镜监测。 目前，通过白色光内窥镜和随机四象限活检进行监测。然而，在这方面， 在食管中，癌前病变是平坦的并且在内窥镜下与肠化生难以区分， 因此该方法的灵敏度和特异性是有限的。此外，这种方法还没有被证明 降低肠上皮化生向腺癌进展的速度（7），因此， 巴雷特食管的监测是非常必要的。 虽然胃食管反流病（GERD）和化生变化的发展， 公认的关键事件，许多特定的分子事件，腺癌的发展 不完全理解（2-6）。家族因素可能作为常染色体显性模式与不完全遗传有关的观点被假设为遗传易感性的基础（8）。 GERD的发展也可能反映了家族遗传的另一个方面， 与配偶对照组相比，在受影响的兄弟姐妹和父母对中频繁发生（9）。的巨大 在食管腺癌中检测到的分子异质性提示多种途径或转化事件的特定组合可能发生在Barrett化生个体中并导致腺癌。与食管下段腺癌和胃食管连接部腺癌发生相关的事件似乎相似，可能反映了相似的病因。 本申请中提出的研究纳入了两种综合方法，以解决 食管腺癌的临床问题日益突出。使用我们的SNP阵列分析食管癌 我们将首先寻找发生在这种腺癌中的非随机DNA扩增的所有组成部分。 肿瘤类型然后，我们将确定定位在每个扩增的核心区域内的基因和编码表面蛋白的基因。使用我们的基因表达数据库，巴雷特化生无异型增生，巴雷特的低度异型增生，高度异型增生和浸润性腺癌，我们将定义那些基因，特别是在癌细胞中过表达。将使用组织微阵列和免疫组织化学来验证候选基因蛋白的过表达。验证的候选基因将被转染并在模型细胞系中过表达，以允许我们选择针对这些基因产物的特异性肽。当荧光标记时，这些肽可以在常规内窥镜检查期间局部施用到高危患者的食管上，以观察大的表面积并引导组织活检。将按照项目2所述进行体内确认。这种方法有可能通过在最有效治疗的时候检测癌症来提高患者的生存率。我们现在相信，我们已经具备了制定一项合理的计划来应对日益严重的健康问题的所有要素。

The incidence of adenocarcinoma of the distal esophagus and gastroesophageal junction has increased at an alarming rate of over 350% in the last few decades (1). This is among the greatest increase in incidence observed for any cancer type. When these cancers are detected early, patients have a relatively good outcome following surgical resection. The rapid vascular and lymphatic spread of this disease and the poor clinical response to radiation and chemotherapy, results in a poor outcome for most patients. Despite multimodal therapy, the 5-year survival rate for esophageal cancer remains dismal at only 5-15% (2,3). This highlights the importance of developing new methods for early cancer detection in patients with premalignant lesions such as Barrett's metaplasia and refining new treatment modalities to improve the survival of patients with this deadly disease. The presence of intestinal (Barrett's) metaplasia, a condition in which the normal squamous mucosa is replaced by columnar epithelium, is associated with esophageal adenocarcinoma (2-6). This disease is increased in obese individuals, a population rapidly expanding in the US and other Western societies. In patients with symptoms of acid reflux, Barrett's mucosa is found in ~10-12% at the time of initial endoscopy (5,6). Patients with Barrett's metaplasia have an estimated 30 to 40-fold greater risk of adenocarcinoma than the general population (3,4). The malignant potential of this condition is evidenced by the progression of non-dysplastic Barrett's metaplasia to low-grade dysplasia, high-grade dysplasia and finally to invasive adenocarcinoma. For this reason, patients with known Barrett's have regular endoscopic surveillance. Currently, surveillance is performed by white light endoscopy and random four-quadrant biopsy. However, in the esophagus, precancerous lesions are flat and endoscopically Indistinct from intesinal metaplasia, thus the sensitivity and specificity of this method is limited. Moreover, this method has not been shown to reduce the rate of progression of intestinal metaplasia to adenocarcinoma (7), thus, a new strategy for surveillance of Barrett's esophagus is critically needed. Although gastroesophageal reflux disease (GERD) and the development of metaplastic changes are recognized critical events, many specific molecular events underlying adenocarcinoma development remain incompletely understood (2-6). The idea that familial factors may be involved as an autosomaldominant pattern with incomplete penetrance has been hypothesized to underlie genetic predisposition (8). The development of GERD may also reflect another aspect of familial inheritance, as it is much more frequent in both affected sibling and parent pairs when compared to spouse controls (9). The tremendous molecular heterogeneity detected in esophageal adenocarcinomas suggests multiple pathways, or specific combinations of transforming events, may be occurring in individuals with Barrett's metaplasia and that lead to adenocarcinoma. The events associated with the development of both adenocarcinoma of the lower esophagus and the gastroesophageal junction, appear to be similar and may reflect the similar efiologies. The studies proposed in this application incorporate two integrated approaches to address the increasing clinical problem of esophageal adenocarcinoma. Using our SNP-array analyses of esophageal adenocarcinoma we will first search for the repertoire of nonrandom DNA amplification that occurs in this tumor type. We will then identify the genes localized within each amplified core region and those genes that encode surface proteins. Using our gene expression database for Barrett's metaplasia without dysplasia, Barrett's with low-grade dysplasia, high-grade dysplasia and invasive adenocarcinoma we will define those genes that are specifically over-expressed in cancer cells. Tissue microarrays and immunohistochemistry will be used to validate candidate gene protein over-expression. Validated candidate genes will be transfected and over-expressed in model cell lines to allow us to select specific peptides directed against these gene products. When fluorescently-labeled, these peptides can be topically administered onto the esophagus of high-risk patients during routine endoscopy to view large surface areas and to guide tissue biopsy. In vivo validation will be performed as described in Project 2. This approach has the potential to increase patient survival by detecting cancer at a point when it is most effectively treated. We now believe we have all of the components in place to make a rational plan of attack against a growing health problem.