Colon cancer biomarkers

Abstract

The present invention provides a method for the diagnosis in vitro of colon cancer in a subject that comprises (i) determining in a sample isolated from the subject the expression level of at least one gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1, and RNF31, (ii) calculating a variable S, wherein S= A +B + C – D – E, and wherein: A is the expression level of gene LNC-TBP-2 when the expression level of gene LNC-TBP-2 is determined in step (i), or is 0 when the expression level of gene LNC-TBP-2 is not determined in step (i); B is the expression level of gene CCL5 when the expression level of gene CCL5 is determined in step (i), or is 0 when the expression level of gene CCL5 is not determined in step (i); C is the expression level of gene DENND2B when the expression level of gene DENND2B is determined in step (i), or is 0 when the expression level of gene DENND2B is not determined in step (i); D is the expression level of gene LRRC52-AS1 when the expression level of gene LRRC52-AS1 is determined in step (i), or 0 when the expression level of gene LRRC52-AS1 is not determined in step (i); and E is the expression level of gene RNF31 when the expression level of gene RNF31 is determined in step (i), or is 0 when the expression level of gene RNF31 is not determined in step (i); and (iii) diagnosing the subject as having colon cancer if S calculated in step (ii) is equal or lower than a corresponding reference value. The present invention also provides the use of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1, and RNF31 as biomarkers of CRC.

Description

[0001] Colon cancer biomarkers

[0002] This application claims the benefit of European Patent Application 24382185.7 filed February 21st2024.

[0003] Technical Field

[0004] The present invention is related to the field of diagnosis. In particular, the present invention provides non- invasive methods for the diagnosis of colon cancer, based on measuring the amount of specific cell-free RNAs in a bodily fluid sample.

[0005] Background Art

[0006] Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer deaths worldwide. Early diagnosis of CRC allows saving lives and is generally associated with better outcomes. However, diagnosis of CRC is challenging and involves a complex process that usually starts with the detection of the first symptoms by the patient, followed by clinical diagnostic procedures, mainly based on colonoscopy. Colonoscopy is an invasive, expensive and time-consuming procedure. Thus, current CRC screening has been generally implemented as a two-step procedure with a first non-invasive test (most commonly a fecal immunochemical test (FIT) allowing quantification of occult hemoglobin in the stool) followed by colonoscopy if the first test is positive. However, the sensitivity of fecal blood tests is low, given that not all CRC show bleeding and, importantly, other gastrointestinal (Gl) malignancies or benign disorders can also cause bleeding. Thus, this approach provides many false positive results, leading to several unnecessary colonoscopies, which as indicated above, are invasive, expensive and time-consuming. Additionally, FIT has limitations in accurately identifying early stages 1 and 2 CRC cases, which is crucial for reducing mortality, saving lives and improving patient outcomes.

[0007] Therefore, there is a need in the state of the art for alternative methods allowing an accurate identification of CRC patients, at an early stage, in a non-invasive, time and cost effective manner.

[0008] Summary of Invention

[0009] The authors of the present invention have surprisingly found that different genes or combination of genes selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1, and RNF31 show an expression level in blood samples that allows to classify subjects into CRC or non-CRC subjects.

[0010] As disclosed in the examples below, the authors of the present invention have determined the expression level of genes LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1, and RNF31 in blood samples from healthy and CRC subjects and have analyzed their diagnostic potential for the detection of CRC based on a receiver operating characteristic (ROC) curve for each gene or combination of genes. As shown in table 1 below, the Area Under the Curve (AUG) thus obtained for several of said genes or combination of genes is higher than 0.7, higher than 0.8 or even almost of 0.9. Thus, the authors of the present invention have identified several genes or combinations of genes whose expression level determined in blood samples allows to differentiate subjects suffering CRC from those not suffering the disease, in an efficient, or even outstanding manner, and with a time and cost-effective approach.

[0011] Thus, in a first aspect, the invention relates to a method for the diagnosis in vitro of colon cancer in a subject that comprises: (i) Determining in a sample isolated from the subject the expression level of at least one gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31 ,

[0012] (II) Calculating a variable S, wherein:

[0013] S= A +B + C - D - E, wherein:

[0014] - A is the expression level of gene LNC-TBP-2 when the expression level of gene LNC-TBP-2 is determined in step (I), or is 0 when the expression level of gene LNC-TBP-2 is not determined in step (I),

[0015] - B is the expression level of gene CCL5 when the expression level of gene CCL5 is determined in step (I), or is 0 when the expression level of gene CCL5 is not determined in step (I),

[0016] - C is the expression level of gene DENND2B when the expression level of gene DENND2B is determined in step (I), or is 0 when the expression level of gene DENND2B is not determined in step (I),

[0017] - D is the expression level of gene LRRC52-AS1 when the expression level of gene LRRC52-AS1 is determined in step (I), or 0 when the expression level of gene LRRC52-AS1 is not determined in step (I),

[0018] - E is the expression level of gene RNF31 when the expression level of gene RNF31 is determined in step (I), or is 0 when the expression level of gene RNF31 is not determined in step (I) and

[0019] (ill) Diagnosing the subject as having colon cancer if S calculated in step (II) is equal or lower than a corresponding reference value and as not having colon cancer if S calculated in step (II) is higher than said corresponding reference value.

[0020] In a second aspect, the invention relates to an in vitro method for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer wherein the method comprises steps (I) and (II) as defined in the first aspect of the invention, and (ill) if S calculated in step (II) is equal or lower than the corresponding reference value, it is indicative that the individual has to start a therapeutic intervention.

[0021] In a third aspect, the invention relates to an in vitro method for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer, wherein the method comprises performing steps (I) and (ii) as defined in the first aspect of the invention before and after starting the therapeutic intervention, and if the level of S calculated in step (ii) after starting the therapeutic intervention is higher than the level of S calculated in step (ii) before starting the therapeutic intervention, it is indicative that the therapeutic intervention is effective in the treatment of colon cancer.

[0022] In a fourth aspect, the invention relates to a method for classifying a subject into a subject cohort, wherein the method comprises steps (I) and (ii) as defined in in the first aspect of the invention and (ill) classifying the subject into:

[0023] - a cohort of subjects with colon cancer if S calculated in step (ii) is equal or lower than the corresponding reference value, or

[0024] - a cohort of subjects with no colon cancer if S calculated in step (ii) is higher than the corresponding reference value.

[0025] In a fifth aspect the invention relates to the use of at least one gene selected from the group consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31, as a biomarker for the diagnosis of colon cancer; or as a biomarker for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer; or as a biomarker for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer; or as a biomarker for classifying a subject into a cohort of subjects with colon cancer or of subjects with no colon cancer.

[0026] In a sixth aspect, the invention relates to the use of a kit for the diagnosis of colon cancer in an individual, or for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer, or for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer, or for classifying a subject into a subject cohort of subjects with colon cancer or of subjects with no colon cancer, the kit comprising means for detecting the level of expression of at least one gene selected from the list consisting of LNC-TBP-2 CCL5, DENND2B, LRRC52-AS1 and RNF31 .

[0027] Detailed description of the invention

[0028] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0029] As used herein, the indefinite articles "a” and "an” are synonymous with "at least one” or "one or more.” Unless indicated otherwise, definite articles used herein, such as "the” also include the plural of the noun.

[0030] For purposes of the present invention, any ranges given include both the lower and the upper end-points of the range. Ranges given, such as concentrations and the like, should be considered approximate, unless specifically stated. The term "about" refers to a deviation of plus / minus 10 %, preferably plus / minus 5 %.

[0031] 1 . Methods of the invention

[0032] A. Method for the diagnosis in vitro of colon cancer in a subject

[0033] In a first aspect, the invention relates to a method for the diagnosis in vitro of colon cancer in a subject that comprises:

[0034] (I) Determining in a sample isolated from the subject the expression level of at least one gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31 ,

[0035] (ii) Calculating a variable S, wherein:

[0036] S= A +B + C - D - E, wherein:

[0037] - A is the expression level of gene LNC-TBP-2 when the expression level of gene LNC-TBP-2 is determined in step (I), or is 0 when the expression level of gene CCL5 is not determined in step (I),

[0038] - B is the expression level of gene CCL5 when the expression level of gene CCL5 is determined in step (I), or is 0 when the expression level of gene CCL5 is not determined in step (I)

[0039] - C is the expression level of gene DENND2B when the expression level of gene DENND2B is determined in step (I), or is 0 when the expression level of gene DENND2B is not determined in step (I).

