Non-invasive methods for diagnosing or screening for colorectal cancer and / or its precancerous stages

Abstract

Non-invasive method for the diagnosis or screening of colorectal cancer and / or pre-cancerous stages thereof The present invention relates to an in vitro method for the diagnosis of colorectal cancer and / or pre-cancerous stages thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from European Patent Application No. 19382597.3 filed on July 15, 2019.

[0002] The present invention can be included in the medical field. Specifically, the present invention relates to an in vitro method for diagnosing or screening colorectal cancer and / or its precancerous stages. [Background technology]

[0003] Colorectal cancer (CRC), also known as colon cancer, rectal cancer, or colorectal cancer, is the development of cancer in the colon or rectum (part of the large intestine). The majority of colorectal cancers are adenocarcinomas. This is because the colon contains numerous glands within its tissue. These glands undergo certain genetic changes and progress in a predictable manner, transitioning from benign to invasive, malignant colon cancer. Colon adenomas, particularly advanced colorectal adenomas (AA), are benign versions of malignant adenocarcinomas but still have the potential to become malignant if not removed (they are usually removed due to their tendency to become malignant and lead to colon cancer).

[0004] Screening is an effective method for preventing and reducing deaths from colorectal cancer and is recommended starting between the ages of 50 and 75. The most well-known and most frequently used screening test for colorectal cancer is called the fecal immunochemical test (FIT). FIT is used to screen for CRC by detecting small amounts of blood in stool samples using antibodies specific to human hemoglobin. FIT detects blood in stool samples, which may be a sign of precancerous or cancerous lesions. If abnormal results are obtained, a colonoscopy is usually recommended, allowing a doctor to view the colon and rectum for diagnosis. If small polyps are found during a colonoscopy, they can be removed. If larger polyps or tumors are found, a biopsy may be performed to determine whether they are cancerous. Gastroenterologists use colonoscopy to detect and remove these adenomas and polyps, preventing them from continuing to develop genetic changes that lead to invasive adenocarcinoma.

[0005] As explained above, FIT is currently used to screen for colorectal cancer. However, it should be noted that FIT has low sensitivity for AA (approximately 20–30% according to the literature), meaning that most of these types of patients may be erroneously classified as disease-free. Therefore, FIT cannot identify adenomas due to its low sensitivity. On the other hand, colonoscopy is an invasive technique, and the most serious complication is generally gastrointestinal perforation. Furthermore, colonoscopy is nowadays a procedure that requires anesthesia, and the laxatives typically administered to prepare the bowel for colonoscopy can cause several digestive problems.

[0006] It should be noted that the methods currently used to screen the general population at risk for CRC or AA are associated with high false-positive rates, resulting in a large number of unnecessary follow-up colonoscopies today. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides a clear solution to the above-cited problems, since it focuses on an in vitro method for identifying or screening human subjects at risk of developing colorectal cancer or colorectal adenoma (particularly advanced colorectal adenoma) starting from miRNA expression levels. Furthermore, the method of the present invention provides high sensitivity and specificity, which means it is a powerful and cost-effective method for detecting both colorectal cancer and colorectal adenoma. [Means for solving the problem]

[0008] The present invention relates to an in vitro method for diagnosing, identifying or screening human subjects at risk of suffering from colorectal cancer and / or advanced colorectal adenoma, starting from the expression levels of miRNAs isolated from non-invasive samples such as stool samples. The method of the present invention provides high sensitivity and specificity, which means it is a powerful and cost-effective method for detecting both colorectal cancer and colorectal adenoma.

[0009] The method of the present invention has higher sensitivity and specificity than the method (FIT) currently used to screen the general population at risk of CRC or AA, leading to a lower rate of false positives.As a result, the method described in the present invention is obviously useful for reducing the number of follow-up colonoscopies, thus improving the current screening or diagnostic method for patients.In carrying out the method of the present invention, if it is determined that a patient may have colorectal cancer and / or precancerous stage, this result will be confirmed by colonoscopy.However, if it is not determined that a patient may have colorectal cancer and / or precancerous stage, colonoscopy is not necessary, and routine testing using the method of the present invention as defined below is recommended. DETAILED DESCRIPTION OF THE INVENTION

[0010] Specifically, the study involved the following four phases: Discovery stage by genome-wide miRNA expression profiling in 124 paired normal tumor tissues (30 CRC; 32 AA); technical validation of miRNA candidates by qRT-PCR in fecal samples from a subset of patients included in the discovery phase (n=39), as well as control individuals (n=39); Clinical validation of the most significantly upregulated miRNAs by qRT-PCR in an independent set of fecal samples (n=767) obtained from FIT-positive participants in a CRC screening program; and Development of a miRNA-based predictive model to identify patients with advanced neoplasms (i.e., CRC or AA).

[0011] Of the 200 and 324 miRNAs significantly deregulated in CRC and AA tissue samples, respectively, 7 and 5 were technically validated in stool samples. Among them, miR-421, miR-130b-3p, and miR-27a-3p were confirmed to be upregulated in patients with advanced neoplasia. Thus, a stool miRNA signature including miR-421 and miR-27a-3p was more accurate than stool hemoglobin concentration (AUC = 0.63 vs. 0.59, respectively) in distinguishing these patients from individuals with normal colonoscopy results, while a combination of both approaches provided the highest accuracy for this purpose (AUC = 0.67).

