Protein signature for diagnosis of colorectal cancer and / or pre-cancerous stage thereof

Abstract

To provide an in vitro method for diagnosing, identifying or screening human subjects at risk of developing colorectal cancer and / or advanced colorectal adenoma, which deviate from a concentration level of a protein biomarker isolated from a minimally invasive sample such as blood, serum or plasma.SOLUTION: An in vitro method for diagnosing colorectal cancer and / or pre-cancerous stages thereof, comprises: a) measuring a concentration level of at least Flt3L in a biological sample obtained from a subject; and b) when identifying a deviation or variation in the concentration level of at least Flt3L compared to a reference concentration level measured in a healthy subject, indicating that the subject is suffering from colorectal cancer and / or pre-cancerous stages thereof.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention can be included in the medical field. In particular, the present invention relates to an in vitro method for diagnosing colorectal cancer and / or its precancerous stages. [Background technology]

[0002] Colorectal cancer (CRC) (also known as colon cancer, rectal cancer, or intestinal cancer) Colon cancer (advanced colorectal adenoma), also known as advanced colorectal adenoma, 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 there are many glands within the tissue of the colon. These glands undergo a number of changes at the genetic level that lead to a predictable progression from benign to invasive malignant colon cancer. Adenomas of the colon, particularly advanced colorectal adenomas (AA), are benign versions of malignant adenocarcinomas, but can become malignant if not removed (commonly called advanced colorectal adenomas). They are usually removed because of their tendency to become malignant and lead to colon cancer.

[0003] Screening is an effective way to prevent colorectal cancer and reduce deaths from it, and is recommended to begin between the ages of 50 and 75. The best-known and most frequently used screening test for colorectal cancer is called the fecal immunochemical test (FIT). FIT detects blood in a stool sample, which may be a sign of pre-cancer or cancer. If an abnormal result is obtained, a colonoscopy is usually recommended, which allows a doctor to see the inside of the colon and rectum to make a diagnosis. During a colonoscopy, if small polyps are found, they can be removed. If larger polyps or tumors are found, a biopsy can be done to see if they are cancerous. Gastroenterologists use colonoscopy to find and remove these adenomas and polyps to prevent them from continuing to acquire genetic changes that lead to invasive adenocarcinoma.

[0004] As explained above, FIT is currently used to screen for colorectal cancer, but it is important to note that FIT has a low sensitivity for AA (approximately 20%-30% depending on the literature), meaning that most of this type of patient may be erroneously classified as disease-free. As a result, FIT fails to identify adenomas due to its low sensitivity. Furthermore, FIT uses fecal samples, which leads to low compliance. On the one hand, colonoscopy is an invasive technique, and the most severe complication is generally gastrointestinal perforation. On the other hand, colonoscopy is today a procedure that involves anesthesia, and the laxatives that are usually administered during bowel preparation for colonoscopy are associated with several digestive problems.

[0005] It is important to note that the methods used today to screen the general population at risk for CRC or AA are associated with a high rate of false positives, resulting in a large amount of unnecessary follow-up colonoscopies being performed today.

[0006] The present invention provides a clear solution to the above mentioned problems by focusing on an in vitro method to identify or screen human subjects at risk of suffering from colorectal cancer or colorectal adenoma (especially advanced colorectal adenoma) from deviating concentration levels of protein biomarkers isolated from minimally invasive samples such as blood, serum or plasma. The method of the present invention is expected to improve compliance to colorectal cancer screening since it is based on blood, serum or plasma samples. Furthermore, the method of the present invention provides high sensitivity and specificity, which means it is a powerful and cost-effective method for the detection of both colorectal cancer and colorectal adenoma. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to an in vitro method of diagnosing, identifying or screening human subjects at risk of suffering from colorectal cancer and / or advanced colorectal adenomas, based on deviating concentration levels of protein biomarkers isolated from minimally invasive samples such as blood, serum or plasma. The method of the present invention provides high sensitivity and specificity, which means that it is a robust and cost-effective method for the detection of both colorectal cancer and colorectal adenomas.