[0040] - D is the expression level of gene LRRC52-AS1 when the expression level of gene LRRC52-AS1 is determined in step (I), or 0 when the expression level of gene LRRC52-AS1 is not determined in step (I),

[0041] - E is the expression level of gene RNF31 when the expression level of gene RNF31 is determined in step (I), or is 0 when the expression level of gene RNF31 is not determined in step (I) and

[0042] (ill) Diagnosing the subject as having colon cancer if S calculated in step (ii) is equal or lower than a corresponding reference value and as not having colon cancer if S calculated in step (ii) is higher than said corresponding reference value.

[0043] The term "diagnose", "diagnosing” or "diagnosis", as used herein, relates to the evaluation of the probability according to which a subject suffers a specific pathology (in this case, suffering from colon cancer). As the skilled in the art will understand, such evaluation may not be correct for 100% of the subjects to be diagnosed, although it preferably is. The term, however, requires being able to identify a statistically significant part of the subjects. Whether a subject is statistically significant can be determined without further ado by the person skilled in the art using various well known statistical evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. The p-values are, particularly, 0.05, 0.01, 0.005 or lower.

[0044] The term "colon cancer”, "colorectal cancer”, "CRC”, "bowel cancer”, or "rectal cancer” as used herein refers to a cancer developed from the colon or rectum. Signs and symptoms may include blood in the stool, a change in bowel movements, weight loss, and fatigue. This malignant disease develops from the pathological transformation of normal colonic epithelium to adenomatous polyps, which ultimately leads to invasive cancer. This process is gradual and is considered to involve the accumulation of genetic and / or epigenetic alterations. Depending on the stage of progression, colon cancer can be classified as follows:

[0045] Stage 0: This is called cancer in situ. The cancer cells are only in the mucosa, or the inner lining, of the colon or rectum.

[0046] Stage I: The cancer has grown through the mucosa and has invaded the muscular layer of the colon or rectum. It has not spread into nearby tissue or lymph nodes.

[0047] Stage 11 A: The cancer has grown through the wall of the colon or rectum but has not spread to nearby tissue or to the nearby lymph nodes.

[0048] Stage II B: The cancer has grown through the layers of the muscle to the lining of the abdomen, called the visceral peritoneum. It has not spread to the nearby lymph nodes or elsewhere.

[0049] Stage IIC: The tumor has spread through the wall of the colon or rectum and has grown into nearby structures. It has not spread to the nearby lymph nodes or elsewhere.

[0050] Stage IIIA: The cancer has grown through the inner lining or into the muscle layers of the intestine. It has spread to 1 to 3 lymph nodes or to a nodule of tumor cells in tissues around the colon or rectum that do not appear to be lymph nodes but has not spread to other parts of the body.

[0051] Stage II IB: The cancer has grown through the bowel wall or to surrounding organs and into 1 to 3 lymph nodes or to a nodule of tumor in tissues around the colon or rectum that do not appear to be lymph nodes. It has not spread to other parts of the body.

[0052] Stage I IIC: The cancer of the colon, regardless of how deep it has grown, has spread to 4 or more lymph nodes but not to other distant parts of the body.

[0053] Stage IVA: The cancer has spread to a single distant part of the body, such as the liver or lungs.

[0054] Stage I VB: The cancer has spread to more than 1 part of the body.

[0055] Stage IVC: The cancer has spread to the peritoneum. It may also have spread to other sites or organs.

[0056] In a particular embodiment, the term colon cancer as used herein, refers to CRC in any stage l-IV indicated in the definition of CRC above. In another embodiment, the term refers to CRC in stage I as defined above. In another embodiment, the term refers to CRC in stage II, particularly to any substage therein, as defined above. In another embodiment, the term refers to CRC in stage III, particularly any substage therein, as defined above. In another embodiment, the term refers to CRC in stage IV, particularly any substage therein, as defined above. In another embodiment, the term refers to CRC in any stage l-ll or l-lll indicated in the definition of CRC above. In another embodiment, the term refers to CRC in any stage ll-IV indicated in the definition of CRC above. In another embodiment, the term refers to CRC in any stage Il-Ill, or lll-IV indicated in the definition of CRC above.

[0057] The term "subject" or "individual" or "animal" or "patient" or "mammal" means any subject, particularly a mammalian subject, for whom diagnosis or prognosis is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on. In a particular embodiment of the invention, the subject is a mammal. In a more particular embodiment of the invention, the subject is a human. In an even more particular embodiment, the subject is a male or female human of any age or ethnicity. In a particular embodiment, the subject has shown signs to be suffering from colon cancer. Well known signs of a subject to be suffering colon cancer include the presence of blood in the stool, determined by eye, or even by fecal immunochemical test (FIT), or signs determined from the results of a colonoscopy.

[0058] Thus, in a particular embodiment, the subject has been considered to show signs of suffering colon cancer, particularly after fecal immunochemical test (FIT) or a colonoscopy. In a particular embodiment, the subject has already been subjected to a fecal immunochemical test (FIT) or a colonoscopy.

[0059] The term "variable S”, “S” or "subject's score”, as used herein, derives from the expression level of a gene or combination of genes in an isolated sample, and is obtained applying the formula: S= A+B+C-D-E, wherein:

[0060] - A is the expression level of gene LNC-TBP-2 when the expression level of gene LNC-TBP-2 is determined in step (I) of the methods of the invention, or is 0 when the expression level of gene LNC-TBP-2 is not determined in step (I) of the methods of the invention,

[0061] - B is the expression level of gene CCL5 when the expression level of gene CCL5 is determined in step (I) of the methods of the invention, or is 0 when the expression level of gene CCL5 is not determined in step (I) of the methods of the invention,

[0062] - C is the expression level of gene DENND2B when the expression level of gene DENND2B is determined in step (I) of the methods of the invention, or is 0 when the expression level of gene DENND2B is not determined in step (I) of the methods of the invention,

[0063] - D is the expression level of gene LRRC52-AS1 when the expression level of gene LRRC52-AS1 is determined in step (I) of the methods of the invention, or 0 when the expression level of gene LRRC52-AS1 is not determined in step (I) of the methods of the invention,

[0064] - E is the expression level of gene RNF31 when the expression level of gene RNF31 is determined in step (I) of the methods of the invention, or is 0 when the expression level of gene RNF31 is not determined in step (I) of the methods of the invention.

[0065] In a particular embodiment, the expression level of at least gene LNC-TBP-2 is determined in step (I) of the methods of the invention. Accordingly, in a particular embodiment, at least the value of variable A in the formula of S is different from 0, i.e. the value of variable A in the formula of S is the expression level of gene LNC-TBP-2 as determined in step (I) of the methods of the invention.

[0066] In a particular embodiment, the method of the first aspect of the invention comprises:

[0067] (I) Determining in a sample isolated from the subject the expression level of gene LNC-TBP-2, and optionally, of at least one gene selected from the list consisting of CCL5, DENND2B, LRRC52-AS1 , and RNF31 ,

[0068] (II) Calculating a variable S, wherein:

[0069] S= A +B + C - D - E, wherein:

[0070] - A is the expression level of gene LNC-TBP-2 determined in step (I),

[0071] - B is the expression level of gene CCL5 when the expression level of gene CCL5 is determined in step (I), or is 0 when the expression level of gene CCL5 is not determined in step (I)

[0072] - C is the expression level of gene DENND2B when the expression level of gene DENND2B is determined in step (I), or is 0 when the expression level of gene DENND2B is not determined in step (i)C is said expression level of gene DENND2B, and when it is not determined in step (I), C is 0,

[0073] - D is the expression level of gene LRRC52-AS1 when the expression level of gene LRRC52-AS1 is determined in step (I), or 0 when the expression level of gene LRRC52-AS1 is not determined in step (I), - E is the expression level of gene RNF31 when the expression level of gene RNF31 is determined in step (I), or is 0 when the expression level of gene RNF31 is not determined in step (I), and

[0074] (ill) Diagnosing the subject as having colon cancer if S calculated in step (II) is equal or lower than a corresponding reference value and as not having colon cancer if S calculated in step (II) is higher than said corresponding reference value.