[0012] Accordingly, a first embodiment of the present invention provides an in vitro method for diagnosing or screening for colorectal cancer and / or precancerous stages thereof (hereinafter "method of the present invention"), comprising: a) measuring the expression level of at least miR-421 and / or miR-130b and / or miR-27a in a biological sample obtained from the subject; b) the overexpression of miR-421 and / or miR-130b and / or miR-27a compared to reference expression levels measured in healthy control subjects is indicative that the subject is suffering from colorectal cancer and / or a precancerous stage thereof.

[0013] In a preferred embodiment, the method comprises: a) measuring the expression levels of a combination of at least [miR-421 and miR-27a], [miR-421 and miR-25], [miR-421 and miR-221], [miR-421 and miR-34a], [miR-421 and miR-29a], or [miR-421 and miR-130b] in a biological sample obtained from the subject; b) Overexpression of at least one of the miRNA combinations compared to a reference expression level measured in a healthy control subject is indicative of the subject suffering from colorectal cancer and / or a precancerous stage.

[0014] The statistical results of the logistic regression obtained using miR-421 or the combinations [miR-421 and miR-27a], [miR-421 and miR-25], [miR-421 and miR-221], [miR-421 and miR-34a], [miR-421 and miR-29a] or [miR-421 and miR-130b] are summarized in Table 1.

[0015] In a preferred embodiment, the method comprises: a) measuring the expression level of at least the combination of [miR-130b and miR-27a], [miR-130b and miR-25], [miR-130b and miR-221], [miR-130b and miR-34a] or [miR-130b and miR-29a] in a biological sample obtained from the subject; and b) overexpression of at least one of the miRNA combinations compared to a reference expression level measured in a healthy control subject is indicative of the subject being affected with colorectal cancer and / or a precancerous stage.

[0016] The statistical results of the logistic regression obtained using miR-130b or the combinations [miR-130b and miR-27a], [miR-130b and miR-25], [miR-130b and miR-221], [miR-130b and miR-34a] or [miR-130b and miR-29a] are summarized in Table 2.

[0017] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below: miR-27a is miR-27a-3p, and / or miR-25 is miR-25-3p, and / or miR-221 is miR-221-3p, and / or miR-34a is miR-34a-5p, and / or miR-29a is miR-29a-3p, and / or miR-130b, miR-130b-3p, or any combination thereof.

[0018] For example, as shown in Tables 1 and 2, in a preferred embodiment, the method further includes determining the age and sex of the subject, preferably prior to measuring the expression level of the miRNA. Thus, in a preferred embodiment, the method of the present invention is carried out on subjects at risk of developing CRC or AA, preferably in the 50-75 year old population.

[0019] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method is performed on a male or female of any age, for example at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 years of age.

[0020] In a preferred embodiment, the method further comprises measuring the presence or concentration of hemoglobin, preferably before measuring the expression level of the miRNA, wherein the presence or higher concentration of hemoglobin compared to a healthy control subject indicates that the subject is suffering from colorectal cancer and / or a precancerous stage.

[0021] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the biological sample is a stool, blood, serum or plasma sample.

[0022] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the biological sample is a stool sample.

[0023] In a preferred embodiment, the method of the present invention is carried out with a stool sample, preferably less than 10 mg, preferably less than 5 mg, more preferably 2.5 mg.In fact, the obvious advantage of the method of the present invention is that it can be carried out by using a small amount of stool sample.In addition, commercially available tests such as Cologuard® typically require the use of a large amount of stool sample, about 50 g.Since the method of the present invention can be carried out using a small amount of stool sample, the measurement of miRNA expression level can be carried out in the remaining stool sample that was previously used to measure the presence or concentration of hemoglobin.

[0024] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises: a) measuring the expression level of at least miR-421 and measuring the presence or concentration of hemoglobin in a stool sample obtained from the subject; b) Overexpression of miR-421 compared to a reference expression level measured in a healthy control subject, and the presence or elevated concentration of hemoglobin compared to a healthy control subject, indicates that the subject is suffering from colorectal cancer and / or a precancerous stage thereof.

[0025] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises: a) measuring the expression levels of a combination of at least [miR-421 and miR-27a], [miR-421 and miR-25], [miR-421 and miR-221], [miR-421 and miR-34a], [miR-421 and miR-29a], or [miR-421 and miR-130b] in a stool sample obtained from the subject, and measuring the presence or concentration of hemoglobin; b) Overexpression of at least one of the miRNA combinations compared to a reference expression level measured in a healthy control subject, and the presence or elevated concentration of hemoglobin compared to a healthy control subject, is indicative that the subject is suffering from colorectal cancer and / or a precancerous stage thereof.

[0026] In a preferred embodiment, the pre-cancerous stage of colorectal cancer is advanced colorectal adenoma.

[0027] In a preferred embodiment, the diagnosis of colorectal cancer and / or its precancerous stages is confirmed by imaging techniques, preferably colonoscopy.

[0028] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method of the invention is carried out by using any algorithm known to those skilled in the art, such as an algorithm described in Friedman JH, "Stochastic gradient boosting" Comput Stat Data Anal 2002 38:367-378, for example a gradient boosting machine algorithm, a C-tree, a random forest, linear discriminant analysis, a support vector machine, a k-nearest neighbor algorithm or logistic regression.

