A MicroRNA Approach to Screen for Sporadic Colon Cancer Exfoliately in Human Stoo

描述（由申请人提供）：本申报的目的是开发一种定量的microRNA检测方法，用于筛查患者粪便中的左右结肠癌，特别是在早期腺瘤阶段（例如，31厘米息肉伴高度不典型增生），该方法比FOBT灵敏度更高，依从性更好，而且比侵入性结肠镜检查更经济。我们通过茎环RT引物和TaqMan minor grove结合探针在30例（5例正常，10例IBD和15例结肠癌）的粪便和组织中研究了15种成熟mirna的表达。一些mirna在粪便和组织中表现出优先表达，在晚期结直肠癌Dukes期更为明显。基于这些重要且已发表的结果，我们提出了一项随机巢式病例对照研究，从500名受试者中随机选择60名受试者[20名对照组和40名结直肠癌患者{20名癌前息肉（0-1期）和20名结肠癌（2 - 4期）}]，对粪便中的15种mirna进行盲测。为了建立临床敏感性和特异性，miRNA结果将与结肠镜检查、FOBT和病理数据相关联。该方法的数值基础将来自分离的结肠细胞和粪便的细胞学和分子方法，以确定转化的miRNA的比例作为总mRNA表达的函数。标准化将建立测试的性能标准（最佳的取样准备、保存、储存和运行条件），以确保该分析在任何实验室都能以相同的方式进行。，由受过训练的人员负责。在这一步成功之后，将进行更大样本的验证研究。1993年，人们设想基于人群的结直肠癌（CRC）筛查已经到来(1)。16年后，尽管有多种筛查试验，我们仍未实现筛查大多数符合条件的美国人口的目标。在美国，结直肠癌（CRC）被认为是男性和女性中第二和第三常见的恶性肿瘤，分别占癌症发病率和癌症死亡人数的10%。大约6%的人会在一生中患上结直肠癌。2007年，估计有153,760例新病例和52 180例死亡(2)。在全球范围内，每年约有100万新病例和约50万例死亡(3)，由于世界范围内采用西方饮食，这些数字注定会增加(4)。结直肠癌是美国白人、非裔美国人、亚洲/太平洋岛民和印第安/阿拉斯加原住民男性癌症死亡的第三大原因，但在西班牙裔男性中排名第二(5)。结直肠癌发病率从1975年的60 / 10万下降到2004年的50 / 10万，美国死亡率的下降最近也在加速（即，死亡率从1970年的29 / 10万下降到2004年的18 / 10万，白人男性和女性的下降幅度相似，尽管男性的下降开始于女性几年之后），但非洲裔美国男性和女性的变化不大。自1990年以来，非洲裔美国人的死亡率从每10万人约30人下降到25人，男性死亡率高于女性，大约自1980年以来，非洲裔美国人的死亡率高于白人。不同阶段的5年生存率差别很大，局部疾病的存活率为90%，远处疾病的存活率为10%，这就明确了早期发现的必要性(5)。三项随机临床试验（rct）证明，愈创木粪便潜血试验是唯一一种有效降低结直肠癌死亡率的筛查试验（6-8）。其他测试，如基于免疫化学（IMC）的粪便血液测试，虽然从未在随机对照试验中进行过测试，但已经与基于愈创木的测试进行了评估，并且至少表现得同样好，符合率更高（9-13）。结直肠癌是唯一一种被认为是金标准的结肠镜诊断试验推荐作为筛查试验的癌症（14.15）。还有其他几种可用于结肠癌筛查的方法[例如，柔性乙状结肠镜检查（16），CT结肠镜检查（虚拟结肠镜检查）（17），胶囊内镜检查（18），双重对比钡灌肠（19），粪便和血液分子DNA检测（20-25）；然而，没有一种是最理想的，在某些人群中发病率很低（26,27）。最近，越来越多的病变发生在结肠的更近端区域，并且随着年龄的增长，右侧CRC病变的发病率也在增加（28,29），这就需要使用柔性光纤乙状结肠镜。然而，在美国，对7000万50岁以上的老年人进行结肠镜检查每年可能要花费100亿美元，并且超出了医生的能力（30,31）。显然，需要一种简单、廉价、无创、敏感和特异性的筛查试验来识别有发展为晚期腺瘤或结直肠癌风险的人，这些人将从随后的结肠镜检查中受益。目前，男女CRC筛查参与率均低于30%，而乳腺癌和宫颈癌筛查参与率分别为70%至80%（32）。可以通过使用不那么不舒服、成本更低、准确性更高（灵敏度和特异性更高）的分子测试来提高参与度。然而，需要更大规模的精心设计的临床研究来证实初步结果。

DESCRIPTION (provided by applicant): The aim of this submission is to develop a quantitative microRNA assay for screening right and left colon cancer in stool of patients, particularly at the early adenoma stage (e.g., polyps 3 1 cm with high grade dysplasia), which shows higher sensitivity than FOBT, and results in better compliance & is more economical than invasive colonoscopy. We studied the expression of 15 mature miRNAs by stem-loop RT primers and TaqMan minor grove binding probes in stool and tissue of 30 individuals (5 normal, 10 IBD & 15 with colon cancer). Several miRNAs showed preferential expression in stool and tissue, which was more pronounced in later CRC Dukes' stages. Based on these significant and published results, we propose a randomized nested case-control study to test the 15 miRNAs blindly in stool of 60 individuals chosen randomly from 500 subjects [20 controls & 40 CRC patients {20 precancerous polyps (stage 0-1), and 20 colon cancer (stages 2 to 4)}]. To establish clinical sensitivity and specificity, the miRNA results will be correlated with colonoscopy, FOBT and pathology data. A numerical underpinning of the method will be derived from cytological and molecular methods on isolated colonocytes and stool to determine the fraction of transformed miRNA as a function of total mRNA expression. Standardization will establish test's performance criteria (optimal sampling preparation, preservation, storage & running conditions) to ensure that the assay will perform the same way in any Lab., by any trained personnel. Success in this step will be followed by a validation study on larger sample. In 1993 it was envisioned that population-based colorectal cancer (CRC) screening had arrived (1). Sixteen years later we