Patent Publication Number: US-2022220562-A1

Title: In vitro method for identifying colorectal adenomas or colorectal cancer

Description:
FIELD OF THE INVENTION 
     The present invention can be included in the field of personalized medicine, wherein specific biomarkers are used for identifying a given disease or disorder. Specifically, some microRNAs (also named miRNAs or miR-) are used in the present invention for identifying human subjects at risk of developing colorectal cancer (CRC) and/or colorectal adenomas (CA), preferably advanced colorectal adenomas (AA). 
     PRIOR ART 
     Colorectal cancer (also known as colon cancer, rectal cancer, or bowel cancer) is the development of cancer in the colon or rectum (parts of the large intestine). The vast majority of colorectal cancers are adenocarcinomas. This is because the colon has numerous glands within the tissue. When these glands undergo a number of changes at the genetic level, they proceed in a predictable manner as they move from benign to an invasive, malignant colon cancer. The adenomas of the colon, also called adenomatous polyps, are a benign version of the malignant adenocarcinomas but still with malignant potential if not removed (they are usually removed because of their tendency to become malignant and to lead to colon cancer). 
     Screening is an effective way for preventing and decreasing deaths from colorectal cancer and is recommended starting from the age of 50 to 75. The best known and most frequently used screening test for colorectal cancer is called Fecal Immunochemical Test (FIT). FIT detects blood in the stool samples which can be a sign of pre-cancer or cancer. If abnormal results are obtained, usually a colonoscopy is recommended which allows the physician to look at the inside of the colon and rectum to make a diagnosis. During colonoscopy, small polyps may be removed if found. If a large polyp or tumor is found, a biopsy may be performed to check if it is cancerous. The gastroenterologist uses a colonoscopy to find and remove these adenomas and polyps to prevent them from continuing to acquire genetic changes that will lead to an invasive adenocarcinoma. 
     Although, as explained above, FIT is nowadays used for screening colorectal cancer, it is important to note that FIT offers a low sensitivity for adenomas (around 20-30% depending on literature) which means that most of said kind of patients can be wrongly classified as not having the disease. 
     Consequently, FIT is not able to identify adenomas due to its low sensitivity. Moreover, since FIT uses stool samples, it offers a low compliance (less than 50%). On the other hand, the colonoscopy is an invasive technique wherein the most severe complication generally is the gastrointestinal perforation (1% of the cases). Moreover, colonoscopy is nowadays a procedure involving anesthesia, and the laxatives which are usually administered during the bowel preparation for colonoscopy are associated with several digestive problems. 
     The present invention offers a clear solution to the problems cited above because it is focused on an in vitro method for identifying or screening human subjects at risk of suffering from colorectal cancer or colorectal adenomas (preferably advanced colorectal adenomas), departing from the level of expression of microRNAs isolated from minimally-invasive samples such as blood, serum or plasma. Moreover, the method of the invention offers high sensitivity and specificity (see the Examples shown below), which means that it is a strong and cost-effective method for the detection of both colorectal cancer or colorectal adenomas (preferably advanced colorectal adenomas). 
     It is important to note that the methods used today for screening general population at risk of suffering for CRC or AA are associated with a high rate of false positives. Consequently a high amount of unnecessary follow-up colonoscopies are nowadays performed. However, since the method of the invention has higher sensitivity and specificity as compared which the method used today for screening general population at risk of suffering from CRC or AA, it is associated with a lower percentage of false positives. Consequently, the method described in the present invention clearly helps in reducing the number of follow-up colonoscopies, thus improving the way that the patients are nowadays screened or diagnosed. Once the method of the invention is performed, if we see that the miRNAs are overexpressed, the result needs to be confirmed by colonoscopy. However, if we obtain a negative result because the miRNAs are down-regulated, there is no need to perform a colonoscopy and the patients go home. 
     BRIEF DESCRIPTION OF THE INVENTION 
     The present invention offers a clear solution for accurately screening and diagnosing subjects at risk of suffering from colorectal cancer and/or colorectal adenomas, by means of a minimally-invasive method able to detect not only patients at risk of suffering from colorectal cancer but also patients which could suffer from colorectal adenomas, preferably advanced colorectal adenomas. In particular, the present invention refers to an in vitro method for screening for subjects at average risk of developing colorectal adenomas and/or colorectal cancer by measuring the expression pattern or level of at least miR-15b in plasma samples. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1 . Receiver-operating-characteristic (ROC) curve for miR-15b in colorectal cancer. Area Under Curve (AUC)=0.833. X axis represents Specificity. Y axis represents Sensitivity. 
         FIG. 2 . Receiver-operating-characteristic (ROC) curve for miR-15b in colorectal advanced adenoma. Area Under Curve (AUC)=0.798. X axis represents Specificity. Y axis represents Sensitivity. 
         FIG. 3 . Receiver-operating-characteristic (ROC) curve for miR-29a+miR-15b in colorectal cancer. Area Under Curve (AUC)=0.878. X axis represents Specificity. Y axis represents Sensitivity. 
         FIG. 4 . Receiver-operating-characteristic (ROC) curve for miR-29a+miR-15b in colorectal advanced adenoma. Area Under Curve (AUC)=0.811. X axis represents Specificity. Y axis represents Sensitivity. 
         FIG. 5 . Receiver-operating-characteristic (ROC) curve for miR-29a+miR-15b+miR-18a in colorectal cancer. Area Under Curve (AUC)=0.880. X axis represents Specificity. Y axis represents Sensitivity. 
         FIG. 6 . Receiver-operating-characteristic (ROC) curve for miR-29a+miR-15b+miR-18a in colorectal advanced adenoma. Area Under Curve (AUC)=0.812. X axis represents Specificity. Y axis represents Sensitivity. 
         FIG. 7 . Receiver-operating-characteristic (ROC) curve for miR-29a+miR-15b+miR-19b in colorectal cancer. Area Under Curve (AUC)=0.876. X axis represents Specificity. Y axis represents Sensitivity. 
         FIG. 8 . Receiver-operating-characteristic (ROC) curve for miR-29a+miR-15b+miR-19b in colorectal advanced adenoma. Area Under Curve (AUC)=0.813. X axis represents Specificity. Y axis represents Sensitivity. 
         FIG. 9 . Receiver-operating-characteristic (ROC) curve for miR-29a+miR-15b+miR-19a in colorectal cancer. Area Under Curve (AUC)=0.880. X axis represents Specificity. Y axis represents Sensitivity. 
         FIG. 10 . Receiver-operating-characteristic (ROC) curve for miR-29a+miR-15b+miR-19a in colorectal advanced adenoma. Area Under Curve (AUC)=0.808. X axis represents Specificity. Y axis represents Sensitivity. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Description of the Invention 
     The present invention refers to an in vitro method for screening or identifying subjects at risk of suffering from colorectal cancer and/or colorectal adenomas, preferably advanced colorectal adenomas, based on measuring the expression profile or level of some miRNAs which are up-regulated or over-expressed in patients suffering from said diseases. The present invention also refers to an in vitro method for obtaining useful data for the diagnosis of colorectal cancer and/or colorectal adenomas, preferably advanced colorectal adenomas, in a subject, preferably in a human subject. 
     In particular, the present invention is based on the discovery that miR-15b is confirmed to be significantly up-regulated in plasma samples of patients with colorectal cancer. Moreover, remarkably as shown in Tables 6, 7 and 8, the results provided herein also show that high circulating levels of miR-15b in plasma are significantly associated with the presence of advanced colorectal adenoma, wherein miR-15b offers the best results in comparison to the rest of the miRNAs tested herein. In fact, the results obtained with miR-15b by itself are even better than those shown for miR-29a by itself for screening for the presence of advanced colorectal adenoma or for obtaining useful data for the diagnosis of advanced colorectal adenoma in a subject, preferably in a human subject. 
     In addition, as shown in Table 8, certain specific combinations of biomarkers departing from miR-15b, such as the combination of at least (miR-15b and miR-29a), are significantly up-regulated in plasma samples of subjects suffering from colorectal cancer and advanced adenomas. In this sense, as shown in Table 8, the combination of at least miR-15b and miR-29a provides significantly better results in terms of AUC, sensitivity and specificity values for both detecting the presence of advanced colorectal adenoma and for detecting the presence of colorectal cancer in comparison to the use of miR-15b or miR-29a by themselves. 