[0029] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method for diagnosis or screening is performed by a Gradient Boosting Machine algorithm.

[0030] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, an in vitro method for diagnosing or screening for colorectal cancer and / or precancerous stages thereof comprises: a) measuring the expression level of at least miR-421 and / or miR-130b and / or miR-27a in a biological sample obtained from the subject (in one example, the biological sample is a stool sample); b) overexpression of miR-421 and / or miR-130b and / or miR-27a compared to reference expression levels measured in healthy control subjects is indicative that the subject is suffering from colorectal cancer and / or a precancerous stage thereof; Optionally, the method additionally comprises determining the age and sex of the subject; The method is performed by an algorithm selected from the group of gradient boosting machine algorithms, C-trees, random forests, linear discriminant analysis, support vector machines, k-nearest neighbors and logistic regression.

[0031] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, an in vitro method for diagnosing or screening for colorectal cancer and / or precancerous stages thereof comprises: a) measuring the expression level of at least miR-421 in a biological sample obtained from the subject (in one example, the biological sample is a stool sample); b) overexpression of miR-421 compared to a reference expression level measured in a healthy control subject is indicative of the subject suffering from colorectal cancer and / or a precancerous stage thereof; Additionally, the method includes determining the age and sex of the subject; The method is implemented by a gradient boosting machine algorithm. This embodiment can optionally include in step a) further measuring the presence or concentration of hemoglobin in a stool sample obtained from the subject, and in step b) additionally, the presence or higher concentration of hemoglobin compared to a healthy control subject indicates that the subject is suffering from colorectal cancer and / or its precancerous stage.

[0032] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises: a) measuring the expression levels of a combination of at least [miR-421 and miR-27a], [miR-421 and miR-25], [miR-421 and miR-221], [miR-421 and miR-34a], [miR-421 and miR-29a], or [miR-421 and miR-130b]; b) overexpression of at least one of the combination of miRNAs, compared to a reference expression level measured in a healthy control subject, is indicative that the subject is suffering from colorectal cancer and / or a precancerous stage thereof; Additionally, the method includes determining the age and sex of the subject; The method is implemented by a gradient boosting machine algorithm. This embodiment can optionally include in step a) further measuring the presence or concentration of hemoglobin in a stool sample obtained from the subject, and in step b) additionally, the presence or higher concentration of hemoglobin compared to a healthy control subject indicates that the subject is suffering from colorectal cancer and / or its precancerous stage.

[0033] A second embodiment of the present invention relates to the in vitro use of at least miR-421 and / or miR-130b and / or miR-27a for screening or diagnosing colorectal cancer and / or precancerous stages thereof.

[0034] In a preferred embodiment, the present invention relates to the in vitro use of at least [miR-421 and miR-27a], [miR-421 and miR-25], [miR-421 and miR-221], [miR-421 and miR-34a], [miR-421 and miR-29a], [miR-421 and miR-130b], [miR-130b and miR-27a], [miR-130b and miR-25], [miR-130b and miR-221], [miR-130b and miR-34a] or [miR-130b and miR-29a] for screening or diagnosing colorectal cancer and / or precancerous stages thereof.

[0035] In a preferred embodiment, the present invention relates to the in vitro use of at least [miR-130b and miR-27a], [miR-130b and miR-25], [miR-130b and miR-221], [miR-130b and miR-34a] or [miR-130b and miR-29a] for screening or diagnosing colorectal cancer and / or precancerous stages thereof.

[0036] In one embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the in vitro use is carried out in a stool, blood, serum or plasma sample.

[0037] In one embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the in vitro use is carried out in a stool sample.

[0038] In a preferred embodiment, the present invention relates to the in vitro use of at least the above-cited miRNAs in combination with determining the age and sex of a subject and / or measuring the presence or concentration of hemoglobin.

[0039] A third embodiment of the present invention relates to a kit comprising: means or reagents for determining the presence or concentration of hemoglobin, and · Means or reagents for determining the expression levels of miR-421 and / or miR-130b and / or miR-27a.

[0040] In a preferred embodiment, the kit comprises: means or reagents for determining the presence or concentration of hemoglobin, and Means or reagents for measuring the expression levels of [miR-421 and miR-27a], [miR-421 and miR-25], [miR-421 and miR-221], [miR-421 and miR-34a], [miR-421 and miR-29a], [miR-421 and miR-130b], [miR-130b and miR-27a], [miR-130b and miR-25], [miR-130b and miR-221], [miR-130b and miR-34a] or [miR-130b and miR-29a].

[0041] In a preferred embodiment, the kit comprises: means or reagents for determining the presence or concentration of hemoglobin, and · Means or reagents for measuring the expression levels of [miR-130b and miR-27a], [miR-130b and miR-25], [miR-130b and miR-221], [miR-130b and miR-34a] or [miR-130b and miR-29a].

[0042] In a preferred embodiment, measuring the presence or concentration of hemoglobin is performed by using an antibody specific for human hemoglobin, and determining the expression level of miRNA is performed by PCR.