[0075] In a particular embodiment, the expression level of at least gene CCL5 is determined in step (I) of the methods of the invention, more particularly of gene CCL5 and of at least one gene selected from the list consisting of LNC-TBP-2, DENND2B, LRRC52-AS1, and RNF31. In another particular embodiment, the expression level of at least gene DENND2B is determined in step (I) of the methods of the invention, more particularly of gene DENND2B and of at least one gene selected from the list consisting of LNC-TBP-2, CCL5, LRRC52-AS1, and RNF31. In another particular embodiment, the expression level of at least gene LRRC52-AS1 is determined in step (I) of the methods of the invention, more particularly of gene LRRC52-AS1 and at least one gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, and RNF31 . In another particular embodiment, the expression level of at least gene RNF31 is determined in step (I) of the methods of the invention, more particularly of gene RNF31 and at least one gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1 .

[0076] In another particular embodiment of the first aspect, the term "at least one gene” or "at least one additional gene” consist of "one gene” or "one additional gene”, respectively. In another particular embodiment of the first aspect, the term "at least one gene” or " at least one additional gene” consist of "two genes” or "two additional genes”, respectively. In another particular embodiment of the first aspect, the term "at least one gene” or "at least one additional gene” consist of "three genes” or "three additional genes”, respectively. In another particular embodiment of the first aspect, the term "at least one gene” or "at least one additional gene” consist of "four genes” or "four additional genes”, respectively. In another particular embodiment of the first aspect, the term "at least one gene” or "at least one additional gene” consist of "five genes” or "five additional genes”, respectively.

[0077] In a particular embodiment, step (I) further comprises determining the expression level of at least one additional gene (i.e. further comprises determining the expression level of at least one additional gene not selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1, and RNF31, or in other words, further comprises determining the expression level of at least one additional gene different from any of the genes disclosed in the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1, and RNF31). More particularly, when step (I) further comprises determining the expression level of at least one additional gene (i.e. further comprises determining the expression level of at least one additional gene not selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1, and RNF31, or in other words, further comprises determining the expression level of at least one additional gene different from any of the genes disclosed in the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1, and RNF31), the variable S is obtained applying the formula:

[0078] N

[0079] S= A +B + C - D - E + al Fl, wherein i=i

[0080] F is the expression level of the said at least one additional gene, N is the number of additional genes whose expression level is determined in step (I) and a is (-1) if an increased expression level of the corresponding gene is associated to having CRC or is (1) if a decreased expression level of the corresponding gene or miRNA is associated to having CRC.

[0081] In other words, S could also be obtained applying the formula S= A +B + C - D - E + G, wherein:

[0082] A, B, C, D and E are as defined above; and:

[0083] N - G is obtained applying formula G= £ a; Fi , when step (I) further comprises determining the i=i expression level of at least one gene (i.e. further comprises determining the expression level of at least one additional gene not selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31 , or in other words, further comprises determining the expression level of at least one additional gene different from any of the genes disclosed in the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52- AS1 , and RNF31), wherein: F, N and a are as defined in the previous paragraphs;

[0084] Or

[0085] - G is 0 when step (I) does not further comprise determining the expression level of at least one additional gene (i.e. does not comprise determining the expression level of a gene not selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31, or in other words, does not comprises determining the expression level of a gene different from any of the genes disclosed in the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31).

[0086] Thus, the first aspect of the invention could also be formulated as a method for the diagnosis in vitro of colon cancer in a subject that comprises:

[0087] (I) Determining in a sample isolated from the subject the expression level of at least one gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31 ,

[0088] (II) Calculating a variable S, wherein:

[0089] S= A +B + C - D - E + G, wherein:

[0090] - A is the expression level of gene LNC-TBP-2 when the expression level of gene LNC-TBP-2 is determined in step (I), or is 0 when the expression level of gene CCL5 is not determined in step (I),

[0091] - B is the expression level of gene CCL5 when the expression level of gene CCL5 is determined in step (I), or is 0 when the expression level of gene CCL5 is not determined in step (I)

[0092] - C is the expression level of gene DENND2B when the expression level of gene DENND2B is determined in step (I), or is 0 when the expression level of gene DENND2B is not determined in step (I).

[0093] - D is the expression level of gene LRRC52-AS1 when the expression level of gene LRRC52-AS1 is determined in step (I), or 0 when the expression level of gene LRRC52-AS1 is not determined in step (I),

[0094] - E is the expression level of gene RNF31 when the expression level of gene RNF31 is determined in step (I), or is 0 when the expression level of gene RNF31 is not determined in step (I),

[0095] N

[0096] - G is obtained applying formula G= a; R , when step (I) further comprises determining the expression level i=i of at least one additional gene (i.e. further comprises determining the expression level of at least one additional gene not selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31), wherein F is the expression level of the at least one additional gene, N is the number of additional genes whose expression level is determined in step (I) and a is (-1) if an increased expression level of the corresponding gene is associated to having CRC or is (1) if a decreased expression level of the corresponding gene is associated to having CRC, or G is 0 when step (I) does not further comprise determining the expression level of at least one additional gene (i.e. does not comprise determining the expression level of a gene not selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31), and

[0097] (ill) Diagnosing the subject as having colon cancer if S calculated in step (II) is equal or lower than a corresponding reference value and as not having colon cancer if S calculated in step (II) is higher than said corresponding reference value.

[0098] In a particular embodiment, the diagnostic method of the first aspect is preliminary or complementary to a different CRC diagnostic method. In other words, in a particular embodiment, in case the subject is diagnosed to have CRC with the method of the first aspect, the subject is selected to additionally perform a different diagnostic method for CRC. Different methods for the diagnosis of colon cancer in a subject are well-known by an expert in the field. Non-limitative examples of such methods include a colonoscopy, or fecal immunochemical test (FIT). In a particular embodiment, the method of the first aspect of the invention is preliminary or complementary to another method for the diagnosis of CRC, wherein said additional method comprises or consists of a colonoscopy, fecal immunochemical test (FIT), or the combination of both, particularly comprises or consists of a colonoscopy.

[0099] Thus, in a particular embodiment of the first aspect, step (ill) further comprises selecting the subject to additionally perform a different diagnostic method of CRC, if S calculated in step (II) is equal or lower than a corresponding reference value, and more particularly, not selecting the subject to additionally perform a different diagnostic method of CRC, if S calculated in step (II) is higher than said corresponding reference value.

[0100] Thus, in a more particular embodiment, step (ill) of the method of the first aspect can be reformulated as follows: (ill) Diagnosing the subject as having colon cancer and selecting the subject to additionally perform a different diagnostic method of CRC, if S calculated in step (II) is equal or lower than the corresponding reference value, particularly wherein the said different diagnostic method of CRC comprises or consists of a colonoscopy, a fecal immunochemical test (FIT), or the combination of both, and more particularly, diagnosing the subject as not having colon cancer and not selecting the subject to additionally perform a different diagnostic method of CRC as just defined, if S calculated in step (II) is higher than the corresponding reference value.

[0101] In a further particular embodiment of the first aspect, the method comprises an additional step (iv) in which the subject is selected to additionally perform a different diagnostic method of CRC, if S calculated in step (II) is equal or lower than the corresponding reference value, particularly wherein said different diagnostic method of CRC comprises or consists of a colonoscopy, a FIT, or the combination of both, and more particularly, not selecting the subject to additionally perform a different diagnostic method of CRC as just defined, if S calculated in step (II) is higher than the corresponding reference value.