[0008] The method of the present invention has higher sensitivity and specificity compared to the method (FIT) used today to screen the general population at risk of suffering from CRC or AA, and is therefore associated with a lower percentage of false positives. As a result, the method described in the present invention clearly helps to reduce the number of follow-up colonoscopies, thus improving the screening or diagnostic method for today's patients. If it is determined that the patient may suffer from colorectal cancer and / or precancerous stage after the method of the present invention is carried out, the result is confirmed by colonoscopy. However, if it is not determined that the patient may suffer from colorectal cancer and / or precancerous stage, it is not necessary to carry out colonoscopy, and routine examination by the method of the present invention as defined below is recommended. [Means for solving the problem]

[0009] In particular, in a first embodiment of the present invention, the present invention relates to an in vitro method for diagnosing colorectal cancer and / or precancerous stages thereof (hereinafter "method of the present invention"), comprising a) measuring a concentration level of at least Flt3L in a biological sample obtained from a subject, and b) identifying a deviation or variation in the concentration level of at least Flt3L compared to a reference concentration level measured in a healthy control subject, which indicates that the subject is affected by colorectal cancer and / or precancerous stages.

[0010] In a particularly preferred embodiment, the method of the invention comprises measuring the concentration levels of at least the combination [Flt3L and CYFRA21-1] in a biological sample obtained from a subject.

[0011] In this regard, it is important to consider that all the most reliable signatures claimed in the present invention comprise Flt3L, preferably [Flt3L and CYFRA21-1], and thus various biomarker signatures comprising Flt3L, preferably [Flt3L and CYFRA21-1], such as [Flt3L and CYFRA21-1 and AREG], [Flt3L and CYFRA21-1 and AREG and ErbB4], or [Flt3L and CYFRA21-1 and AREG and CLEC2C], are obtained with an area under the curve (AUC) of approximately 0.9 for the detection of CRC and with good performance for the detection of AA (see Table 12). According to the present invention, any of the signatures comprising Flt3L, preferably [Flt3L and CYFRA21-1], can be effectively used, however, the following signatures comprising Flt3L, preferably [Flt3L and CYFRA21-1]: [Flt3L and CYFRA21-1 and AREG and ErbB4] (AUC for CRC=0.931) or [Flt3L and CYFRA21-1 and AREG and CLEC2C] (AUC for CRC=0.915) (see Table 12) are particularly preferred as they provide AUC values ​​of more than 0.9 for the detection of CRC and good performance for the detection of AA.

[0012] A second embodiment of the present invention relates to a kit of parts comprising reagents for determining the concentration levels of any of the signatures mentioned above. In a preferred embodiment, the present invention relates to a kit of parts comprising reagents for determining the concentration levels of Flt3L, or the combination [Flt3L and CYFRA21-1], preferably [Flt3L and CYFRA21-1 and AREG], [Flt3L and CYFRA21-1 and AREG and ErbB, for the diagnosis of colorectal cancer and / or precancerous stages thereof. 4] or the in vitro use of a kit comprising reagents for determining the concentration levels of [Flt3L and CYFRA21-1 and AREG and CLEC2C].

[0013] According to the method of the present invention, after measuring the concentration level of any of the combinations of biomarkers mentioned above, a score value is obtained for the signature and this score value is compared with a threshold value that defines a 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 affected by colorectal cancer and / or its precancerous stage. The threshold value is defined to optimize the sensitivity and specificity values. Thus, in a preferred embodiment, the method of the present invention comprises: a) measuring the concentration level of any of the combinations of biomarkers mentioned above in a biological sample obtained from a subject; b) processing the concentration value to obtain a risk score; and c) if a deviation or variation of the risk score value obtained for any of the combinations of biomarkers mentioned above is identified compared to a reference value, this indicates that the subject is affected by colorectal cancer and / or a precancerous condition.

[0014] A third embodiment of the present invention relates to the in vitro use of any of the above mentioned biomarkers or signatures for the diagnosis of colorectal cancer and / or its precancerous stages.

[0015] In a preferred embodiment, the precancerous stage of colorectal cancer is advanced colorectal adenoma.

[0016] In a preferred embodiment, the diagnosis of colorectal cancer and / or its precancerous stages is confirmed by imaging techniques, preferably colonoscopy.