[0043] In one embodiment of the third aspect of the invention, optionally in combination with any of the embodiments provided above or below, the means or reagents for measuring the expression level of any miRNA are for reverse transcription quantitative polymerase chain reaction (qRT-PCR).

[0044] A fourth embodiment of the present invention relates to the use of the above cited kit for screening or diagnosing colorectal cancer and / or its precancerous stages.

[0045] In one embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the use is in a stool sample of a subject.

[0046] According to the method of the present invention, after measuring the expression level of any combination of biomarkers mentioned above, obtain a score value for the signature, and compare this score value with the threshold value that defines the diagnostic rule.If this score value is higher than the threshold value, the corresponding sample is classified as a positive sample, which indicates that the patient may be suffering from colorectal cancer and / or its precancerous stage.The threshold value is defined to optimize the sensitivity and specificity value.Therefore, in a preferred embodiment, the method of the present invention comprises: a) measuring the concentration level of any of the above-cited biomarker combinations in a biological sample obtained from the subject; b) processing the expression values ​​to obtain a risk score; c) If a deviation or variation in the value of the risk score obtained for any of the above-cited biomarker combinations is identified compared to the reference value, this is indicative that the subject is suffering from colorectal cancer and / or pre-cancerous stages.

[0047] All embodiments of the first aspect of the invention are also embodiments of the second, third and fourth aspects of the invention.

[0048] In one embodiment of the first, second, third or fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, miRNA expression levels are measured by qRT-PCR.

[0049] A final embodiment of the present invention is a method of treating colorectal cancer or a pre-cancerous stage thereof, comprising: a) diagnosing a patient with colorectal cancer or a precancerous stage thereof according to any of the above embodiments; b) treating the patient by performing a colonoscopy, which may include removing colorectal cancer lesions or polyps; Hereinafter, this method for treating colorectal cancer or its precancerous stage is referred to as the "therapeutic method of the present invention."

[0050] All embodiments of the first, second, third and fourth aspects of the invention are also embodiments of the method of treatment of the invention.

[0051] In one embodiment of the treatment method of the present invention, optionally in combination with any of the embodiments provided above or below, in step b), patients diagnosed with colorectal cancer are treated, e.g., by endoscopic polypectomy, surgical resection, chemotherapy (e.g., fluoropyrimidines, oxiplatin and / or irinotecan) and / or radiation therapy, depending on the tumor stage, while patients diagnosed with advanced adenoma are treated, e.g., by endoscopic polypectomy or surgical resection, depending on the size of the polyps.

[0052] For purposes of the present invention, the following terms are defined:

[0053] 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), which includes the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum).

[0054] The term "colorectal adenoma" refers to adenomas of the colon, also known as adenomatous polyps, which are benign and precancerous stages of colorectal cancer but still pose a high risk of progression to colorectal cancer.

[0055] The term "advanced colorectal adenoma" refers to an adenoma that is at least 10 mm in size or has histologically high-grade dysplasia or a villous component of more than 20%.

[0056] The expression "non-invasive biological sample" refers to any sample taken from a patient's body without the need for any harmful instruments other than the fine needles used to draw blood from the patient, and therefore without harming the patient. Specifically, non-invasive biological samples in the context of the present invention refer to stool, blood, serum or plasma samples.

[0057] The expression "reference expression level measured in healthy control subjects" refers to a "reference value" of the expression level of a biomarker. If a deviation in the expression level of a biomarker is measured with respect to said "reference expression level measured in healthy control subjects", this is indicative of colorectal cancer or a precancerous stage thereof. In particular, if the expression level of a biomarker or signature of the present invention is significantly higher or lower with respect to said "reference value", this is indicative of colorectal cancer or a precancerous stage thereof.

[0058] The expression "risk score" refers to a risk value obtained after processing one or more concentration values ​​into a single value (or risk value) that represents the probability of disease for an individual. This risk value is compared with a reference value to assess whether a patient is likely to suffer from colorectal cancer and / or its precancerous stages.

[0059] The "reference value" can be a threshold or cutoff value. Typically, the "threshold" or "cutoff value" can be determined experimentally, empirically, or theoretically. The threshold can also be arbitrarily selected based on existing experimental and / or clinical conditions, as will be recognized by those skilled in the art. The threshold must be determined to obtain optimal sensitivity and specificity according to the test's function and the benefit / risk balance (false-positive and false-negative clinical outcomes). Preferably, those skilled in the art can compare the biomarker level (or score) obtained according to the method of the present invention with a defined threshold. Typically, optimal sensitivity and specificity (as well as the threshold) can be determined using a receiver operating characteristic (ROC) curve based on experimental data. For example, after determining the biomarker level in a reference group, algorithmic analysis can be used to statistically process the measured biomarker concentrations in the biological sample being tested, thereby obtaining classification criteria important for sample classification. The full name of the ROC curve is the receiver operator characteristic curve, also known as the receiver operating characteristic curve. It is primarily used in clinical biochemical diagnostic tests. The ROC curve is a comprehensive index that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (specificity). It reveals the relationship between sensitivity and specificity through image synthesis. A series of different cutoff values ​​(thresholds or critical values, the boundary between normal and abnormal results of a diagnostic test) are set as continuous variables, and a series of sensitivity and specificity values ​​are calculated. The curve is then drawn using sensitivity as the vertical coordinate and specificity as the horizontal coordinate. The larger the area under the curve (AUC), the higher the diagnostic accuracy. On the ROC curve, the point closest to the upper left on the coordinate diagram is the critical point where both sensitivity and specificity are high. The AUC value of the ROC curve is between 1.0 and 0.5. If AUC > 0.5, the diagnostic result is better as the AUC approaches 1.Existing software or systems in the art can be used to plot ROC curves, such as, for example: MedCalc 9.2.0.1 medical statistics software, SPSS 9.0 or pROC R package.