     Consequently, a first embodiment of the present invention refers to an in vitro method for screening for subjects, preferably human subjects, at risk of developing colorectal adenomas and/or colorectal cancer comprising: (a) measuring the expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) obtained from a minimally-invasive biological sample of the human subjects to be screened and (b) comparing said expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) of the human subjects to be screened with an already established expression pattern or level, wherein overexpression of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) is indicative of colorectal adenomas and/or colorectal cancer. In a preferred embodiment, the minimally-invasive biological sample obtained in the step (a) comprises: blood sample, plasma sample or serum sample. In a preferred embodiment, colorectal adenoma is advanced colorectal adenoma. In a preferred embodiment, the step (a) which comprises measuring the expression pattern or level of one microRNAs, is carried out by using a detectably labeled probe that hybridizes to a least one of the miRNAs described above. 
     A second embodiment of the present invention refers to an in vitro method for the diagnosis of colorectal adenoma and/or colorectal cancer in a subject, preferably in a human subject, comprising: (a) measuring the expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) obtained from a minimally-invasive biological sample of the subjects, preferably human subjects, suspected of suffering from colorectal adenoma and/or colorectal cancer, (b) comparing the expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) obtained from a minimally-invasive biological sample of the subjects, preferably human subjects, suspected of suffering from colorectal adenoma and/or colorectal cancer with the expression pattern or level of a normal subject, wherein the normal subject is a healthy subject not suffering from colorectal adenoma and/or colorectal cancer, and wherein over-expression of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) is indicative of colorectal adenoma and/or colorectal cancer, and optionally (c) confirming the diagnosis by means of the examination of the bowel by any means, preferably using colonoscopy. In a preferred embodiment, the minimally-invasive biological sample obtained in the step (a) comprises: blood sample, plasma sample or serum sample. In a preferred embodiment, colorectal adenoma is advanced colorectal adenoma. In a preferred embodiment, the step (a) which comprises measuring the expression pattern or level of one microRNAs, is carried out by using a detectably labeled probe that hybridizes to a least one of the miRNAs described above. 
     A third embodiment of the present invention refers to an in vitro method for obtaining useful data for the diagnosis of colorectal adenoma and/or colorectal cancer in subjects, preferably human subjects, comprising: (a) measuring the expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) obtained from a minimally-invasive biological sample of the subjects, preferably human subjects, suspected of suffering from colorectal cancer and/or colorectal adenoma and (b) comparing said expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) of the subjects, preferably human subjects, suspected of suffering from colorectal cancer and/or colorectal adenoma with an already established expression pattern or level, wherein overexpression of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) is indicative of colorectal adenomas and/or colorectal cancer. In a preferred embodiment, the minimally-invasive biological sample obtained in the step (a) comprises: blood sample, plasma sample or serum sample. In a preferred embodiment, colorectal adenoma is advanced colorectal adenoma. In a preferred embodiment, the step (a) which comprises measuring the expression pattern or level of one microRNAs, is carried out by using a detectably labeled probe that hybridizes to a least one of the miRNAs described above. 
     A fourth embodiment of the present invention refers to the use of a kit comprising biomarker detecting reagents for determining a differential expression level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a), wherein overexpression of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) is indicative of colorectal adenoma and/or colorectal cancer, for diagnosing in vitro the risk for colorectal adenoma and/or colorectal cancer. In a preferred embodiment, colorectal adenoma is advanced colorectal adenoma. In a preferred embodiment, the step which comprises measuring the expression pattern or level of one microRNA, is carried out by using a detectably labeled probe that hybridizes to a least one of the miRNAs described above. 
     A fifth embodiment of the present invention refers to an in vitro method for classifying subjects, preferably human subjects, as healthy subjects or as subjects suffering from colorectal adenoma and/or colorectal cancer comprising: (a) measuring the expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) obtained from a minimally-invasive biological sample of the subjects, preferably human subjects, to be classified and (b) comparing said expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) of the subjects, preferably human subjects, to be classified with an already established expression pattern or level, wherein overexpression of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) is indicative of colorectal adenomas and/or colorectal cancer. In a preferred embodiment, the minimally-invasive biological sample obtained in the step (a) comprises: blood sample, plasma sample or serum sample. In a preferred embodiment, colorectal adenoma is advanced colorectal adenoma. In a preferred embodiment, the step (a) which comprises measuring the expression pattern or level of one microRNAs, is carried out by using a detectably labeled probe that hybridizes to a least one of the miRNAs described above. 
     A sixth embodiment of the present invention refers to a method for detecting any possible combination of at least two, three, four or five biomarkers from the following miRNAs: miR-29a, miR-15b, miR-18a, miR-19a or miR-19b. In a preferred embodiment the biomarkers detected in the present invention are: at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a). 
     A seventh embodiment of the present invention refers to a method for treating subjects, preferably human subjects, suffering from colorectal adenoma and/or colorectal cancer comprising: (a) measuring in vitro the expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) obtained from a minimally-invasive biological sample of the subjects, preferably human subjects, to be treated, (b) comparing said expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) of the subjects, preferably human subjects, to be treated with an already established expression pattern or level, wherein overexpression of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) is indicative of colorectal adenomas and/or colorectal cancer and (c) treating the patient diagnosed with colorectal adenoma and/or colorectal cancer. In a preferred embodiment, the minimally-invasive biological sample obtained in the step (a) comprises: blood sample, plasma sample or serum sample. In a preferred embodiment, colorectal adenoma is advanced colorectal adenoma. In a preferred embodiment, the step (a) which comprises measuring the expression pattern or level of one microRNAs, is carried out by using a detectably labeled probe that hybridizes to a least one of the miRNAs described above. In a preferred embodiment the method comprises confirming the diagnosis by means of the examination of the bowel by any means, preferably using colonoscopy. Since colorectal adenoma can be seen as a precursor of colorectal cancer, because of the acknowledged adenoma-carcinoma sequence, and the notion that advanced colorectal adenomas are more likely to transition to cancer, it is well established that colorectal adenomas, and preferably colorectal advanced adenomas, should be treated, preferably, by being removed through colonoscopy (subsequent surveillance could be performed). Treatment of colorectal cancer depends on the stage at which cancer was discovered. Early stage colorectal cancer is best treated with surgery. Approximately 95% of Stage I and 65-80% of Stage II colorectal cancers are curable with surgery. Rectal cancer, however, may require additional radiation therapy to minimize the risk of recurrence. Advanced stage (Stage III and Stage IV) treatment often comprises combination of therapies, including: surgery, chemotherapy, treatment with antibodies, therapies anti-VEGF/R and radiation. 