[0017] In a preferred embodiment, the present invention relates to an in vitro method for detecting colorectal cancer and / or its precancerous stages, comprising: a) obtaining a plasma sample from a human patient; and b) contacting the plasma sample with an antibody against the above mentioned protein biomarkers or signatures and detecting whether any of said protein biomarkers or signatures are present in the plasma sample by detecting binding between the protein and the antibody.

[0018] A fourth embodiment of the present invention relates to a method for diagnosing and treating colorectal cancer or precancerous stages thereof comprising: a) obtaining a plasma sample from a human patient; b) detecting whether any of the above mentioned protein biomarkers or signatures are present in the plasma sample; and c) diagnosing the patient as having colorectal cancer or precancerous stages thereof if the presence of said protein biomarkers or signatures in the plasma sample is detected, and performing a colonoscopy on the patient to subsequently remove colorectal cancer or polyps.

[0019] Alternatively, a fifth embodiment of the present invention relates to an in vitro method for diagnosing colorectal cancer and / or precancerous stages thereof (hereinafter "the method of the present invention"), comprising a) measuring a concentration level of at least AREG in a biological sample obtained from a subject, and b) identifying a deviation or variation in the concentration level of at least AREG compared to a reference concentration level measured in a healthy control subject, which indicates that the subject is suffering from colorectal cancer and / or a precancerous stage thereof.

[0020] In a particularly preferred embodiment, the method of the invention comprises measuring the concentration levels of at least the combination [AREG and CYFRA21-1] in a biological sample obtained from a subject.

[0021] In this regard, the most reliable signature of all that is claimed in this invention is the ARE It is important to consider that a different biomarker signature including AREG, preferably including [AREG and CYFRA21-1], is thus obtained with an area under the curve (AUC) of approximately 0.9 for the detection of CRC and also with good performance for the detection of AA (see Table 12, 2). As a result, according to the present invention, any signature comprising AREG, preferably comprising [AREG and CYFRA21-1], can be effectively used, however, the following signatures comprising AREG, preferably comprising [AREG and CYFRA21-1]: [AREG and CYFRA21-1 and Flt3L and ErbB4] (AUC for CRC = 0.931) or [AREG and CYFRA21-1 and Flt3L and CLEC2C] (AUC for CRC = 0.915) (see Table 12, item 2) are particularly preferred as they provide AUC values ​​of greater than 0.9 for detecting CRC and good performance for detecting AA.

[0022] Therefore, in a particularly preferred embodiment, the method of the present invention comprises measuring the concentration levels of at least the combination [AREG and CYFRA21-1 and Flt3L], or the combination [AREG and CYFRA21-1 and CLEC2C], or the combination [AREG and CYFRA21-1 and ErbB4], or the combination [AREG and CYFRA21-1 and FasL], or the combination [AREG and CYFRA21-1 and CD147], or the combination [AREG and CYFRA21-1 and HGFR], or the combination [AREG and CYFRA21-1 and Flt3L and ErbB4], or the combination [AREG and CYFRA21-1 and Flt3L and CLEC2C], or the combination [AREG and CYFRA21-1 and HGFR and CD147] in a biological sample obtained from a subject.

[0023] In a particularly preferred embodiment, the method of the present invention comprises measuring the concentration levels of at least the combination [AREG and CD147], or the combination [AREG and CLEC2C], or the combination [AREG and HGFR], or the combination [AREG and CD147 and HGFR] in a biological sample obtained from a subject.

[0024] A sixth embodiment of the invention relates to the in vitro use of any of the above mentioned signatures for the diagnosis of colorectal cancer and / or its precancerous stages.

[0025] According to the method of the present invention, after measuring the concentration level of any of the combinations of biomarkers mentioned above, a score value is obtained for the signature and this score value is compared with a threshold value that defines a 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 affected by colorectal cancer and / or its precancerous stage. The threshold value is defined to optimize the sensitivity and specificity values. Thus, in a preferred embodiment, the method of the present invention comprises: a) measuring the concentration level of any of the combinations of biomarkers mentioned above in a biological sample obtained from a subject; b) processing the concentration value to obtain a risk score; and c) if a deviation or variation of the risk score value obtained for any of the combinations of biomarkers mentioned above is identified compared to a reference value, this indicates that the subject is affected by colorectal cancer and / or a precancerous condition.