[0060] "Comprising" means including, but not limited to, what follows the word "comprising." Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present.

[0061] "Consisting of" means "including, and limited to" whatever follows the word "consisting of." Thus, the word "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0062] Throughout the specification and claims, the term "comprise" encompasses the term "consisting of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the detailed description of the invention or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration and are not intended to limit the invention. Furthermore, the present invention covers all possible combinations of the specific preferred embodiments described herein. [Brief explanation of the drawings]

[0063] [Figure 1] Study summary. CRC: colorectal cancer, AA: advanced adenoma, NAA: non-advanced adenoma, FDR: false discovery rate, FC: fold change. [Figure 2]Panel A: Between-group analysis plot showing clustering of samples based on miRNA expression profiles. CRC: colorectal cancer, AA: advanced adenoma, C: paired normal tissue. Panel B: Venn diagram constructed from NGS results of 124 tissue samples. The inner circle indicates miRNAs with a false discovery rate <0.05 and a fold change ≥1.5 or ≤-1.5. Convergence between the circles indicates common significantly deregulated miRNAs in both neoplastic lesions. Red: number of upregulated miRNAs, green: number of downregulated miRNAs. [Figure 3] The receiver operating characteristic (ROC) curve of the best miRNA-fecal prediction model (miR-421, miR-27a, age, sex) calculated by the GBM algorithm shows a high ability to distinguish between: 1) aggressive colorectal neoplasia (CRC + AA) vs. non-aggressive colorectal neoplasia (NAA + healthy controls); 2) aggressive colorectal neoplasia (CRC + AA) vs. healthy controls; 3) CRC vs. healthy controls; and 4) AA vs. healthy controls. Area under the curve (AUC). The X-axis represents specificity. The Y-axis represents sensitivity. [Example]

[0064] Detailed Description of the Invention Example 1 Materials and Methods. Example 1.1 Patients and Methods.

[0065] The study included four main phases: 1) miRNA discovery phase by next-generation sequencing (NGS) in tissue sets of CRC and AA samples and their paired normal mucosa; 2) technical validation of candidate miRNAs by quantitative reverse transcription PCR (qRT-PCR) in fecal samples from a subset of patients included in the discovery phase as well as control individuals; 3) clinical validation of the most significantly upregulated miRNAs by qRT-PCR in an independent set of fecal samples obtained from participants in the Barcelona CRC screening program; 4) Development of a miRNA-based predictive model to identify patients with advanced neoplasia (i.e., CRC or AA) among patients with unrelated colonoscopy findings. An overview of the study is shown in Figure 1.

[0066] For the miRNA discovery phase, 124 fresh colorectal tissue samples were collected in advance at the Hospital Clinic in Barcelona. Paired neoplastic and normal mucosal samples were obtained from 30 patients with CRC who underwent surgery, none of whom had received neoadjuvant chemotherapy or radiation therapy. In addition, paired neoplastic and normal mucosal samples were obtained from 32 patients with AA after endoscopic resection. All tissue samples were stored in RNAlater® (Invitrogen, Carlsbad, CA) and frozen at -80°C until RNA extraction.

[0067] For technical validation of miRNA candidates in fecal samples, a subset of 39 patients included in the previous stage (11 with CRC and 28 with AA) was analyzed, as well as 39 control individuals with normal colonoscopy.

[0068] Finally, clinical validation of the selected miRNAs was performed in 767 fecal samples collected between March 2011 and May 2017 among FIT-positive participants in the Barcelona CRC screening program. Each participant provided one fecal sample using a specimen collection device (OC-Sensor, Eiken Chemical Co., Ltd., Japan), which collected 10 mg of feces in 2 mL of buffer solution with a serrated probe attached to the cap. All samples were stored at -80 °C until RNA extraction. The characteristics of this population-based organized screening program have been described elsewhere.

[0069] The clinicopathological characteristics of all individuals included in this study are shown in Table 3. The study was approved by the Ethics Committee of the Barcelona Hospital Clinic and was performed in accordance with the Declaration of Helsinki with written informed consent from all participants.

[0070] Example 1.2 MicroRNA extraction.

[0071] Total RNA, including miRNAs, was isolated from tissues or feces (500 μL buffer) using the miRNeasy® Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. For tissue samples, after RNA extraction, the RNeasy MinElute Cleanup® Kit (Qiagen, Valencia, CA) was used to ensure removal of contaminants and concentrate the sample to a final elution volume of 12 μL. The purity of RNA tissue samples was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), and the concentration was measured using a NanoDrop® 1000 Spectrophotometer (Wilmington, DE). The concentration of total miRNA present in fecal samples was measured using the Quant-iT® microRNA Assay Kit (Invitrogen, Carlsbad, CA).

[0072] Example 1.3 Genome-wide miRNA profiling by NGS.