     An eight embodiment of the present invention refers to an in vitro method for assessing or monitoring the response to a therapy in a subject suffering from colorectal adenoma and/or colorectal cancer comprising: (a) measuring the expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) obtained from a minimally-invasive biological sample of the subjects, preferably human subjects, to be monitored and (b) comparing said expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) of the subjects, preferably human subjects, to be monitored with an already established expression pattern or level, wherein overexpression of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) is indicative of colorectal adenomas and/or colorectal cancer. A significant decrease or lack of change in the miRNA levels after the administration of said therapy in comparison with the level of the miRNAs prior to the administration of the therapy in the subject sample is indicative that the therapy administered to the subject is efficacious. A significant increase in the miRNA levels after the administration of said therapy in comparison with the level of expression of each of the miRNAs prior to the administration of the therapy in the subject sample is indicative that the therapy administered to the subject is inefficacious. In a preferred embodiment, the minimally-invasive biological sample obtained in the step (a) comprises: blood sample, plasma sample or serum sample. In a preferred embodiment, colorectal adenoma is advanced colorectal adenoma. In a preferred embodiment, the step (a) which comprises measuring the expression pattern or level of one microRNAs, is carried out by using a detectably labeled probe that hybridizes to a least one of the miRNAs described above. 
     A ninth embodiment of the present invention refers to an in vitro method for monitoring the progression of colorectal adenoma and/or colorectal cancer in a subject comprising: (a) measuring the expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) obtained from a minimally-invasive biological sample of the subjects, preferably human subjects, to be monitored and (b) comparing said expression pattern or level of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) of the subjects, preferably human subjects, to be monitored with an already established expression pattern or level, wherein overexpression of at least (miR-15b) or of at least (miR-15b and miR-29a) or of at least (miR-15b and miR-18a), or of at least (miR-15b and miR-19b), or of at least (miR-15b and miR-19a), or of at least (miR-15b, miR-29a and miR-18a), or of at least (miR-15b, miR-19b and miR-18a), or of at least (miR-15b, miR-29a and miR-19b), or of at least (miR-15b, miR-19a and miR-18a), or of at least (miR-15b, miR-29a and miR-19a), or of at least (miR-15b, miR-19a and miR-19b), or of at least (miR-15b, miR-29a, miR-19a, miR-19b and miR-18a) is indicative of colorectal adenomas and/or colorectal cancer. A significant decrease or a lack of change in the miRNA levels in comparison with the level of expression of said miRNAs at the earlier time point is indicative that the colorectal adenoma and/or colorectal cancer shows a good progression. A significant increase in the miRNA levels in comparison with the level of expression of said miRNAs at the earlier time point is indicative that the colorectal adenoma and/or colorectal cancer shows a bad progression. In a preferred embodiment, the minimally-invasive biological sample obtained in the step (a) comprises: blood sample, plasma sample or serum sample. In a preferred embodiment, colorectal adenoma is advanced colorectal adenoma. In a preferred embodiment, the step (a) which comprises measuring the expression pattern or level of one microRNAs, is carried out by using a detectably labeled probe that hybridizes to a least one of the miRNAs described above. 
     A tenth embodiment of the invention refers to a kit comprising biomarker detecting reagents for determining a differential expression level of one or more miRNAs selected from the group consisting of: miR15b; miR-15b and miR-29a; miR-15b and miR-29a and miR-18a; miR-15b and miR-29a and miR-19a; miR-15b and miR-29a and miR-19b; miR-15b and miR-29a and miR-18a and miR-19a and miR-19b. Please note that this specific embodiment of the invention refers to a numerous clauses of miRNAs. In addition, preferably the aforesaid kit comprises reagents selected from the group consisting of all or of at least one of the following: i) specific primers to the miRNA or combinations of miRNAs as defined in claim  13  capable of producing primer-ligated miRNA sequences; ii) reverse transcribing means to produce cDNAs from the primer-ligated miRNA sequences of i); iii) means such as primers capable of amplifying the cDNAs derived from the primer-ligated miRNA sequences as defined in i); iv) means to transcribe the amplified cDNAs to produce sense target RNAs; and v) a population of miRNA antisense probes capable of detecting the sense target RNAs of iv). 
     An eleventh embodiment of the invention refers to the use of the kit of the tenth embodiment of the invention, for determining a differential expression level of at least miR15b, wherein overexpression of at least miR15b is indicative of colorectal adenomas or colorectal cancer, for screening or obtaining useful data for diagnosing in vitro the risk for colorectal adenomas, preferably advanced colorectal adenoma, and/or colorectal cancer. 
     On the other hand, it is worth mentioning that the present invention is preferably carried out in plasma or serum samples obtained from the patients, and that it is well known in the prior art that the fact that a given biomarker is suitable for the diagnosis of a given disease departing from tissue samples, does not directly mean that the same positive result will be reproduced when the sample used for implementing the method is serum or plasma. On the other hand, it is important to emphasize that obtaining serum or plasma preparations from blood comprises several steps carried out by technicians: in the case of serum, allowing the blood to clot by leaving it undisturbed at room temperature, removing the clot by centrifuging and isolating the supernatant which is designed as serum. In the case of plasma, centrifugation is also needed. Moreover, after the centrifugation process, it is important to immediately transfer the liquid component (serum or plasma) into a clean tube. The samples are maintained at 2-8° C. while handling. If the serum or plasma is not analyzed immediately, it should be apportioned into aliquots, stored, and transported at −80° C. or lower. It is important to avoid freeze-thaw cycles because this is detrimental to many serum components. Samples which are hemolyzed, icteric or lipemic can invalidate certain tests. 
     For the purpose of the present invention, the following definitions are included below:
         The term “screening” is understood as the examination or testing of a group of individuals pertaining to the general population, at risk of suffering from colorectal cancer or colorectal adenoma, with the objective of discriminating healthy individuals from those who are suffering from an undiagnosed colorectal cancer or colorectal adenoma or who are at high risk of suffering from said indications.   The term “colorectal cancer” is a medical condition characterized by cancer of cells of the intestinal tract below the small intestine (i.e., the large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum).   The expression “colorectal adenoma” refers to adenomas of the colon, also called adenomatous polyps, which is a benign and pre-cancerous stage of the colorectal cancer but still with high risk of progression to colorectal cancer.   The expression “advanced colorectal adenoma” refers to adenomas having a size of at least 10 mm or histologically having high grade dysplasia or a villous component higher than 20%.   The expression “minimally-invasive biological sample” refers to any sample which is taken from the body of the patient without the need of using harmful instruments, other than fine needles used for taking the blood from the patient, and consequently without being harmfully for the patient. Specifically, minimally-invasive biological sample refers in the present invention to: blood, serum, or plasma samples.   The term “up-regulated” or “over-expressed” of any of the micro-RNAs or combinations thereof described in the present invention, refers to an increase in their expression level with respect to a given “threshold value” or “cutoff value” by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%&gt;, by at least 65%&gt;, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%, by at least 110%, by at least 120%, by at least 130%, by at least 140%, by at least 150%, or more.   The term “threshold value” or “cutoff value”, when referring to the expression levels of the miRNAs described in the present invention, refers to a reference expression level indicative that a subject is likely to suffer from colorectal cancer or colorectal adenoma with a given sensitivity and specificity if the expression levels of the patient are above said threshold or cut-off or reference levels.   The term “comprising” it is meant including, but not limited to, whatever follows the word “comprising”. Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.   By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.   It is also noted that the term “kit” as used herein is not limited to any specific device and includes any device suitable for working the invention such as but not limited to microarrays, bioarrays, biochips or biochip arrays.       

