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

描述（由申请人提供）：本申请的目的是开发一种定量microRNA测定法，用于筛查患者粪便中的右结肠癌和左结肠癌，特别是在早期腺瘤阶段（例如，息肉3 - 1cm，高度异型增生），其显示出比FOBT更高的灵敏度，并导致更好的依从性，比侵入性结肠镜检查更经济。我们用茎环RT引物和TaqMan minor格罗夫结合探针研究了30名个体（5名正常人、10名IBD和15名结肠癌患者）粪便和组织中15种成熟miRNA的表达。几种miRNA在粪便和组织中显示出优先表达，这在CRC Dukes分期后期更为明显。基于这些显著的和已发表的结果，我们提出了一项随机巢式病例对照研究，以在从500名受试者[20名对照和40名CRC患者{20名癌前息肉（0-1期）和20名结肠癌（2 - 4期）}]中随机选择的60名个体的粪便中盲测15种miRNA。为了建立临床灵敏度和特异性，miRNA结果将与结肠镜检查、FOBT和病理学数据相关联。该方法的数值基础将来自于对分离的结肠细胞和粪便的细胞学和分子学方法，以确定转化的miRNA的分数作为总mRNA表达的函数。标准化将建立测试的性能标准（最佳的样品制备、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，任何受过训练的人。这一步骤成功后，将进行更大样本的验证研究。 1993年，人们设想以人群为基础的结直肠癌（CRC）筛查已经到来（1）。16年后，尽管有多种筛查方法，我们仍然没有实现对大多数符合条件的美国人口进行筛查的目标。 在美国，结直肠癌（CRC）被认为是第二和第三最常见的恶性肿瘤，分别在和妇女，占10%的发病率和癌症死亡。大约6%的人会在一生中发展为CRC。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50万人死亡（3），由于全世界采用西式饮食，这些数字注定会增加（4）。CRC是白色、非裔美国人、亚洲/太平洋岛民和印第安人/阿拉斯加原住民男性癌症死亡的第三大原因，但在美国西班牙裔男性中排名第二（5）。CRC发病率从1975年的约60/100，000下降到2004年的约50/100，000，并且美国死亡率的下降最近加速（即，这一比率从1970年的每100 000人中约29人下降到2004年的每100 000人中约18人，白色男子和妇女的下降幅度相似，尽管男子的下降比妇女晚几年开始），但非裔美国男子和妇女的变化不大。自1990年以来，非裔美国人的死亡率从每10万人中约30人下降到25人，男性死亡率高于女性，自1980年以来，非裔美国人的死亡率一直高于白人。5年生存率在不同阶段之间存在显著差异，从局部疾病的90%到远处疾病的10%，明确主张早期检测（5）。 基于愈创木脂的粪便潜血试验是唯一被三项随机临床试验（RCT）证明有效降低CRC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RCT中进行过测试，但已对基于愈创木脂的测试进行了评估，并且至少具有较高的依从率（9-13）。结肠直肠癌是唯一一种推荐使用诊断性结肠镜检查作为筛查试验的癌症，结肠镜检查被认为是金标准（14.15）。有几种其他方法可用于结肠癌筛查[例如，乙状结肠软镜检查（16）、CT结肠造影（虚拟结肠镜检查）（17）、胶囊内镜检查（18）、双重对比钡灌肠（19）、粪便和血液中的分子DNA检测（20-25）;然而，这些检查都不是最佳的，并且在某些人群中的发生率很低（26，27）。最近，来自结肠近端区域的病变数量有所增加，并且随着年龄的增长，右侧CRC病变的发生率也有所增加（28，29），因此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花费100亿美元，超过了医生执行该程序的能力（30，31）。显然，需要一种简单、廉价、无创、敏感和特异的筛查试验来识别有发展为晚期腺瘤或CRC风险的人，这些人将从随后的结肠镜检查中受益。 目前参与CRC筛查的男女比例均低于30%，而乳腺癌和宫颈癌筛查的比例分别为70%至80%（32）。可以通过使用不那么不舒服、更便宜和提供更高准确性（更敏感和特异性）的分子测试来加强参与。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.