have not yet achieved the goal of screening most of the eligible USA population despite the availability of multiple screening tests. In the USA, colorectal cancer (CRC) considered the second and third most common malignancy in and women, respectively, represents 10% of incident cancers and cancer deaths. About 6% of the population will develop CRC in their lifetime. In 2007, 153,760 new cases and 52,180 deaths were estimated (2). Globally, there are about 1 million new cases and about 500,000 deaths per year (3), and these numbers are destined to increase because of worldwide adoption of a Western-type diet (4). CRC is the third leading cause of cancer death among white, African American, Asian/Pacific Islander and Indian/Alaska Native men, but second among Hispanic men in the USA (5). CRC incidence has declined from about 60 per 100,000 in 1975 to ~ 50 per 100,000 in 2004, and the decrease in USA mortality has recently accelerated (i.e., the rate decreased from about 29 per 100,000 population in 1970 to about 18 per 100,000 in 2004, with similar decline for white men and women, although the decline for men began several years after women), but it has changed little for African American men and women. Since 1990, the mortality in African Americans has decreased from about 30 to 25 per 100,000 populations, with higher mortality in men than women, and since about 1980 the mortality has been higher in African Americans than whites. Five-year survival rates are strikingly different by stage ranging from 90% for localized disease to 10% for distant disease, clearly arguing for early detection (5). The guaiac-based fecal occult blood test is the only screening test proved to be effective in reducing CRC mortality by three randomized clinical trials (RCTs) (6-8). Other tests such as an immunochemical (IMC)-based fecal blood test although never tested in a RCT, have been evaluated against guaiac-based tests and have performed at least as well with higher compliance rates (9-13). CRC is the only cancer for which the diagnostic test colonoscopy, considered to be the gold standard, is recommended as a screening test (14.15). There are several other methods available for colon cancer screening [e.g., flexible sigmoidoscopy (16), CT colonography (virtual colonoscopy) (17), capsule endoscopy (18), double contrast barium enema (19), molecular DNA tests in stool and blood (20-25); however, none is optimal, and with poor rates in some segments of the population (26,27). Recently, there has been an increase in the number of lesions arising from more proximal regions of the colon, and increasing incidence of right sided CRC lesions has been reported with increasing age (28,29), necessitating the use of flexible, fiber optic sigmoidoscopes. However, colonoscopy screening for the 70 million people older than 50 years of age in the USA could cost $10 billion per year and exceed the physician capacity to perform this procedure (30,31). Clearly, a simple, inexpensive, noninvasive, sensitive and specific screening test is needed to identify people at risk for developing advanced adenomas or CRC who would benefit from subsequent colonoscopy. Current participation rates in CRC screening are less than 30% for both genders, compared to rates of 70 to 80% for breast and cervical cancer screening, respectively (32). Participation could be enhanced by use of molecular tests that are less uncomfortable, less expensive and offer greater accuracy (more sensitivity and specificity). However, larger well designed clinical studies are needed to corroborate initial results.