[0073] Small RNA libraries were generated from 1 μg of total RNA from tissues using the TruSeq® Small RNA Sample Preparation Kit (Illumina, San Diego, CA) according to the manufacturer's protocol. First, 3' and 5' RNA adapters were ligated to the ends of the small RNAs. Then, cDNA constructs were synthesized by reverse transcription using SuperScript® II Reverse Transcriptase (Invitrogen, Carlsbad, CA) with a specific primer complementary to the 3' RNA adapter. The cDNA was further amplified by PCR using the indexed adapters provided with the kit. This step selectively enriches for RNA fragments with adapter molecules at both ends. Finally, the amplified cDNA constructs from the library were isolated on a 6% Novex® TBE gel (Life Technologies, Carlsbad, CA). Regions showing band sizes between 18 and 36 base pairs (bp) were excised from the gel, and the DNA was precipitated and eluted in 10 μL of elution buffer. The generated cDNA libraries were analyzed using the Agilent High Sensitivity DNA® Kit (Agilent Technologies, CA) to ensure acceptable quantities and confirm size distribution. High-throughput sequencing of the cDNA libraries was performed on a HiSeq® 2000 (Illumina, CA) using 1x50bp single-end reads, yielding >15M reads per sample. Quality control was performed by image analysis and base assignment using real-time analysis software. Reads with low confidence values ​​in the first 25 bases were discarded. Quality control reads were aligned to the reference genome using the GEM program. The error percentage calculated from the genome of PhiX spiked into the samples before sequencing was less than 2.5%.

[0074] Example 1.4 Analysis of fecal miRNA expression by qRT-PCR.

[0075] qRT-PCR was performed using singleplex TaqMan® microRNA assays (Applied Biosystems Inc., Foster City, CA). Briefly, reverse transcription was performed using 5 ng of total miRNA. For some miRNAs, a preamplification step was then performed prior to quantitative qPCR due to the low fecal miRNA levels. Finally, qPCR was performed using a Viia7® Real-Time PCR System (Applied Biosystems Inc., Foster City, CA) using 2 μL of cDNA in a final volume of 10 μL. Each point was evaluated in triplicate.

[0076] Absolute quantification was performed because there was no reliable endogenous control for normalizing fecal miRNAs. Therefore, the average Ct for each sample was converted to ng specific miRNA / g total miRNA using a standard curve generated by serial dilution of known amounts of each specific synthetic miRNA (Integrated DNA Technologies, IA).

[0077] Example 1.5 Bioinformatics and statistical analysis.

[0078] Sequencing analysis was performed using the sRNAbench package. Briefly, after adapter trimming and grouping of unique reads, Bowtie 1.1.2 was used to align reads to the human genome (UCSC hg19), allowing one mismatch. miRBase (version 21) for mature and pre-miRNA sequences was used to provide annotation for RNA elements mapped to the human genome. Count data were transformed to log2-counts per million (logCPM) and normalized by the cyclic-loess method. A moderated t-test was applied to identify differentially expressed miRNAs. Differential expression, fold change, and mean expression as log2 of the difference were analyzed between colorectal neoplastic tissues and paired normal mucosa. p-values ​​were adjusted for multiple testing by the method of Benjamini and Hochberg. miRNAs with a false discovery rate <0.05 were considered significant. A principal component analysis plot was created to visualize high-dimensional data in a 2D graph where the area bounded by the ellipse represents 95% of the binomial distribution of sample scores on the first and second axes. A Venn diagram was also created considering significant miRNAs. Selection of miRNA candidates from the NGS results was based on the following criteria:

[0079] False discovery rate <0.05, fold change ≥1.5, mean expression >3.5, and upregulated in both CRC and AA.

[0080] For quantitative variables, Student's t-test was used. The discriminatory ability of individual miRNAs was assessed by multivariate logistic regression adjusted for age and sex. A P value of ≤0.05 was considered significant. The pROC R package was used to calculate the area under the receiver operating characteristic curve (AUC) and the resulting cutpoints, considering each miRNA as a continuous variable. Sensitivity and specificity were calculated from the optimal cutpoint associated with the lowest error rate.

[0081] Example 1.6 Predictive modeling based on fecal miRNAs.

[0082] A predictive model for distinguishing different individual groups was developed taking into account age and gender, as well as the miRNA results obtained during the clinical validation phase. The primary endpoint of this analysis was to distinguish patients with advanced neoplasia (i.e., CRC or AA) among patients with unrelated findings on colonoscopy (i.e., NAA or normal). Secondary endpoints were to distinguish patients with advanced neoplasia, CRC, and AA, respectively, from individuals with normal colonoscopy.

[0083] To generate predictive models, samples were randomly divided into training and test sets at 75% and 25% ratios. Numerical data were then preprocessed by centering and scaling. To address sample imbalance, a synthetic minority oversampling technique was used. 10-fold cross-validation was performed on the training set. The following algorithms were tested to generate models: C-tree, random forest, linear discriminant analysis, gradient boosting machine, support vector machine, and K-nearest neighbor. A P value of <0.05 was considered significant. Discriminant measures included area under curve (AUC), sensitivity, specificity, and positive and negative predictive values. Postprocessing calibration was performed using 15 predicted versus observed probability bins to analyze the error distribution of the predictive models. All analyses were performed using R with the CARET package.