[0102] In a particular embodiment, the term "different diagnostic method of CRC” as used herein, refers to any CRC diagnostic method known by an expert in the field different from that of aspect 1 , in particular a diagnostic method comprising or consisting of colonoscopy, analysis of a biopsy sample, Fecal Immunochemical Test (FIT), well-known imaging tests, or combinations thereof. In a more particular embodiment, the term refers to diagnostic methods comprising or consisting of a colonoscopy, a FIT, or the combination of both, yet more particularly comprising or consisting of a colonoscopy.

[0103] The term "sample”, or "biological sample”, as used herein, refers to biological material isolated from a subject. In a particular embodiment, the sample is isolated from any suitable biological fluid, particularly from blood. The blood sample can be whole blood, plasma or serum, particularly the blood sample is a plasma sample. Thus, in a particular embodiment of the invention, the isolated sample used in the methods of the invention is a fluid biological sample. In a more particular embodiment, the isolated sample used in the method of the first aspect of the invention is a blood sample, a plasma sample, or a serum sample, more particularly, a plasma sample. The sample comprises cell and / or non-cell material of the subject, particularly non-cell material. In the present invention, the sample comprises genetic material, e.g., RNA, cell-free RNA, heterogeneous nuclear RNA (hnRNA), mRNA, DNA, genomic DNA (gDNA), complementary DNA (cDNA) etc., from the subject under study. In a particular embodiment, the genetic material is RNA, more particularly cell-free RNA. In a more particular embodiment, the sample comprises RNA, particularly C-RNA, but does not comprise DNA. Methods for obtaining samples from a subject, including blood, plasma or serum samples, are well known to those skilled in the art. The term "circulating RNA”, "C-RNA”, "circulating cell-free RNA”, or "cell-free (cfRNA)”, as used herein, refers to RNA released by many tissues into the circulation via different cellular processes, such as apoptosis, microvesicle shedding, or exosome signaling. Because of these diverse origins, C-RNA measurements reflect tissue-specific changes in gene expression, intercellular signaling, as well as the degree of cell death occurring within different tissues throughout the body, including, but not limited to, some tumors. C-RNA is composed of full-length RNA and fragmented RNA, and is stable due to, for instance, its containment within vesicles or forming ribonucleoprotein complexes, protecting it from degradation by nucleases. However, it is of low abundance and warrants careful processing for effective sequencing library preparation. C-RNA also predominantly consists of ribosomal RNA, which is not informative, and generally co-purifies with cell-free DNA (cfDNA). Thus, generally, C-RNA sequencing workflow involves extracting nucleic acids from the body sample of interest, generally from plasma, followed by DNase treatment to remove cfDNA from the sample. Because the input amounts are low, ribosomal RNA is generally retained in the sample and can act as a carrier through the library preparation. This ribosomal RNA can be later removed through the use of ribosomal RNA specific probes .

[0104] The term "expression level of a gene”, "level of gene expression product from a gene”, "amount of gene expression product from a gene”, as used herein has the same meaning and refer to the level or amount of the protein or RNA product (mRNA, IncRNA, miRNA etc) encoded by the indicated gene, particularly in an indicated sample or unit of biological material. In a particular embodiment, the term "expression level of a gene” refers to the level or amount of the RNA product transcribed from said gene, particularly in a certain volume of body fluid comprising this RNA product, or in a sample of said body fluid, more particularly in a sample of said body fluid, yet more particularly in an isolated blood sample, even yet more particularly in an isolated plasma sample.

[0105] The level of expression of a gene can be provided as a normalized value, an absolute value, a relative value, a value that has an upper and / or a lower limit, a series of values, an average value, a median, a mean value or values expressed by referring to the expression level of a control gene. In a particular embodiment, the expression level of a gene is herein provided as a normalized gene expression value. Several methods can be used to normalize the expression level of a gene and a normalized expression level of a gene can be provided in different units. Non-limiting examples of said units include TPM (Transcripts Per Million, also referred to as Transcripts Per Kilobase Million), RPKM (Reads Per Kilobase Million), or FPKM (Fragments Per Kilobase Million). Methods to obtain the normalized expression level of a gene in any of said units are well known by an expert in the field. Particularly, to obtain the normalized expression level of a gene in TPM, the following steps are generally followed: 1) The read counts obtained for each gene are divided by the length of the corresponding gene in kilobases, thus obtaining reads per kilobase (RPK), 2) All the RPK values in a sample are counted up and this number is divided by 1,000,000, thus obtaining a "per million” scaling factor, 3) The RPK obtained for each gene is divided by the "per million” scaling factor, thus obtaining the TPM value for each gene.

[0106] In an embodiment, the expression level of the gene or combination of genes used in the methods of the invention is provided as a normalized gene expression level for each gene, particularly in TPM for each gene. Thus, in a particular embodiment, the expression level of the gene or combination of genes determined in step (I) of the methods of the invention is provided as a normalized expression level, particularly in TPM. As well understood by a skilled person, the units of variable S calculated in step (II) of the method of the invention correspond to the units in which the gene expression level values are provided in step (I). Thus, in a particular embodiment, the units of variable S calculated in step (II) of the methods of the invention are those of the normalized expression level used in step (I), particularly TPM as defined herein, more particularly wherein the expression level determined in step (I) is provided in TPM. Methods to determine the expression level of a gene in a sample are well-known by an expert in the field. Non-limitative examples of such methods include the use of reverse transcription polymerase chain reaction (RT-PCR), wherein extracted RNA can be reverse-transcribed, for example, using a GeneAmp® RNA PCR kit (Perkin Elmer, Calif., USA), following the manufacturer's instructions. In some embodiments, gene expression levels are determined using a gene expression analysis technology that measures RNA in solution. Examples of such gene expression analysis technologies include, but are not limited to, RNAscope™, RT-PCR, quantitative reverse transcription polymerase chain reaction (RT-qPCR), Nanostring®, QuantiGene®, gNPA®, microarray, use of Next Generation Sequencing (NGS) techniques, or RNA sequence (RNA-seq). RNA sequencing can for instance be performed using a Lexogen CORALL RNA-Seq Library Prep Kit to obtain libraries that are then sequenced (for instance with the Illumina® NovAseq sequencer). The sequencing data obtained can then be analyzed with known bioinformatics tools, such as those described in the examples below, to obtain the expression level of the genes of interest. Thus, in a particular embodiment, gene expression levels are determined in the methods of the invention by methods based on RT-qPCR, RT-PCR, RNA sequencing, or Next Generation Sequencing (NGS) techniques, particularly are determined using RT- qPCR, RNA sequencing, or Next Generation Sequencing (NGS) techniques. In another embodiment, gene expression levels are determined using an RNAscope™, Nanostring®, QuantiGene®, gNPA®., or a microarray. In an even more particular embodiment, the gene expression levels determined in step (I) of the method of the invention, are provided as normalized gene expression levels as defined above, which as well understood by a skilled person, derive from the gene expression data obtained of the corresponding genes, particularly obtained with the techniques just mentioned. Thus in a particular embodiment, the normalized gene expression levels mentioned above, are determined by methods that use data of gene expression levels obtained from RT-qPCR, RT-PCR, RNA sequencing, Next Generation Sequencing (NGS) techniques, RNAscope™, Nanostring®, QuantiGene®, gNPA®, or a microarray techniques, particularly from RT-qPCR or RNA sequencing.

[0107] In a particular embodiment, the expression level of a gene, as used herein, corresponds to the amount of RNA transcribed from said gene, wherein said RNA comprises any of the RNA isoforms encoded by said gene, particularly comprises at least one RNA isoform transcribed from said gene. In another particular embodiment, the expression level of a gene, as used herein, corresponds to the amount of RNA transcribed from said gene, wherein said RNA comprises at least the most abundant RNA isoform transcribed from said gene. In a particular embodiment, it comprises all the RNA isoforms transcribed from said gene. In a particular embodiment, the expression level of the gene or combination of genes determined in step (I) of the methods of the invention is determined using RNA sequencing techniques. In other particular embodiments, the expression level of the gene or combination of genes determined in step (I) of the methods of the invention is determined using RT-qPCR techniques.