     A variety of statistical and mathematical methods for establishing the threshold or cutoff level of expression are known in the prior art. A threshold or cutoff expression level for a particular biomarker may be selected, for example, based on data from Receiver Operating Characteristic (ROC) plots, as described in the Examples and Figures of the present invention. One of skill in the art will appreciate that these threshold or cutoff expression levels can be varied, for example, by moving along the ROC plot for a particular biomarker or combinations thereof, to obtain different values for sensitivity or specificity thereby affecting overall assay performance. For example, if the objective is to have a robust diagnostic method from a clinical point of view, we should try to have a high sensitivity. However, if the goal is to have a cost-effective method we should try to get a high specificity. The best cutoff refers to the value obtained from the ROC plot for a particular biomarker that produces the best sensitivity and specificity. Sensitivity and specificity values are calculated over the range of thresholds (cutoffs). Thus, the threshold or cutoff values can be selected such that the sensitivity and/or specificity are at least about 70%, and can be, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% in at least 60% of the patient population assayed, or in at least 65%, 70%, 75% or 80% of the patient population assayed. 
     Consequently, each of the above cited embodiments of the present invention is preferably carried out by determining the expression levels of at least the micro-RNAs previously cited in a minimally-invasive sample isolated from the subject to be diagnosed or screened, and comparing the expression levels of said micro-RNAs with predetermined threshold or cutoff values, wherein said predetermined threshold or cutoff values correspond to the expression level of said micro-RNAs which correlates with the highest specificity at a desired sensitivity in a ROC curve calculated based on the expression levels of the micro-RNAs determined in a patient population being at risk of suffering colorectal cancer or colorectal adenoma, wherein the overexpression of at least one of said micro-RNAs with respect to said predetermined cutoff value is indicative that the subject suffers from colorectal cancer or colorectal adenoma with said desired sensitivity. 
     The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. 
     The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. 
     Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the group. 
     Examples 
     Example 1. Population of Study 
     A total of 300 subjects from eight Spanish hospitals (Hospital Clinic de Barcelona, Hospital de Burgos, Hospital de Vigo, Hospital de Tenerife, Hospital de Alicante, Hospital de Donosti, Hospital de Ourense and Hospital de Zaragoza) were prospectively included in this study: 193 patients newly diagnosed with sporadic colorectal neoplasia (92 with CRC and 101 with AA) and 100 healthy individuals without personal history of any cancer and with a recent colonoscopy confirming the lack of colorectal neoplastic lesions. Patients with AA were those with adenomas having a size of at least 10 mm or histologically having high grade dysplasia or &gt;20% villous component. The characteristics of participants are shown in Table 1. Blood samples were collected prior to endoscopy or surgery in all individuals. 
     The study was approved by the Institutional Ethics Committee of Hospital Clinic of Barcelona (approval date: Mar. 11, 2014), and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                 Total  
                 Control 
                 AA 
                 CRC 
               