[0026] A seventh embodiment of the present invention relates to a kit of parts comprising reagents for determining the concentration levels of any of the above mentioned signatures. In a preferred embodiment, the present invention relates to the in vitro use of a kit comprising reagents for determining the concentration levels of any of the above mentioned combinations of biomarkers for the diagnosis of colorectal cancer and / or precancerous stages thereof.

[0027] In a preferred embodiment, the precancerous stage of colorectal cancer is advanced colorectal adenoma.

[0028] In a preferred embodiment, the diagnosis of colorectal cancer and / or its precancerous stages is confirmed by imaging techniques, preferably colonoscopy.

[0029] In a preferred embodiment, the present invention relates to an in vitro method for detecting colorectal cancer and / or its precancerous stages, comprising: a) obtaining a plasma sample from a human patient; and b) contacting the plasma sample with an antibody against the above mentioned protein biomarkers or signatures and detecting whether any of said protein biomarkers or signatures are present in the plasma sample by detecting binding between the protein and the antibody.

[0030] A final embodiment of the present invention relates to a method for diagnosing and treating colorectal cancer or precancerous stages thereof, comprising: a) obtaining a plasma sample from a human patient; b) detecting whether any of the above mentioned protein biomarkers or signatures are present in the plasma sample; and c) diagnosing the patient as having colorectal cancer or precancerous stages thereof if the presence of said protein biomarkers or signatures in the plasma sample is detected, and performing a colonoscopy on the patient followed by removal of colorectal cancer or polyps.

[0031] For purposes of the present invention, the following terms are defined: The term "colorectal cancer" refers to 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). The expression "colorectal adenoma" refers to adenomas of the colon, also called adenomatous polyps, which are a benign, precancerous stage of colorectal cancer but which nevertheless pose an increased risk of progressing to colorectal cancer. The term "advanced colorectal adenoma" refers to an adenoma at least 10 mm in size or one with histologically high grade dysplasia or villous component of more than 20%. The expression "minimally invasive biological sample" refers to any sample taken from a patient's body that does not require the use of harmful instruments other than a thin needle used to draw the patient's blood, and is therefore not harmful to the patient. In particular, a minimally invasive biological sample in the present invention refers to a blood, serum, or plasma sample. The expression "reference concentration level measured in healthy control subjects" refers to a "reference value" of the concentration level of a protein. If a deviation of the concentration level of a protein is determined with respect to said "reference concentration level measured in healthy control subjects", this is indicative of colorectal cancer or a precancerous stage thereof. In particular, if the concentration level of a biomarker or signature of the invention is significantly higher or lower with respect to said "reference value", this is indicative of colorectal cancer or a precancerous stage thereof. 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 to a reference value to assess whether a patient is likely to suffer from colorectal cancer and / or its precancerous stages. The "reference value" can be a threshold or cut-off value. Typically, the "threshold" or "cut-off value" can be experimentally, empirically, or theoretically determined. As will be appreciated by the skilled artisan, the threshold value can also be arbitrarily selected based on existing experimental and / or clinical conditions. The threshold value must be determined to obtain optimal sensitivity and specificity depending on the function of the test and the benefit / risk balance (false positive and false negative clinical outcomes). Preferably, the skilled artisan may compare the biomarker level (or score) obtained according to the method of the present invention with a defined threshold value. Typically, the optimal sensitivity and specificity (and thus the threshold value) can be determined using a receiver operating characteristic (ROC) curve based on experimental data. For example, after determining the level of the biomarker in the reference group, an algorithmic analysis can be used for the statistical treatment of the measured concentrations of the biomarker in the biological sample of the test subject to determine the sample fraction. The ROC curve is a receiver operating characteristic curve, also known as the receiver operation characteristic curve. It is a receiver operator characteristic curve. ROC curve is mainly used in clinical biochemical diagnostic tests, and is a comprehensive index that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). ROC curve reveals the relationship between sensitivity and specificity by image synthesis method. A series of different cut-off values ​​(threshold or critical value, the boundary value between normal and abnormal results of diagnostic tests) are set as continuous variables to calculate a series of sensitivity and specificity values. Then, the curve is drawn using sensitivity as the vertical coordinate and specificity as the horizontal coordinate. The larger the area under the curve (AUC), the higher the accuracy of diagnosis. In the ROC curve, the point closest to the upper left end of the coordinate diagram is the critical point with both high sensitivity and high specificity values. The AUC value of the ROC curve is 1.0 to 0.5. When the AUC is more than 0.5, the diagnostic result is better and better as the AUC approaches 1. When the AUC is 0.5 to 0.7, the accuracy is low. When AUC is 0.7-0.9, the accuracy is good. When AUC is greater than 0.9, the accuracy is very high. This algorithmic method is preferably performed using a computer. Existing software or systems in the art, such as MedCalc 9.2.0.1 medical statistics software, SPSS 9.0, etc., can be used to draw the ROC curve. "Including" includes, but is not limited to, what follows the word "including." 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. "Consisting only of" means "including and limiting" what precedes the phrase "consisting only of." Thus, the phrase "consisting only of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0032] For the purposes of the present invention, the following proteins are identified according to the Uniprot database:

[0033] TIFF2025038186000002.tif49170 [Brief description of the drawings]

[0034] [Figure 1] FIG. 1 shows the receiver operating characteristic (ROC) curves: A) [Flt3L and CYFRA21-1] in colorectal cancer. Area under the curve (AUC)=0.865. B) [Flt3L and CYFRA21-1] in advanced colorectal adenoma. Area under the curve (AUC)=0.606. The X-axis represents specificity. The Y-axis represents sensitivity. [Diagram 2] FIG. 1 shows the receiver operating characteristic (ROC) curves: A) [Flt3L and CYFRA21-1 and AREG] in colorectal cancer. Area under the curve (AUC)=0.899. B) [Flt3L and CYFRA21-1 and AREG] in advanced colorectal adenoma. Area under the curve (AUC)=0.720. The X-axis represents specificity. The Y-axis represents sensitivity. [Diagram 3] Receiver operating characteristic (ROC) curves: A) [Flt3L and CYFRA21-1 and AREG and ErbB4] in colorectal cancer. Area under the curve (AUC)=0.931. B) [Flt3L and CYFRA21-1 and AREG and ErbB4] in advanced colorectal adenoma. Area under the curve (AUC)=0.707. X-axis represents specificity. Y-axis represents sensitivity. [Figure 4] Receiver operating characteristic (ROC) curves: A) [Flt3L and CYFRA21-1 and AREG and CLEC2C] in colorectal cancer. Area under the curve (AUC)=0.915. B) [Flt3L and CYFRA21-1 and AREG and CLEC2C] in advanced colorectal adenoma. Area under the curve (AUC)=0.727. X-axis represents specificity. Y-axis represents sensitivity. [Diagram 5]Receiver operating characteristic (ROC) curves: A) [AREG and CYFRA21-1] in colorectal cancer. Area under the curve (AUC) = 0.878. B) [AREG and CYFRA21-1] in advanced colorectal adenoma. Area under the curve (AUC) = 0.722. X-axis represents specificity. Y-axis represents sensitivity. [Figure 6] Receiver operating characteristic (ROC) curves: A) [AREG and CYFRA21-1 and Flt3L and ErbB4] in colorectal cancer. Area under the curve (AUC) = 0.931. B) [AREG and CYFRA21-1 and Flt3L and ErbB4] in advanced colorectal adenoma. Area under the curve (AUC) = 0.707. X-axis represents specificity. Y-axis represents sensitivity. [Figure 7] Receiver operating characteristic (ROC) curves: A) [AREG and CYFRA21-1 and Flt3L and CLEC2C] in colorectal cancer. Area under the curve (AUC) = 0.915. B) [AREG and CYFRA21-1 and Flt3L and CLEC2C] in advanced colorectal adenoma. Area under the curve (AUC) = 0.727. X-axis represents specificity. Y-axis represents sensitivity. [Figure 8] Receiver operating characteristic (ROC) curve diagram: A) [AREG and CYFRA21-1 and CD147 and HGFR] in colorectal cancer. Area under the curve (AUC) = 0.888. B) [AREG and CYFRA21-1 and CD147 and HGFR] in advanced colorectal adenoma. Area under the curve (AUC) = 0.769. X-axis represents specificity. Y-axis represents sensitivity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0035] Example 1. Materials and Methods Example 1.1. Study population A total of 96 subjects from eight Spanish hospitals (Burgos, Vigo, Donosti, Ourense, Bierzo, Bertovigte, and Zaragoza Hospitals) were prospectively included in the study: 64 patients with newly diagnosed sporadic colorectal neoplasia (32 with CRC and 32 with AA) and 32 healthy individuals without any personal history of cancer and confirmed by recent colonoscopy to be free of colorectal neoplastic lesions. Patients with AA were those with adenomas at least 10 mm in size or histologically with high-grade dysplasia or villous component >20%. Participant characteristics are shown in Table 1. Blood samples were collected before endoscopy or surgery in all individuals.