[0108] In the present invention, the term "reference value”, or "reference level”, as used herein in any of the methods of the invention, is to be understood as a predefined value for variable S for the gene or combination of genes whose expression level is determined in step (I) of the methods of the invention. The reference value derives from the expression level in a sample or group of samples (also referred to as control sample / s), of the gene or combination of genes whose expression level is determined in step (I) of the methods of the invention. The samples are taken from a subject or group of subjects wherein the presence, absence, stage, or course of the disease has been properly determined previously (also referred to as control subject / s). This value is used as a threshold to discriminate subjects wherein the condition to be analyzed is present from those wherein such condition is absent, to determine the stage of the disease, the risk of developing or of being suffering from CRC, among others. This reference control level is also useful for determining whether the subject has to initiate a medical regimen. In a particular embodiment, the threshold value, as well as the expression level as defined above, can be a normalized value, an absolute value, a relative value, a value that has an upper and / or a lower limit, a series of values, an average value, a median, a mean value or values expressed by referring to the expression level of a control gene. In a more particular embodiment, the threshold value is provided as a range of values. In a particular embodiment of this specific case, a subject is diagnosed as having colon cancer in the method of the first aspect if S calculated in step (ii) of the method is equal or lower than the higher value of a range of values provided as a corresponding reference value, more particularly if S calculated in step (ii) of the method is equal or lower than the lower value of a range of values provided as a corresponding reference value, and as not having colon cancer if S calculated in step (ii) is higher than the upper value of said range of values provided as the corresponding reference value.

[0109] Methods for obtaining the reference value from the group of subjects selected are well-known in the state of the art (Burtis C. A. et al., 2008, Chapter 14, section "Statistical Treatment of Reference Values”). In a particular case the "reference value” is a cut-off value defined by means of a conventional ROC analysis. As the skilled person will appreciate, optimal cut-off value will be defined according to the particular applications of the diagnostic or prognostic method: purpose, target population for the diagnosis or prognosis, balance between specificity and sensibility, etc. Methods to determine the cut-off value by means of conventional ROC curves are well-known by an expert in the field and include those disclosed in the examples below.

[0110] The subject or subjects from whom the "reference value” is derived (i.e. the control subject / s) may include subject / s wherein the CRC is absent, subject / s wherein CRC is present, particularly at different stages, or both. The skilled person in the art, making use of the general knowledge, is able to choose the subject or group of subjects more adequate for obtaining the reference control level for each of the methods of the present invention. In an embodiment of the present invention, optionally in combination with any of the embodiments provided above or below, the reference value referred in any of the methods of the invention is taken from a group of subjects (i.e. group of control subjects) which do not suffer CRC. In one embodiment of the present invention, the reference value referred to in any of the methods of the invention is taken from a group of subjects (i.e. control subjects) which have never been diagnosed as suffering CRC. In one embodiment of the present invention, the reference value referred to in any of the methods of the invention is taken from a group of subjects (i.e. control subjects) which have been diagnosed to not suffer CRC. Clinical routine techniques such as colonoscopy, analysis of a biopsy sample, Fecal Immunochemical Test (FIT), or well-known imaging tests, can be used to that end.

[0111] In one embodiment of the methods of the invention, the reference level is obtained prior to performing the methods of the invention, from the analysis of sets of samples from control subjects as just defined above, particularly from a set of samples from subjects determined to not suffer CRC, more particularly that have never suffered CRC, and from a set of samples from subjects known to suffer CRC. Particularly, the number of samples in each set can be of at least 10, at least 20, at least 40, at least 44, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 500, at least 750, at at least 1000, at least 5000, or at least 10000. Non-limiting examples of the analysis of both sets of samples to obtain the reference value as defined herein include estimation of distribution algorithms, determination of confidence intervals, computer intensive methods, and machine learning methods, including for instance bagging procedures, boosting procedures, random forest algorithms, and / or combinations thereof. In a particular embodiment, the reference value is defined by means of a conventional ROC analysis of the results obtained, particularly of the results obtained with any of the previous methods, more particularly of the results obtained with machine learning methods, applied to the data obtained from the two sets of samples. In another particular embodiment, the reference value is provided as a range of values, or as a value comprised in a range of reference values, wherein the lower limit of said range of reference values corresponds to the higher value S, as defined above, obtained from the samples of subjects known to have CRC as just defined, and the upper limit of the range is the lower value S, as defined above, obtained from the samples of subjects determined to not suffer CRC, particularly that have never suffered CRC.

[0112] In a particular embodiment, the units of the reference value correspond to those of variable S as calculated in step (II) of the methods of the invention. Thus, in a particular embodiment, the reference value used in the methods of the invention is provided in TPM.

[0113] The term "LNC-TBP-2” or "LNC-TBP-6”, as used herein refers to the gene with gene ID ENSG00000230423 in the Ensembl database, release version 110, of July 2023 (gene ID version in the Ensembl database: ENSG00000230423.3). As indicated therein, said gene encodes an RNA classified as IncRNA with unidentified function.

[0114] The term "CCL5”, or "C-C Motif Chemokine Ligand 5”, as used herein, refers to the gene with gene ID ENSG00000271503 in the Ensembl database, release version 110, of July 2023 (gene ID version in the Ensembl database: ENSG00000271503.7).

[0115] The term "DENND2B”, or "DENN domain containing 2B ", as used herein , refers to the gene with gene ID ENSG00000166444 in the Ensembl database, release version 110, of July 2023 (gene ID version in the Ensembl database: ENSG00000166444.19).

[0116] The term “LRRC52-AS1” or "LRRC52 antisense RNA 1” as used herein, refers to the gene with gene ID ENSG00000237463 in the Ensembl database, release version 110, of July 2023 (gene ID version in the Ensembl database: ENSG00000237463.7).

[0117] The term “RNF31” or "ring finger protein 31” as used herein, refers to the gene with gene ID ENSG00000092098 in the Ensembl database, release version 110, of July 2023 (gene ID version in the Ensembl database: ENSG00000092098.18).

[0118] The term "lower than a corresponding reference value”, as used herein, refers to the fact that the value of variable S calculated in step (II) of the method (for the gene or combination of genes whose expression level is determined in step (I)), is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 90%, 95%, 100%, 125%, 150%, 175%, 200% or more than 200% lower than the reference value for the corresponding gene or combination of genes. As well understood by a skilled person, the term "corresponding reference value” as used herein in the methods of the invention, refers to the reference value for the gene or group of genes whose expression level is determined in step (I) of the methods of the invention. Thus, when in step (I) the expression level of genes LNC-TBP2 and of CCL5 is determined, the corresponding reference value, as defined herein, is the reference value as indicated in the definition of "reference value”, for genes LNC-TBP2 and CCL5. The term "lower than the higher value of a range of values provided as a corresponding reference value” as used herein, refers to the fact that the value of variable S calculated in step (II) of the method (for the gene or combination of genes whose expression level is determined in step (I)), is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 90%, 95%, 100%, 125%, 150%, 175%, 200% or more than 200% lower than the higher value of a range of values provided as a corresponding reference level. The term "lower than the lower value of a range of values provided as a corresponding reference value” as used herein, refers to the fact that the value of variable S calculated in step (II) of the method (for the gene or combination of genes whose expression level is determined in step (I)), is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 90%, 95%, 100%, 125%, 150%, 175%, 200% or more than 200% lower than the lower value of a range of values provided as a corresponding reference level. The term "higher to a corresponding reference value”, as used herein, refers to the fact that the value of variable S calculated in step (ii) of the method (for the gene or combination of genes whose expression level is determined in step (I)), is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200% higher than the value of the the reference value for the corresponding gene or combination of genes. The term "higher than the higher value of a range of values provided as the corresponding reference value” as used herein, refers to the fact that the value of variable S calculated in step (II) of the method (for the gene or combination of genes whose expression level is determined in step (I)), is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 90%, 95%, 100%, 125%, 150%, 175%, 200% or more than 200% higher than the higher value of a range of values provided as a corresponding reference level.