               
                   
                 (n = 293) 
                 (n = 100) 
                 (n = 101) 
                 (n = 92) 
               
               
                   
               
             
            
               
                 Mean age (SD) 
                 65.7 (11.5) 
                 60.7 (11.1) 
                 63.8 (9.4) 
                 73.1 (10.6) 
               
            
           
           
               
            
               
                 GENDER 
               
            
           
           
               
               
               
               
               
            
               
                 Male 
                 170 
                 51 
                 73 
                 46 
               
               
                 Female 
                 123 
                 49 
                 28 
                 46 
               
            
           
           
               
            
               
                 COLORECTAL FEATURES 
               
               
                 TNM stage 
               
            
           
           
               
               
               
               
               
            
               
                 I 
                 — 
                 — 
                 — 
                 20 
               
               
                 II 
                 — 
                 — 
                 — 
                 23 
               
               
                 III 
                 — 
                 — 
                 — 
                 32 
               
               
                 IV 
                 — 
                 — 
                 — 
                 12 
               
               
                 Unknown 
                 — 
                 — 
                 — 
                 5 
               
            
           
           
               
            
               
                 Location 
               
            
           
           
               
               
               
               
               
            
               
                 Ascending colon  
                 — 
                 — 
                 — 
                 30 
               
               
                 and cecum 
                   
                   
                   
                   
               
               
                 Descending colon 
                 — 
                 — 
                 — 
                 38 
               
               
                 and sigma 
                   
                   
                   
                   
               
               
                 Transverse colon 
                 — 
                 — 
                 — 
                 6 
               
               
                 Rectum 
                 — 
                 — 
                 — 
                 18 
               
            
           
           
               
            
               
                 Proximal/Distal 
               
            
           
           
               
               
               
               
               
            
               
                 Proximal 
                 — 
                 — 
                 — 
                 36 
               
               
                 Distal 
                 — 
                 — 
                 — 
                 56 
               
            
           
           
               
            
               
                 ADVANCED COLORECTAL ADENOMA FEATURES 
               
            
           
           
               
               
               
               
               
            
               
                 Size = &gt;10 mm 
                 — 
                 — 
                 93 
                 — 
               
               
                 Mean size (mm) (SD) 
                 — 
                 — 
                 20.2 (11.8) 
                 — 
               
               
                 Small AA (&lt;= 15 mm) 
                 — 
                 — 
                 51 
                 — 
               
               
                 Big AA (&gt;15 mm) 
                 — 
                 — 
                 47 
                 — 
               
               
                 No. AAs Mean (SD) 
                 — 
                 — 
                 3 (3) 
                 — 
               
               
                 Stage 0 
                 — 
                 — 
                 6 
                 — 
               
            
           
           
               
            
               
                 High-grade dysplasia 
               
            
           
           
               
               
               
               
               
            
               
                 Yes 
                 — 
                 — 
                 38 
                 — 
               
               
                 No 
                 — 
                 — 
                 63 
                 — 
               
            
           
           
               
            
               
                 Villous component 
               
            
           
           
               
               
               
               
               
            
               
                 Yes 
                 — 
                 — 
                 41 
                 — 
               
               
                 No 
                 — 
                 — 
                 55 
                 — 
               
               
                 Unknown 
                 — 
                 — 
                 5 
                 — 
               
               
                   
               
            
           
         
       
     
     Example 2. RNA Extraction 
     Ten mL of whole blood from each participant were collected in EDTA K3 containing tubes. Blood samples were placed at 4° C. until plasma separation. Samples were centrifuged at 1,600×g for 10 min at 4° C. to spin down blood cells, and plasma was transferred into new tubes, followed by further centrifugation at 16,000×g for 10 minutes at 4° C. to completely remove cellular components. Plasma was then aliquoted and stored at −80° C. until use. miRNA isolation was performed using mirVana™ PARIS™ kit (Ambion by Life Technologies). 
     The miRNAs were extracted from all plasma samples, retro-transcribed, pre-amplified and analyzed by Real-Time quantitative PCR. For each RNA sample, 3 control miRNAs (cel-miR-39-3p as “spike-in”, and hsa-miR-1228-3p) and 5 candidate miRNAs (hsa-miR-15b-5p, hsa-miR-18a-5p, hsa-miR-29a-3p, hsa-miR-19a-3p and hsa-miR-19b-3p) were analyzed separately and in combination. Each miRNA in each sample was assessed in triplicate and mean Ct values were normalized in order to obtain −deltaCT values (DCt). 
     MiRNA singleplex Retrotranscription protocol (RT) was performed using RT primers from TaqMan® MicroRNA Assays. (P/N: 4427975. Assay ID: specific for each miRNA-Life Technologies and using TaqMan® MicroRNA Reverse Transcription Kit. Life Technologies): Cel-miR-39-3p (Assay ID: 000200 Exogenous control), Hsa-miR-1228-3p (Assay ID: 002919 Endogenous control), Hsa-miR-15b-5p (Assay ID: 000390), Hsa-miR-18a-5p (Assay ID: 002422), Hsa-miR-29a-3p (Assay ID: 002112), Hsa-miR-19a-3p (Assay ID: 000395) and Hsa-miR-19b-3p (Assay ID: 000396). 
     RT protocol was performed in Veriti® 96 well Thermal Cycler P/N (product number): 4375786 (Life Technologies). MiRNA singleplex Preamplification protocol (Preamp) was performed for all miRNAs analyzed. Real Time primers from TaqMan® MicroRNA Assays (P/N Assay ID: specific for each miRNA. Life Technologies as previously described) were used. TaqMan® Preamp Master Mix was used (Life Technologies). PreAmplification analyses were performed in Veriti® 96 well Thermal Cycler P/N: 4375786 (Life Technologies.) Real Time PCR protocol for miRNA expression analysis were performed using Real Time primer 20× from TaqMan® MicroRNA Assays (P/N Assay ID: specific for each miRNA-Life Technologies as previously described). Expression analyses were performed in a ViiA™ 7 Real-Time PCR System (P/N: 4375786-Life technologies.) Ct values were calculated from automatic threshold. No template controls showed no amplification. Three technical replicates were included for each point of qPCR. Relative expression levels of selected miRNAs were calculated for each sample as DCt values [DCt=Ct of target miRNA−Ct of internal control miRNA]. 
     Example 3. Statistical Analysis 
     Differences in the miRNA expression levels (−DCt values) between different groups of patients were explored using multivariate logistic regression analysis adjusted by age, gender and site computing the odds ratio and intervals and their corresponding p values. ROC analysis plots and derived cut-points, as well as overall discriminative accuracy parameters, have been computed using DiagnosisMed R-package considering each miRNA expression as a continuous variable. Sensitivity (Sn), specificity (Sp), were calculated for several cut-points (the optimum cut-point associated with the minimum distance between the ROC curve and upper left corner, the cut-point associated with 80% Sn, with 85% Sn and with 90% Sn) (see Table 2 for colorectal cancer and Table 3 for advanced colorectal adenoma). 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Colorectal Cancer 
                 Critera 
                 Cut-off 
                 Sn 
                 Sp 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 miR-19a + miR-19b + miR-15b 
               
            
           
           
               
               
               
               
               
            
               
                 AUC = 0.845 
                 Min.ROC distance 
                 0.530 
                 70 
                 86 
               