[0084] We compared the results obtained with the fecal miRNA-based prediction model with those that would be obtained by using age- and sex-adjusted fecal hemoglobin concentrations, which represent the standard of care in most CRC screening programs.

[0085] Finally, we also evaluated the performance of a predictive model that combined both miRNA signatures and fecal hemoglobin concentrations.

[0086] Example 2 Results. Example 2.1 miRNA discovery by NGS.

[0087] In the colorectal tissue set, the expression of 1,640 miRNAs was detected, with 637 miRNAs having counts greater than 100. Of these, 200 and 324 miRNAs were significantly deregulated in CRC and AA tissues, respectively, compared with their paired normal mucosa, with a fold change of ≥ 1.5. Between-group analysis demonstrated that miRNA expression profiling could distinguish CRC or AA tissue samples from their paired normal mucosa (Figure 2A), as well as CRC from AA. Furthermore, we found that 72 and 56 miRNAs were commonly up- or down-regulated in both neoplastic lesions, respectively (Figure 2B).

[0088] According to the selection criteria (i.e., false discovery rate <0.05, fold change ≥1.5, mean expression >3.5, and up-regulation in both CRC and AA), 21 miRNA candidates (Table 4) were selected for technical validation in fecal samples.

[0089] Example 2.2 miRNA analysis of fecal samples by qRT-PCR.

[0090] To determine whether the miRNA patterns in colorectal tissues could be recapitulated in fecal samples, we first analyzed the 21 miRNAs mentioned above by qRT-PCR in a subset of 39 patients included in the discovery phase (11 CRC patients and 28 AA patients) and 39 control individuals with normal colonoscopy.

[0091] The results of this technical validation phase showed that seven miRNAs (miR-130b-3p, miR-21-5p, miR-221-5p, miR-25-3p, miR-27a-3p, miR-34a-5p, and miR-421) were significantly upregulated in fecal samples from patients with CRC. Furthermore, four of them (miR-130b-3p, miR-21-5p, miR-27a-3p, and miR-421) and miR-335-3p were significantly upregulated in fecal samples from patients with AA. These eight miRNAs, along with miR-29a-3p, which was also upregulated in fecal samples from patients with CRC and AA (AUCs of 0.84 and 0.71, respectively, p-values ​​<0.1), were selected for clinical validation in an independent cohort of participants in a CRC screening program.

[0092] Example 2.3 Clinical validation of miRNA candidates.

[0093] The above nine upregulated miRNAs were validated in fecal samples from a prudently collected cohort of 767 FIT-positive individuals, including 67 patients with CRC, 347 patients with AA, 136 patients with NAA, and 217 individuals with normal colonoscopy (Table 3 ).

[0094] In this set of samples, we observed upregulation of miR-25-3p, miR-27a-3p, miR-29a-3p, miR-34a-5p, miR-130b-3p, miR-221-3p, and miR-421 in CRC patients compared to controls (AUCs ranged from 0.69 to 0.77), whereas miR-130b-3p and miR-421 were upregulated in AA patients (AUCs were 0.69 and 0.71, respectively). It is important to note that none of these seven fecal miRNAs showed significant differences between patients with NAA and individuals with normal colonoscopy (Table 5).

[0095] Finally, with regard to the primary endpoint of the study, miR-421, miR-27a-3p, and miR-130b-3p were specifically selected as the most discriminatory fecal miRNAs for distinguishing patients with advanced neoplasia from those with unrelated findings on colonoscopy (Table 6).

[0096] Development and validation of a fecal miRNA-based predictive model for colorectal cancer screening

[0097] Predictive modeling was performed to identify distinct patient groups, taking into account fecal miRNAs significantly upregulated in patients with advanced neoplasms, along with age and gender. Principal component analysis (PCA) demonstrated that miR-421 and miR-27a were ultimately selected for this purpose because they were nonredundant among the three most discriminatory fecal miRNAs. For model generation, individuals were randomly divided into a training set (n = 578) and a test set (n = 189), and 10-fold cross-validation was performed during the development phase to reduce bias and variability. As previously mentioned, different algorithms were tested to select the best fit for the study's primary endpoint, and the gradient boosting machine (GBM) algorithm was ultimately selected based on its highest accuracy.

[0098] As shown in Table 7, the prediction model obtained by combining miR-421, miR-27a, age, and gender was highly accurate (AUC = 0.63) for identifying patients with advanced neoplasms among FIT-positive participants in a CRC screening program. Interestingly, this result was due not only to the high accuracy for recognizing patients with CRC (AUC = 0.74, sensitivity, 96%), but also to distinguishing patients with AA from control individuals (AUC = 0.64, sensitivity, 59%).

[0099] The results obtained with the fecal miRNA-based prediction model [miR-421 and miR-27a] were superior to those obtained using fecal hemoglobin concentration as a classifier for the detection of advanced neoplasia (AUC = 0.62 when compared with subjects with unrelated findings at colonoscopy and AUC = 0.59 when compared with normal individuals who underwent colonoscopy), CRC (AUC = 0.67), and AA (AUC = 0.59) (Table 7).