[0119] The term "equal to a corresponding reference value”, as used herein, refers to the fact that the value of variable S calculated in step (II) of the method (for the gene or combination of genes whose expression level is determined in step (I)), is equal to about 95%, 97%, 98%, 99%, 99.5%, 99.9%, 100%, 101%, 103% 105%, 107% or 110%, particularly to about 98%, the value of the the reference value for the corresponding gene or combination of genes. The term "equal to the higher value of a range of values provided as the corresponding reference value” as used herein, refers to the fact that the value of variable S calculated in step (II) of the method (for the gene or combination of genes whose expression level is determined in step (I)), is equal to the higher value of the range of values provided as reference level, in a percentage selected from those indicated in the previous definition of "equal to a corresponding reference value”. The term "equal to the lower value of a range of values provided as the corresponding reference value” as used herein, refers to the fact that the value of variable S calculated in step (II) of the method (for the gene or combination of genes whose expression level is determined in step (I)), is equal to the lower value of the range of values provided as reference level, in a percentage selected from those indicated in the previous definition of "equal to a corresponding reference value”.

[0120] In an embodiment, step (I) of the method of the first aspect of the invention (or step (I), as defined in this section 1 .A) comprises determining in the isolated sample the expression level of gene LNC-TBP-2. As well understood by a skilled person, and as indicated in the definition of variable S, in this case variable A in the formula of S corresponds to the expression level of gene LNC-TBP-2, as determined in step (I), and each one of the rest of variables in the formula of S (i.e. B-E) has a value, independently selected from the group consisting of: 0 ("zero”) and the expression level of the corresponding gene as determined in step (I).

[0121] In an embodiment, step (I) of the method of the first aspect of the invention comprises or consists of determining the expression level of a gene or combination of genes selected from the list consisting of:

[0122] - LNC-TBP-2,

[0123] - CCL5,

[0124] - RNF31,

[0125] - DENND2B,

[0126] - LRRC52-AS1,

[0127] - DENND2B and RNF31,

[0128] - LNC-TBP-2 and RNF31,

[0129] - RNF31 and LRRC52-AS1,

[0130] - CCL5 and RNF31,

[0131] - DENND2B and LNC-TBP-2,

[0132] - DENND2B and LRRC52-AS1, - DENND2B and CCL5,

[0133] - LNC-TBP and 2-LRRC52-AS1,

[0134] - LNC-TBP-2 and CCL5,

[0135] - CCL5 and LRRC52-AS1,

[0136] - DENND2B, LNC-TBP-2 and RNF31,

[0137] - DENND2B, RNF31 and LRRC52-AS1,

[0138] - DENND2B, CCL5 and RNF31,

[0139] - LNC-TBP-2, RNF31 and LRRC52-AS1,

[0140] - LNC-TBP-2, CCL5 and RNF31,

[0141] - CCL5, RNF31 and LRRC52-AS1,

[0142] - DENND2B, LNC-TBP-2 and LRRC52-AS1,

[0143] - DENND2B, LNC-TBP-2 and CCL5,

[0144] - DENND2B, CCL5 and LRRC52-AS1,

[0145] - LNC-TBP-2, CCL5 and LRRC52-AS1,

[0146] - DENND2B, LNC-TBP-2, RNF31 and LRRC52-AS1,

[0147] - DENND2B, LNC-TBP-2, CCL5 and RNF31,

[0148] - DENND2B, CCL5, RNF31 and LRRC52-AS1,

[0149] - LNC-TBP-2, CCL5, RNF31 and LRRC52-AS1,

[0150] - DENND2B, LNC-TBP-2, CCL5 and LRRC52-AS1 and

[0151] - DENND2B, LNC-TBP-2, CCL5, RNF31 and LRRC52-AS1, particularly wherein the expression level of the selected gene or of the selected combination of genes is provided in TPM.

[0152] In a particular embodiment, step (i) comprises determining in the isolated sample the expression level of gene CCL5. As well understood by a skilled person and as indicated in the definition of variable S, in this case the value of variable B corresponds to the expression level of gene CCL5 as determined in step (i), and each one of the rest of variables in the formula of S (i.e.A, C-E) has a value, independently selected from the group consisting of 0 ("zero”) and the expression level of the corresponding gene as determined in step (i).

[0153] In a particular embodiment, step (i) comprises determining in the isolated sample the expression level of genes LNC-TBP-2 and CCL5. As well understood by a skilled person and as indicated in the definition of variable S, in this case the value of variable A in the formula of S corresponds to the expression level of gene LNC-TBP-2 as determined in step (i), the value of variable B corresponds to the expression level of gene CCL5 as determined in step (i), and each one of the rest of variables in the formula of S (i.e. C-E) has a value, independently selected from the group consisting of 0 ("zero”) and the expression level of the corresponding gene as determined in step (i).

[0154] In another particular embodiment, step (i) comprises determining in the isolated sample the expression level of genes LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1. As well understood by a skilled person and as indicated in the definition of variable S, in this case the value of variable A in the formula of S corresponds to the expression level of gene LNC-TBP-2 as determined in step (i), the value of variable B corresponds to the expression level of gene CCL5 as determined in step (i), variable C corresponds to the expression level of gene DENND2B as determined in step (i), variable D corresponds to the expression level of gene LRRC52- AS1 as determined in step (i), and variable E has a value selected from the group consisting of 0 ("zero”) and the expression level of the corresponding gene as determined in step (i).

[0155] In another particular embodiment, step (i) comprises determining in the isolated sample the expression level of genes LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 and RNF31. As well understood by a skilled person and as indicated in the definition of variable S, in this case the value of variable A in the formula of S corresponds to the expression level of gene LNC-TBP-2 as determined in step (I), the value of variable B corresponds to the expression level of gene CCL5 as determined in step (I), variable C corresponds to the expression level of gene DENND2B as determined in step (I), variable D corresponds to the expression level of gene LRRC52-AS1 as determined in step (I), and variable E corresponds to the expression level of gene RNF31 as determined in step (I).

[0156] In another particular embodiment, step (I) comprises or consists of determining in the isolated sample the expression level of a combination of genes, particularly provided in TPM, wherein the combination of genes is selected from the group consisting of:

[0157] LNC-TBP-2 and CCL5,

[0158] LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1,

[0159] LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 and RNF31,

[0160] LNC-TBP-2, CCL5 and DENND2B

[0161] CCL5, DENND2B and LRRC52-AS1,

[0162] LNC-TBP-2, CCL5 and LRRC52-AS1, and

[0163] LNC-TBP-2, CCL5, DENND2B and RNF31, particularly from the group consisting of:

[0164] LNC-TBP-2 and CCL5,

[0165] LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1 , and

[0166] LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 and RNF31 , more particularly from the group consisting of:

[0167] LNC-TBP-2 and CCL5,

[0168] LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1 , and

[0169] LNC-TBP-2, CCL5, DENND2B and RNF31 , yet more particularly, from the group consisting of:

[0170] LNC-TBP-2 and CCL5, and

[0171] LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1 .

[0172] In an embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for the gene or combination of genes that comprises or consists of RNF31, has a value in the range of about -90 to -40, -85 to -45, -80 to -50, -75 to -55, -71 to -60, -70.1 to -63.1, -70.025 to -63.181 or about -70 to -63, particularly in the range of about -70.025 to - 63.181. In another particular embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) for said gene or combination of genes, has a value of about -90, -85, -80, -75, -74, -73, -72, -71, -70.1, -70.025, -70, -69, -68, -67, -66.603, -66.6, -66.5, -66, -65, - 64, -63.2, -63.181, -63, -62, -61 or about -60, particularly of about -66.603.

[0173] In another embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B, has a value in the range of about 80 to 125, 85 to 120, 90 to 115, 95 to 112, 100 to 110, 102 to 108, 105 to 106.5, 105.1 to 106.41, or about 105.038 to 106.406, particularly in the range of about 105.038 to 106.406. In another particular embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for said gene or combination of genes has a value of about 80, 85, 90, 95, 97, 100, 101, 102, 103, 104, 105, 105.038,105.7, 105.722, 106, 106.4, 107, 108, 110, 112, or about 115, particularly of about 105,722.