               
                 AUC = 0.845 
                 Max.Youden Index 
                 0.618 
                 64 
                 94 
               
            
           
           
               
            
               
                 miR-19a + miR-29a + miR-15b 
               
            
           
           
               
               
               
               
               
            
               
                 AUC = 0.880 
                 Min.ROC distance 
                 0.466 
                 76 
                 88 
               
               
                 AUC = 0.880 
                 Max.Youden Index 
                 0.466 
                 76 
                 88 
               
               
                 AUC = 0.880 
                 Sn = Sp 
                 0.430 
                 79 
                 80 
               
               
                 AUC = 0.880 
                 Sn 80% 
                 0.377 
                 81 
                 75 
               
            
           
           
               
            
               
                 miR-19a + miR-15b + miR-18a 
               
            
           
           
               
               
               
               
               
            
               
                 AUC = 0.842 
                 Max.Youden Index 
                 0.580 
                 66 
                 93 
               
            
           
           
               
            
               
                 miR-19b + miR-29a + miR-15b 
               
            
           
           
               
               
               
               
               
            
               
                 AUC = 0.876 
                 Min.ROC distance 
                 0.524 
                 74 
                 89 
               
               
                 AUC = 0.876 
                 Max.Youden Index 
                 0.524 
                 74 
                 89 
               
               
                 AUC = 0.876 
                 Sn = Sp 
                 0.415 
                 77 
                 77 
               
               
                 AUC = 0.876 
                 Sn = 80% 
                 0.396 
                 80 
                 75 
               
            
           
           
               
            
               
                 miR-19b + miR-15b + miR-18a 
               
            
           
           
               
               
               
               
               
            
               
                 AUC = 0.845 
                 Max.Youden Index 
                 0.543 
                 67 
                 92 
               
            
           
           
               
            
               
                 miR-29a + miR-I5b + miR-18a 
               
            
           
           
               
               
               
               
               
            
               
                 AUC = 0.880 
                 Min.ROC distance 
                 0.504 
                 75 
                 87 
               
               
                 AUC = 0.880 
                 Max.Youden Index 
                 0.549 
                 72 
                 90 
               
               
                 AUC = 0.880 
                 Sp 70% 
                 0.362 
                 80 
                 71 
               
               
                 AUC = 0.880 
                 Sn = 80% 
                 0.333 
                 81 
                 69 
               
            
           
           
               
            
               
                 miR-19a + miR-15b 
               
            
           
           
               
               
               
               
               
            
               
                 AUC = 0.838 
                 Min.ROC distance 
                 0.518 
                 70 
                 88 
               
               
                 AUC = 0.838 
                 Max.Youden Index 
                 0.621 
                 62 
                 95 
               
            
           
           
               
            
               
                 miR-19b + miR-15b 
               
            
           
           
               
               
               
               
               
            
               
                 AUC = 0.839 
                 Min.ROC distance 
                 0.518 
                 70 
                 88 
               
               
                 AUC = 0.839 
                 Max.Youden Index 
                 0.640 
                 64 
                 95 
               
            
           
           
               
            
               
                 miR-29a + miR-15b 
               
            
           
           
               
               
               
               
               
            
               
                 AUC = 0.878 
                 Min.ROC distance 
                 0.478 
                 75 
                 88 
               
               
                 AUC = 0.878 
                 Max.Youden Index 
                 0.478 
                 75 
                 88 
               
               
                 AUC = 0.878 
                 Sn = Sp 
                 0.435 
                 78 
                 79 
               
            
           
           
               
            
               
                 miR-15b + miR-18a 
               
            
           
           
               
               
               
               
               
            
               
                 AUC = 0.836 
                 Max.Youden Index 
                 0.590 
                 64 
                 93 
               
            
           
           
               
            
               
                 miR-15b 
               
            
           
           
               
               
               
               
               
            
               
                 AUC = 0.833 
                 Min.ROC distance 
                 0.514 
                 68 
                 86 
               
               
                 AUC = 0.833 
                 Max.Youden Index 
                 0.621 
                 63 
                 95 
               
               
                 AUC = 0.833 
                 Sn 80% 
                 0.301 
                 80 
                 58 
               
               
                 AUC = 0.833 
                 Sn 85% 
                 0.284 
                 85 
                 58 
               
               
                 AUC = 0.833 
                 Sn 90% 
                 0.215 
                 90 
                 44 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
             
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Advanced 
                   
                   
                   
                   
               
               
                   
                 Colorectal 
                   
                   
                   
                   
               
               
                   
                 Adenoma 
                 Criteria 
                 Cut-off 
                 Sn 
                 Sp 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 miR-19a + miR-19b + miR-15b 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 AUC = 0.799 
                 Min.ROC distance 
                 0.510 
                 67 
                 75 
               
               
                   
                 AUC = 0.799 
                 Max.Youden Index 
                 0.346 
                 87 
                 58 
               
            
           
           
               
            
               
                 miR-19a + miR-29a + miR-15b 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 AUC = 0.808 
                 Min.ROC distance 
                 0.523 
                 71 
                 78 
               
               
                   
                 AUC = 0.808 
                 Max.Youden Index 
                 0.523 
                 71 
                 78 
               
               
                   
                 AUC = 0.808 
                 Sn = Sp 
                 0.472 
                 71 
                 71 
               
            
           
           
               
            
               
                 miR-19a + miR-15b + miR-18a 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 AUC = 0.803 
                 Max.Youden Index 
                 0.290 
                 91 
                 52 
               
            
           
           
               
            
               
                 miR-19b + miR-29a + miR-15b 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 AUC = 0.813 
                 Min.ROC distance 
                 0.574 
                 67 
                 82 
               
               
                   
                 AUC = 0.813 
                 Max.Youden Index 
                 0.574 
                 66 
                 82 
               
               
                   
                 AUC = 0.813 
                 Sn = Sp 
                 0.484 
                 71 
                 71 
               
            
           
           
               
            
               
                 miR-19b + miR-15b + miR-18a 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 AUC = 0.798 
                 Max.Youden Index 
                 0.480 
                 69 
                 75 
               
            
           
           
               
            
               
                 miR-29a + miR-15b + miR-18a 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 AUC = 0.812 
                 Min.ROC distance 
                 0.517 
                 74 
                 73 
               
               
                   
                 AUC = 0.812 
                 Max.Youden Index 
                 0.599 
                 62 
                 86 
               
            
           
           
               
            
               
                 miR-19a + miR-15b 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 AUC = 0.795 
                 Min.ROC distance 
                 0.448 
                 72 
                 71 
               
               
                   