[0036] [Table 1]

[0037] The study was approved by the institutional ethics committee of each hospital, and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki.

[0038] Example 1.2. Sample preparation Ten milliliters of whole blood from each participant was collected into tubes containing EDTA K2. Blood samples were placed at 4°C until plasma separation. Samples were centrifuged at 1600×g for 10 minutes at 4°C to spin down blood cells, and the plasma was transferred to a new tube and then further centrifuged at 16000×g for 10 minutes at 4°C to completely remove cellular components.

[0039] Example 1.3. Molecular Analysis The concentrations of biomarkers in plasma samples were established using commercially available ELISA (enzyme-linked immunosorbent assay) and CLIA (chemiluminescence immunoassay) tests according to the corresponding instructions. HGFR and ErbB4 were analyzed with ELISA kits from Cloud clone Corp. The levels of CD147, CLEC2C, Flt3L, and FasL were measured using Elabscience. IFN-gamma was measured using an ELISA kit from Abcam. In connection with the CLIA study, CYFRA21-1 and AREG were cloned using Cloud Clone. The analysis was performed using a CLIA test manufactured by Corp.

[0040] Example 1.4. Quantification of Data For the protein quantification step, samples were treated with the corresponding kit (ELISA / CLIA) and distributed into experimental plates. Each plate also contained control data used to construct a standard curve. Fluorescence data (expressed as integers) obtained from each run were background corrected for each sample and quantified using a standard curve constructed using a second-order polynomial regression model.

[0041] Example 1.5. Statistical Analysis Three groups of individuals were considered in this analysis: CRC (individuals diagnosed with colorectal cancer), AA (individuals diagnosed with advanced adenoma) and CTL (disease-free individuals).

[0042] The raw quantification data were transformed by applying a square root function followed by centering and scaling so that after transformation, each protein measurement has a mean of 0 and a standard deviation of 1. The quantitative values ​​are summarized in Tables 2 and 3 and each protein is described as the median and interquartile range for the different groups studied.

[0043] Non-normality of the data was confirmed by the Shapiro-Wilk test, and the Wilcoxon rank sum test was then used to compare either CRC or AA cases with CTL individuals.

[0044] The diagnostic performance of the individual proteins and some of their combinations was assessed by the receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC). Furthermore, the sensitivity, specificity, positive predictive value and negative predictive value (PPV and NPV) of the different tests were calculated at the optimal cut-off point defined by the best Youden's Index (or equivalently, the point of the ROC that maximizes the sum of the sensitivity and specificity).

[0045] The scores used to derive the ROC-AUC and residual performance values ​​were obtained using univariate logistic regression models of the individual proteins and multivariate logistic regression models of the various combinations of the proteins considered. The 95% CIs of the AUCs were obtained using the DeLong methodology for both the individual markers and their combinations.

[0046] Example 2. Results Example 2.1. Individual marker results Various metrics were determined to evaluate individual proteins, and the following comparisons were also made between CRC / AA and CTL. It can be seen that AREG, CYFRA21-1, and Flt3L are significantly different between the CRC and CTL groups, and the AUC is significantly different from 0.5 (because the 95% confidence interval does not include 0.5). For the AA group, AREG also shows a statistical difference compared to the CTL group.

[0047] Tables 2 and 3 show the metrics of individual proteins including Wilcoxon p-values ​​(p.Wilc), area under the ROC curve (AUC), and sensitivity (Sens.), specificity (Spec.), and positive predictive value (VPP) and negative predictive value (VPN) calculated at the cutoff point of the ROC curve using the best Youden's index. The sign column indicates whether high levels of the marker increase or decrease the risk of disease (+ and -, respectively) for biomarkers with p-values ​​less than 0.25.