[0174] In another embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of LNC-TBP-2, has a value in the range of about 130 to 200, 135 to 195, 140 to 190, 145 to 185, 150 to 180, 155 to 175, 157 to 172, 160 to 171, 160.246 to 170.766, 162 to 170, 164 to 169, or about 165 to 166, particularly of about 160.246 to 170.766. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes, has a value of about 150, 155, 157, 160, 160.2, 160.246, 161, 162, 163, 164, 165, 165.5, 165,506, 166, 167, 168, 169, 170, or about 170.766, particularly of about 165.506.

[0175] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of LRRC52-AS1, has a value in the range of about -60 to 15, -55 to -20, -50 to -25, -48 to -27, -45 to -30, -42 to -32, -41 to -35, -40.1 to -35, or about -40.061 to -35.075, particularly of about -40.061 to -35.075 In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about -60, -55, -50, -47, -45, -44, -43, -42, -41, -40.061, -40, -39, -38, - 37.6, -37.568, -37, -36, -35.1, -35.075, -35, 34, -33,. -32, -31, -30, -27, or about 25, particularly of about - 37,568.

[0176] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of CCL5, has a value in the range of about 105 to 150, 110 to 145, 112 to 140, 115 to 138, 117 to 135, 120 to 132, 122 to 130, 125 to 129, 125.208 to 128.311 or about 126 to 128, particularly of about 125.208 to128.311 . In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 105, 110, 112, 115, 117, 120, 121, 122, 123, 124, 125, 125.208, 126, 126.76, 127, 128, 128.311, 129, 130, 132, 135, or about 138, particularly of about 126.76.

[0177] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B and RNF31, has a value in the range of about 80 to 125, 85 to 120, 90 to 115, 92 to 112, 95 to 110, 98 to 108, 100 to 106, 103 to 105, 103.264 to 104.329, or about 103 to 104, particularly of about 103.264 to 104.329. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 80, 85, 90, 92, 95, 97, 98, 99, 100, 101, 102, 103, 103.264, 103.796, 104, 104.329, 105, 106, 107, 108, 109, 110, 112, 115, 120, or about 125, particularly of about 103.796 .

[0178] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of LNC-TBP-2 and RNF31, has a value in the range of about 0 to 50, 0 to 45, 0 to 40, 0 to 35, 0 to 34.153, 0 to 34, 0 to 30, 5 to 30, 7 to 27, 10 to 25, 12 to 22, 15 to 20, or about 16 to 18, particularly of about 0 to 34.153. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 2, 5, 7, 10, 12, 13, 14, 15, 16, 17, 17.077, 18, 19, 20, 22, 25, 27, or about 30 particularly of about 1 .077.

[0179] In another embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for the gene or combination of genes that comprises or consists of RNF31 and LRRC52-AS1, has a value in the range of about -110 to -80, -107 to -85, -105 to -87, -102 to -90, -101 to -92, -100 to -94, -99.209 to -94.119, or about -99 to -95, particularly of about - 99.209 to -94.119. In another particular embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for said gene or combination of genes has a value of about -110, -107, -105, -102, -101, -100, -99.209, -99, -98, -97, -96.664, - 96, -95, -94.119, -94, -93, -92, -91, -90, -87, -85, or about -80, particularly of about -96.664 .

[0180] In another embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for the gene or combination of genes that comprises or consists of CCL5 and RNF31, has a value in the range of about 90 to 150, 95 to 145, 100 to 140, 102 to 137, 105 to 135, 107 to 132, 108 to 130, 109 to 129, 109.175 to128.311, 110 to 125, 115 to 120, or about 116 to 119, particularly of about 109.175 to 128.311. In another particular embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for said gene or combination of genes has a value of about 90, 95, 100, 102, 105, 107, 108, 109, 109.175, 110, 112, 115, 117, 120, 122, 125, 126, 127, 128, 128.311, 129, 130, 132,135, 137, 140, 145, or about 150, particularly of about 118.743.

[0181] In another embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B and LNC-TBP-2, has a value in the range of about 315 to 1370, 320 to 365, 325 to 360, 327 to 357, 330 to 355, 332 to 352, 335 to 351, 335.256 to 350.125, 336 to 347, 340 to 345, or about 341 to 344, particularly of about 335.256 to 350.125. In another particular embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for said gene or combination of genes has a value of about 315, 320, 325, 327, 330, 332, 335, 337, 340, 341, 342, 342.69, 343, 344, 345, 347, 348, 349, 350, 352, 355, 357, 360, 365 or about 370, particularly of about 342,69.

[0182] In another embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B and LRRC52-AS1, has a value in the range of about 45 to 85, 50 to 80, 55 to 75, 57 to 72, 60 to 70, 61 to 68, 62 to 67, 63 to 65, or about 63.181 to 64.155, particularly of about 63.181 to 64.155. In another particular embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for said gene or combination of genes has a value of about 45, 50, 52, 55, 57, 60, 61, 62, 63.181, 63.2, 633.668, 63.7, 64, 64.155, 65, 67, 70, 75, 80, or about 85, particularly of about 63.668.

[0183] In another embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B and CCL5, has a value in the range of about 300 to 335, 305 to 330, 307 to 327, 310 to 325, 312 to 322, 315 to 320, or about 315.865 to 319.723, particularly of about 315.865 to 319.723. In another particular embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for said gene or combination of genes has a value of about 300, 305, 307, 310, 312, 315, 315.865, 3159, 316, 317, 317.794, 317.8, 318, 319, 319.723, 320, 322, 325, 327, 330, or about 335 particularly of about 317.794.

[0184] In another embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of LNC-TBP-2 and LRRC52-AS1, has a value in the range of about 35 to 65, 37 to 62, 40 to 60, 42 to 57, 45 to 55, 47 to 53, 50 to 52, 51 .3 to 51 .6, 51 .4 to 51 .5, or about 51 .483 to 51 .499, particularly of about 51 .483 to 51 .499. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 35, 37, 40, 42, 45, 47, 50, 41, 51.3, 51.4, 51 .491 , 51 .5, 51 .55, 51 .6, 51 .7, 52, 53, 54, 55, 57, 60, 62, or about 65 particularly of about 51 ,491 .

[0185] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of LNC-TBP-2 and CCL5, has a value in the range of about 280 to 330, 282 to 327, 285 to 325, 287 to 322, 290 to 320, 292 to 317, 292 to 315, 295 to 312, 300 to 310, 301 to 308, 301.5 to 307.5, 301 .7 to 307.5, or about 302 to 307, particularly of about 301 .73 to 307.379. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 280, 282, 285, 287, 290, 292, 295, 297, 300, 301, 301.7, 301.73, 302, 303, 304, 304.5, 304.55, 304.6,

[0186] 304.7, 305, 306, 307, 307.3, 307.379, 307.5, 3207.7, 308, 309, 310, 312, 315, 317, 320, 322, 325, 327, or about 330, particularly of about 304.555.

[0187] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of CCL5 and LRRC52-AS1, has a value in the range of about 45 to 80, 47 to 77, 50 to 75, 52 to 72, 55 to 70, 57 to 67, 60 to 66, 62 to 65, 62.8 to 64.7, 62.857 to 64.61 or about 63 to 64, particularly of about 62.857 to 64.61. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 45, 47, 50, 52, 55, 57, 60, 62, 62.8, 62.9, 63, 63.2, 63.5,

[0188] 63.7, 63,733, 63.75, 64, 65, 67, 70, 72, 75, 77, or about 80 particularly of about 63,733.

[0189] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B, LNC-TBP-2 and RNF31, has a value in the range of about 55 to 95, 57 to 92, 60 to 90, 62 to 87, 65 to 85, 67 to 83, 70 to 82, 70.3 to 81.5, 70.3 to 81.2, 70.292 to 81.176, 71 to 81, 72 to 80, 73 to 79, or about 74 to 78, particularly of about 70.292 to 81.176. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 55, 57, 60, 62, 65, 67, 70, 70.3, 70.292, 70.3, 70.5, 70.7, 71, 72, 75, 75.5, 75.7, 75.734, 76, 77, 80, 81, 81.176, 82, 85, 87, 90, 92, or about 95 particularly of about 75,734.