                 AUC = 0.795 
                 Max.Youden Index 
                 0.323 
                 90 
                 55 
               
            
           
           
               
            
               
                 miR-19b + miR-15b 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 AUC = 0.798 
                 Min.ROC distance 
                 0.483 
                 68 
                 74 
               
               
                   
                 AUC = 0.798 
                 Max.Youden Index 
                 0.332 
                 88 
                 54 
               
            
           
           
               
            
               
                 miR-29a + miR-15b 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 AUC = 0.811 
                 Min.ROC distance 
                 0.469 
                 74 
                 73 
               
               
                   
                 AUC = 0.811 
                 Max.Youden Index 
                 0.469 
                 74 
                 73 
               
            
           
           
               
            
               
                 miR-15b + miR-18a 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 AUC = 0.805 
                 Max.Youden Index 
                 0.468 
                 74 
                 71 
               
            
           
           
               
            
               
                 miR-15b 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 AUC = 0.798 
                 Min.ROC distance 
                 0.438 
                 76 
                 67 
               
               
                   
                 AUC = 0.798 
                 Max.Youden Index 
                 0.314 
                 90 
                 55 
               
               
                   
                 AUC = 0.798 
                 Sn 80% 
                 0.381 
                 81 
                 63 
               
               
                   
                 AUC = 0.798 
                 Sn 85% 
                 0.351 
                 86 
                 59 
               
               
                   
                 AUC = 0.798 
                 Sn 90% 
                 0.314 
                 90 
                 55 
               
               
                   
                   
               
            
           
         
       
     
     With all Ct values from all samples obtained from the Real-Time PCR we have used different normalization transformations in order to obtain −DCt. We have also selected normalization by multiple housekeeping miRNAs (cel-miR-39, hsa-miR-1228) instead of one. Normalization factor based on expression levels of the housekeeping miRNAs have been calculated by using geometric mean (http://genomebiology.com/2002/3/7/research/0034) of the different housekeeping combinations. All 3 housekeeping miRNAs (cel-miR-39, hsa-miR-1228) showed good Ct levels in all samples and showed high stability between samples, with no differences in their expression between groups. 
     Initially we used different normalization schemes (normalization using cel-miR-39; normalization using hsa-miR-1228, normalization using geometric mean of cel-mir-39, and hsa-miR-1228, normalization using geometric mean of cel-miR-39, and normalization using geometric mean of cel-miR-39 and hsa-miR-1228). 
     Although all six normalization methods gave very similar results, the most accurate was normalization using geometric mean of cel-miR-39 and hsa-miR-1228. 
     Example 4. Comparison of Patients with Colorectal Cancer Versus Control Individuals 
     Before analyzing results we have excluded some Ct values from the analysis for being above the maximum Ct value of the linearity range (obtained from our previous analytical validation). 
     The comparison of colorectal cancer patients versus control individuals was also carried out. Logistic regression results: Odds ratio (OR), 95% confidence interval (CI) and the corresponding p-value from logistic regression are shown for each individual miRNA in the model adjusted by age and gender. The “n” means the number of samples that have been finally analyzed for each miRNA (see Table 4). 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 CRC vs Control 
                   
                   
                   
                   
                   
               
               
                 Individual miRNA 
                 n 
                 OR 
                 Cl low 
                 Cl high 
                 p-value 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 miR-29a 
                 187 
                 1.80 
                 2.36 
                 1.37 
                 0.00002 
               
               
                 miR-15b 
                 171 
                 1.61 
                 2.17 
                 1.20 
                 0.00167 
               
               
                 miR-18a 
                 183 
                 1.26 
                 1.60 
                 0.99 
                 0.06364 
               
               
                 miR-19a 
                 187 
                 1.06 
                 1.32 
                 0.85 
                 0.63336 
               
               
                 miR-19b 
                 190 
                 0.96 
                 1.20 
                 0.77 
                 0.74196 
               
               
                   
               
            
           
         
       
     
     In order to correct any possible influence of the sample origin, logistic regression has also been adjusted by hospital of origin. In this analysis, only hospitals that had samples in the different groups have been included (see Table 5). For this reason the “n” is different from the table shown above (Table 4). 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 CRC vs Control 
                   
                   
                   
                   
                   
               
               
                 Individual miRNA 
                 n 
                 OR 
                 Cl low 
                 Cl high 
                 p-value 
               
               
                   
               
             
            
               
                 miR-29a 
                 173 
                 1.94 
                 2.68 
                 1.40 
                 0.0001 
               
               
                 miR-15b 
                 157 
                 1.53 
                 2.16 
                 1.08 
                 0.0163 
               
               
                 miR-18a 
                 169 
                 1.32 
                 1.73 
                 1.00 
                 0.0497 
               
               
                 miR-19a 
                 173 
                 1.15 
                 1.48 
                 0.89 
                 0.2956 
               
               
                 miR-19b 
                 176 
                 1.03 
                 1.35 
                 0.78 
                 0.8476 
               
               
                   
               
            
           
         
       
     
     Interestingly, miR-18a and miR-15b were confirmed to be significantly up-regulated in patients with colorectal cancer. 
     Example 5. Comparison of Patients with Advanced Colorectal Adenoma Versus Control Individuals 
     The comparison of advanced colorectal adenoma patients versus control individuals was also carried out. Logistic regression results: Odds ratio (OR), 95% confidence interval (CI) and the corresponding p-value from logistic regression are shown in Table 6 for each miRNA in the model adjusted by age and gender. The “n” means the number of samples that have been analyzed for each miRNA. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 AA vs Control 
                   
                   
                   
                   
                   
               
               
                 Individual miRNA 
                 n 
                 OR 
                 Cl low 
                 Cl high 
                 p-value 
               
               
                   
               
             
            
               
                 miR-15b 
                 183 
                 1.78 
                 2.33 
                 1.37 
                 0.00002 
               
               
                 miR-29a 
                 195 
                 1.37 
                 1.69 
                 1.12 
                 0.00235 
               
               
                 miR-18a 
                 194 
                 1.27 
                 1.57 
                 1.03 
                 0.02327 
               
               
                 miR-19a 
                 197 
                 1.10 
                 1.33 
                 0.91 
                 0.32414 
               
               
                 miR-19b 
                 201 
                 1.02 
                 1.24 
                 0.84 
                 0.85322 
               
               
                   
               
            
           
         
       
     
     In order to correct any possible influence of sample origin, logistic regression has also been adjusted by hospital of origin. In this analysis, only hospitals that had samples in the different groups have been included (see Table 7). For this reason the “n” is different from the Table 6 shown above. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 AA vs Control 
                   