[0100] Finally, as can be seen, for example, in Figure 3, the combination of both the miRNA signature [miR-421 and miR-27a] and fecal hemoglobin concentration enabled the highest accuracy for identifying patients with advanced neoplasia (AUC = 0.70 when compared with subjects with unrelated findings on colonoscopy, and AUC = 0.67 when compared with normal individuals who underwent colonoscopy), CRC (AUC = 0.93), and AA (AUC = 0.64) (Table 7). Indeed, the calibration curve of the combined model showed that the predicted results were closer to the observed results and had a lower error distribution compared to the results obtained using fecal hemoglobin concentration alone.

[0101] [Table 1]

[0102] [Table 2]

[0103] [Table 3]

[0104] [Table 4]

[0105] [Table 5]

[0106] [Table 6]

[0107] [Table 7]

[0108] For completeness, various aspects of the invention are described in the following numbered sections. Section 1. An in vitro method for diagnosing or screening for colorectal cancer and / or its precancerous stages, comprising: a) measuring the expression level of at least miR-421 in a biological sample obtained from a subject, wherein said biological sample is a stool sample; and b) overexpression of miR-421 compared to a reference expression level measured in a healthy control subject indicates that said subject is suffering from colorectal cancer and / or its precancerous stages. Section 2. a) measuring the expression level of a combination of at least [miR-421 and miR-27a], [miR-421 and miR-25], [miR-421 and miR-221], [miR-421 and miR-34a], [miR-421 and miR-29a], or [miR-421 and miR-130b] in a biological sample obtained from said subject; and b) comparing the reference expression levels measured in healthy control subjects. 2. The in vitro method of claim 1, wherein overexpression of a combination of at least [miR-421 and miR-27a], [miR-421 and miR-25], [miR-421 and miR-221], [miR-421 and miR-34a], [miR-421 and miR-29a] or [miR-421 and miR-130b] compared to control is indicative of the subject suffering from colorectal cancer and / or a precancerous stage. Item 3. An in vitro method according to any one of items 1 to 2, carried out on a subject aged 50 to 75 years. Item 4. The in vitro method of any of items 1 to 3, further comprising measuring the presence or concentration of hemoglobin, preferably before measuring the expression level of the miRNA, wherein the presence or higher concentration of hemoglobin compared to a healthy control subject indicates that the subject is suffering from colorectal cancer and / or a precancerous stage. Item 5. An in vitro method according to any one of items 1 to 4, wherein the biological sample is a stool sample of less than 5 mg, preferably 2.5 mg. Item 6. An in vitro method described in any of items 1 to 5, wherein the measurement of the expression level of the miRNA is performed in a remaining stool sample previously used to measure the presence or concentration of hemoglobin. Item 7. An in vitro method according to any one of items 1 to 6, wherein the precancerous stage of colorectal cancer is advanced colorectal adenoma. Clause 8. The in vitro method of any of the preceding clauses, wherein the diagnosis of colorectal cancer and / or precancerous stages thereof is confirmed by imaging techniques, preferably colonoscopy. Item 9. In vitro use of at least miR-421 for the diagnosis or screening of colorectal cancer and / or precancerous stages thereof in a stool sample from a subject. Clause 10. In vitro use of at least [miR-421 and miR-27a], [miR-421 and miR-25], [miR-421 and miR-221], [miR-421 and miR-34a], [miR-421 and miR-29a] or [miR-421 and miR-130b] for the diagnosis or screening of colorectal cancer and / or precancerous stages thereof, as described in clause 9. Section 11. a. a means or reagent for determining the presence or concentration of hemoglobin; b. A means or reagent for measuring the expression level of miR-421; Includes kit of parts. Section 12. a. a means or reagent for determining the presence or concentration of hemoglobin; b. A means or reagent for measuring the expression level of [miR-421 and miR-27a], [miR-421 and miR-25], [miR-421 and miR-221], [miR-421 and miR-34a], [miR-421 and miR-29a] or [miR-421 and miR-130b]; 12. The kit-of-parts according to claim 11, comprising: Item 13. The kit of parts according to either item 11 or 12, wherein the measurement of the presence or concentration of hemoglobin is carried out by using an antibody specific to human hemoglobin, and the measurement of the expression level of miRNA is carried out by qRT-PCR. Clause 14. Use of the kit according to any one of clauses 11 to 13 for diagnosing or screening for colorectal cancer and / or precancerous stages thereof. Item 15. Use of the kit according to item 14 for diagnosing or screening for advanced colorectal adenoma.

Claims

1. A kit of parts for screening for either or both of colorectal cancer and advanced colorectal adenoma, comprising: a. an antibody specific for human hemoglobin to measure the presence or concentration of hemoglobin; b. A means or reagent for measuring the expression level of miR-421 by qRT-PCR; Kit of parts including.

2. a. an antibody specific for human hemoglobin to measure the presence or concentration of hemoglobin; b. Means or reagents for measuring the expression levels of [miR-421 and miR-27a], [miR-421 and miR-25], [miR-421 and miR-221], [miR-421 and miR-34a], [miR-421 and miR-29a] or [miR-421 and miR-130b] by qRT-PCR; 2. The kit of parts of claim 1, comprising:

3. Use of the kit of parts described in claim 1 or 2 for screening for colorectal cancer and / or advanced colorectal adenoma in a subject's stool sample.

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