[0048] [Table 2]

[0049] [Table 3]

[0050] Example 2.2. Best combination of biomarkers With the aim of improving the individual diagnostic power, combinations of proteins were investigated as follows: Based on markers with p<0.25 for either the CRC vs. CTL (Table 3) or AA vs. CTL (Table 4) comparisons, all possible combinations of two, three, and four of these proteins were investigated simultaneously using multivariate logistic regression.

[0051] Tables 4, 5, 5.2, 6, and 6.2 show the AUC achieved with combinations of two, three, and four biomarkers, respectively, distinguishing CRC versus CTL.

[0052] [Table 4]

[0053] [Table 5] TIFF2025038186000008.tif70170TIFF2025038186000009.tif241170TIFF2025038186000010.tif100170

[0054] [Table 6] TIFF2025038186000012.tif143170TIFF2025038186000013.tif240170TIFF2025038186000014.tif111170

[0055] Tables 7, 8, and 9 show the AUC achieved with combinations of two, three, and four biomarkers, respectively, distinguishing AA versus CTL.

[0056] [Table 7]

[0057] [Table 8]

[0058] [Table 9]

[0059] The best models were selected based on their AUC. Tables 10 and 10-2 show the best results for CRC. Tables 11 and 11-2 show the best results for AA. ROC curves Metrics of the best combination of proteins are included including area under the line (AUC), sensitivity (Sens.), specificity (Spec.), and positive predictive value (PPV) and negative predictive value (NPV) calculated at the cutoff point of the ROC curve using the best Youden's index.

[0060] [Table 10] TIFF2025038186000019.tif91170

[0061] [Table 11] TIFF2025038186000021.tif87170

[0062] Finally, Tables 12 and 12-2 are designed to show the overlap of the most important signatures claimed in the present invention. It is clearly shown that all the best signatures include [Flt3L and CYFRA21-1] and [AREG and CYFRA21-1].

[0063] [Table 12] TIFF2025038186000023.tif78170

Claims

1. An in vitro method for diagnosing colorectal cancer and / or its precancerous stages, comprising: a) measuring concentration levels of the combination [Flt3L and CYFRA21-1] in a biological sample obtained from a subject; and b) identifying a deviation or variation in the concentration levels of at least the combination [Flt3L and CYFRA21-1] compared to reference concentration levels measured in healthy control subjects, which indicates that the subject is suffering from colorectal cancer and / or its precancerous stages.

2. 2. An in vitro method for diagnosing colorectal cancer and / or precancerous stages thereof according to claim 1, comprising: a) measuring the concentration levels of at least the combination [Flt3L and CYFRA21-1 and AREG], or the combination [Flt3L and CYFRA21-1 and AREG and ErbB4], or the combination [Flt3L and CYFRA21-1 and AREG and CLEC2C] in a biological sample obtained from said subject, and b) if a deviation or variation in the concentration levels of at least one combination mentioned in step a) is identified compared to reference concentration levels measured in healthy control subjects, this indicates that said subject is suffering from colorectal cancer and / or precancerous stages.

3. 3. The method according to claim 1 or 2, wherein the precancerous stage of colorectal cancer is advanced colorectal adenoma. vitro method.

4. In vitro use of the combination [Flt3L and CYFRA21-1] for the diagnosis of colorectal cancer and / or its precancerous stages in biological samples obtained from patients.

5. 5. The in vitro use according to claim 4 of the combination [Flt3L and CYFRA21-1 and AREG], or [Flt3L and CYFRA21-1 and AREG and ErbB4], or [Flt3L and CYFRA21-1 and AREG and CLEC2C] for the diagnosis of colorectal cancer and / or precancerous stages thereof in a biological sample obtained from said patient.

6. A kit comprising a reagent for determining the concentration level of a signature selected from the list of at least [Flt3L and CYFRA21-1], or [Flt3L and CYFRA21-1 and AREG], or [Flt3L and CYFRA21-1 and AREG and ErbB4], or [Flt3L and CYFRA21-1 and AREG and CLEC2C].

7. Use of the kit according to claim 6 for the diagnosis of colorectal cancer and / or its precancerous stages.

8. The use of the kit according to claim 7, wherein the precancerous stage of colorectal cancer is advanced colorectal adenoma.

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