                   
                   
                   
                   
               
               
                 Individual miRNA 
                 n 
                 OR 
                 Cl low 
                 Cl high 
                 p-value 
               
               
                   
               
             
            
               
                 miR-15b 
                 169 
                 1.94 
                 2.66 
                 1.41 
                 0.00004 
               
               
                 miR-29a 
                 181 
                 1.33 
                 1.66 
                 1.07 
                 0.01089 
               
               
                 miR-18a 
                 180 
                 1.33 
                 1.70 
                 1.04 
                 0.02413 
               
               
                 miR-19a 
                 183 
                 1.15 
                 1.42 
                 0.94 
                 0.17758 
               
               
                 miR-19b 
                 187 
                 1.10 
                 1.42 
                 0.85 
                 0.48836 
               
               
                   
               
            
           
         
       
     
     Results show that high circulating levels of miR-15b and miR-18a are significantly associated with the presence of advanced colorectal adenoma, but miR-15b offering the best results. 
     Example 6. miRNA Signatures for Colorectal Cancer and Advanced Colorectal Adenoma Detection Versus Control Population 
     The next step was to analyze any possible combinations of at least two of the five miRNAs to test the best signature to discriminate between colorectal cancer and/or advanced colorectal adenoma vs control individuals. 
     All possible combinations among miR-19a, miR-19b, miR-29a, miR-15b and miR-18a have been assessed and those models showing the highest AUC (= or &gt;0.88) for colorectal cancer vs Controls and the highest AUC (&gt;0.80) for advanced colorectal adenoma vs Controls when adjusted by age, gender and hospital, have been selected. It is worth pointing out all the best signatures for the diagnosis of colorectal cancer and/or advanced colorectal adenoma comprises at least miR-15b. Consequently, we tested the differences between the AUC, Sensitivity (Sn) and Specificity (Sp) comparing the following signature: miR-15b vs miR-29a vs miR-15b+miR-29a vs miR-15b+miR-29a+miR-18a vs miR-15b+miR-29a+miR-19a vs miR-15b+miR-29a+miR-19b. Results are shown in Table 8 below (*Min. Roc distance **Max.Youden Index). 
     
       
         
           
               
               
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 CRC 
                 AA 
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 AUC 
                 Criteria 
                 Sn 
                 Sp 
                 AUC 
                 Criteria 
                 Sn 
                 Sp 
               
               
                   
               
            
           
           
               
            
               
                 miR-29a 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 0.87 
                 * 
                 73 
                 82 
                 0.73 
                 * 
                 76 
                 61 
               
            
           
           
               
            
               
                 miR-15b 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 0.833 
                 * 
                 68 
                 86 
                 0.798 
                 * 
                 76 
                 67 
               
            
           
           
               
            
               
                 miR-29a + miR-15b 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 0.878 
                 * 
                 75 
                 88 
                 0.811 
                 * 
                 74 
                 73 
               
               
                 0.878 
                 ** 
                 75 
                 88 
                 0.811 
                 ** 
                 74 
                 73 
               
            
           
           
               
            
               
                 miR-29a + miR-15b + miR-19b 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 0.876 
                 * 
                 74 
                 89 
                 0.813 
                 * 
                 67 
                 82 
               
               
                 0.876 
                 ** 
                 74 
                 89 
                 0.813 
                 ** 
                 67 
                 82 
               
            
           
           
               
            
               
                 miR-29a + miR-15b + miR-18a 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 0.880 
                 * 
                 75 
                 87 
                 0.812 
                 * 
                 74 
                 73 
               
               
                 0.880 
                 ** 
                 72 
                 90 
                 0.812 
                 ** 
                 63 
                 86 
               
            
           
           
               
            
               
                 miR-29a + miR-15b + miR-19a 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 0.880 
                 * 
                 76 
                 88 
                 0.808 
                 * 
                 71 
                 78 
               
               
                 0.880 
                 ** 
                 76 
                 88 
                 0.808 
                 ** 
                 71 
                 78 
               
               
                   
               
            
           
         
       
     
     ROC curve parameters for the signature comprising miR-15b+miR-29a (area under curve (AUC) and 95% confidence interval (CI) are shown for all CRC cases as well as for different tumor stages (I/II and III/IV) and locations (right and left, with respect to the splenic flexure). In addition, we have analyzed AA taking into account AA&gt;10 mm vs AA&lt;10 mm (see Table 9 for CRC and Table 10 for AA). (*Min.Roc distance **Max.Youden Index). 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 9 
               
               
                   
                   
               
               
                   
                 AUC 
                 Criteria 
                 Sn 
                 Sp 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 CRC vs Control 
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.88 (0.82-0.93) 
                 * 
                 75 
                 88 
               
               
                   
                 0.88 (0.82-0.93) 
                 ** 
                 75 
                 88 
               
            
           
           
               
            
               
                 CRC (I/II) vs Control 
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.89 (0.83-0.96) 
                 * 
                 78 
                 88 
               
               
                   
                 0.89 (0.83-0.96) 
                 ** 
                 78 
                 88 
               
            
           
           
               
            
               
                 CRC (III/IV) vs Control 
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.89 (0.82-0.96) 
                 * 
                 86 
                 77 
               
               
                   
                 0.89 (0.82-0.96) 
                 ** 
                 86 
                 77 
               
            
           
           
               
            
               
                 CRC (Distal/Left) vs Control 
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.88 (0.82-0.94) 
                 * 
                 83 
                 76 
               
               
                   
                 0.88 (0.82-0.94) 
                 ** 
                 73 
                 87 
               
            
           
           
               
            
               
                 CRC (Proximal/Right) vs Control 
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.88 (0.80-0.96) 
                 * 
                 71 
                 94 
               
               
                   
                 0.88 (0.80-0.96) 
                 ** 
                 71 
                 94 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 10 
               
               
                   
                   
               
               
                   
                 AUC 
                 Criteria 
                 Sn 
                 Sp 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 AA vs Control 
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.81 (0.75-0.87) 
                 * 
                 74 
                 73 
               
               
                   
                 0.81 (0.75-0.87) 
                 ** 
                 74 
                 73 
               
            
           
           
               
            
               
                 Small AA vs Control 
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.83 (0.75-0.91) 
                 * 
                 77 
                 77 
               
               
                   
                 0.83 (0.75-0.91) 
                 ** 
                 64 
                 92 
               
            
           
           
               
            
               
                 Big AA vs Control 
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.84 (0.77-0.91) 
                 * 
                 74 
                 80 
               
               
                   
                 0.84 (0.77-0.91) 
                 ** 
                 85 
                 70