[0190] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B, RNF31 and LRRC52-AS1, has a value in the range of about 130 to 200, 135 to 195, 137 to 190, 140 to 185, 142 to 180, 145 to 177, 148 to 176, 148.5 to 175.5, 148.813 to 175.368 , 150 to 170, 160 to 167 or about 161 to 165, particularly of about 148.813 to 175.368. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 130, 135, 140, 145, 147, 148, 148.5, 148.8, 148.813, 148.9, 145, 147, 150, 155, 157, 160, 161, 162, 162.091, 162.1, 162.5, 163, 164, 165, 167, 170, 172, 174, 175, 175.3, 175.37, 175.368, 175.5, 176, 177, 180, 182, 185, 190, 192, 195, or about 200, particularly of about 162,091.

[0191] In another embodiment, when the gene expression level determined in step (I) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B, CCL5 and RNF31, has a value in the range of about 290 to 350, 295 to 345, 300 to 340, 302 to 335, 305 to 332, 307 to 330, 310 to 325, 312 to 322, 315 to 320, 315.5 to 319.8, 315.865 to 319.723, or about 316 to 319, particularly of about 315.865 to 319.723. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 290, 295, 300, 302, 305, 307, 310, 3132, 315, 315.5, 315. 865, 316, 316.5,317, 317.5, 317.7, 317.794, 317.8, 318, 319, 320, 322, 325, 327, 330, 335, 340, 345, or about 350, particularly of about 317.794.

[0192] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of LNC-TBP-2, RNF31 and LRRC52-AS1, has a value in the range of about 0 to 50, 0 to 45, 0 to 40, 0 to 35, 0 to 34.153, 0 to 34, 0 to 30, 5 to 30, 7 to 27, 10 to 25, 12 to 22, 15 to 20, or about 16 to 18, particularly of about 0 to 34.153. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 2, 5, 7, 10, 12, 13, 14, 15, 16, 17, 17.077, 18, 19, 20, 22, 25, 27, or about 30 particularly of about 17.077.

[0193] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of LNC-TBP-2, CCL5 and RNF31, has a value in the range of about 105 to 145, 110 to 140, 112 to 137, 115 to 135, 117 to 132, 120 to 130, 120.184 to 129.219, 120.2 to 129.2, 120.5 to 129, 122 to127 or about 123 to 125, particularly of about 120.184 to 129.219. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 105, 110, 115, 117, 120, 120.184, 120.2, 121, 122, 123, 124, 124.5, 124.7, 124.702, 125, 126, 127, 129, 129.2, 129.219, 130,132, 135, 137, 140, or about 145 particularly of about 124,702.

[0194] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of CCL5, RNF31 and LRRC52-AS1, has a value in the range of about 25 to 100, 30 to 95, 35 to 90, 40 to 85, 45 to 80, 50 to 75, 55 to 70, 60 to 67, 62 to 65, 62.857 to 64.61, or about 63 to 64, particularly of about 62.857 to 64.61 . In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 25, 30, 35, 40, 45, 50, 55, 60, 62, 62.857, 63, 63.5, 63.7, 63.733, 64, 65, 70, 75, 80, 85, 90, 95, or about 100, particularly of about 63,733.

[0195] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B, LNC-TBP-2 and LRRC52-AS1, has a value in the range of about 100 to 160, 105 to 155, 110 to 150, 115 to 150, 117 to 147, 120 to 145, 122 to 140, 125 to 135, 128 to 131, 128.3 to 130.5, or about 128.311 to 130.425, particularly of about 128.311 to 130.425. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (ill) of the methods of the invention for said gene or combination of genes has a value of about 100, 105, 110, 115, 120, 122, 125, 127, 128, 128.311, 128.5, 129, 129.3, 129.366, 129.4, 129.5, 130, 130.4, 130.425, 130.5, 131, 132, 135, 137, 140, 145, 150, 155, or about 160, particularly of about 129.368.

[0196] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B, LNC-TBP-2 and CCL5, has a value in the range of about 520 to 620, 530 to 610, 540 to 600, 545 to 595, 550 to 590, 555 to 585, 560 to 580, 561 to 581, 561 .178 to 580.605, 561 .2 to 580.5, 562 to 580, 565 to 575, or about 567 to 572, particularly of about 561.178 to 580.605 In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 520, 530, 540, 545, 550, 55, 560, 561, 561.178, 561.2, 562, 563, 565, 567, 570, 570.891,570.9, 580, 582, 585, 590, 595, 600, 610, or about 620 particularly of about 570.891.

[0197] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B, CCL5 and LRRC52-AS1, has a value in the range of about 290 to 345, 295 to 340, 297 to 337, 300 to 335, 305 to 330, 307 to 327, 310 to 325, 315 to 320 315.5 to 319.75, 315.865 to 319.723, or about 316 to 319, particularly of about 315.865 to 319.723. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 290, 295, 300, 305, 307, 310, 312, 315, 315.5, 315.865, 316, 317, 317.5, 317.794, .318, 319, 320, 322, 325, 327, 330, 335, 340, or about 345, particularly of about 317.794.

[0198] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of LNC-TBP-2, CCL5 and LRRC52-AS1, has a value in the range of about 150 to 220, 155 to 215, 160 to 215, 165 to 210, 167 to 205, 170 to 200, 172 to 195, 173 to 193, 173.8 to 192.5, 173.853 to 192.466, 175 to 190, or about 180 to 185, particularly of about 173.853 to 192.466. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 150, 155, 160, 165, 170, 175, 180, 183, 183.16, 183.2, 183.5, 184, 185, 190, 195, 200, 205, 210, 215, or about 220, particularly of about 183.16.

[0199] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B, LNC-TBP-2, RNF31 and LRRC52-AS1, has a value in the range of about 190 to 230, 195 to 225, 200 to 220, 202 to 2018, 205 to 215, 207 to 212, 208 to 211, 208.6 to 210.4, or about 208.658 to 210.45, particularly of about 208.658 to 210.45. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 190, 195, 200, 202, 205, 207, 209, 209.5, 209.554, 210, 210.4, 210.45, 210.5, 211, 212, 215, 220, 225, or about 230, particularly of about 209.554.

[0200] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B, LNC-TBP-2, CCL5 and RNF31, has a value in the range of about 225 to 280, 230 to 275, 235 to 270, 240 to 265, 245 to 260, 250 to 258, 253 to 257, 254.7 to 256.7, 254.768 to 256.621 or about 254.8 to 256.6, particularly of about 254.768 to 256.621. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 225, 230, 235, 2340, 245, 250, 253, 254, 254.7, 254.768, 254.8, 255, 255.5, 255.695, 255.7, 256, 257, 260, 265, 270, 275 or about 280, particularly of about 255.695.

[0201] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B, CCL5, RNF31 and LRRC52-AS1, has a value in the range of about 290 to 350, 295 to 345, 295 to 340, 300 to 335, 305 to 330, 310 to 325, 315 to 320, 315.86 to 319.723, 316 to 319.5, or about 316 to 319.5, particularly of about 315.865, 319.723. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 290, 295, 300, 305, 310, 315, 315.5, 315.86, 316, 317, 317.5, 317.794, 317.8, 318, 319, 319.723, 320, 325, 330, 335, 340, 345 or about 350 , particularly of about 317.794.

[0202] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of LNC-TBP-2, CCL5, RNF31 and LRRC52-AS1, has a value in the range of about 100 to 150, 105 to 145, 110 to 140, 115 to 135, 119 to 130, 119.652 to 129.219, 120 to 129, or about 120 to 125, particularly of about 119.652 to 129.219. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 100, 105, 110, 115, 119.5, 119.652, 119.7,120, 122, 123, 124, 124.435, 124.5, 125, 130, 135, 140, 145 or about 150, particularly of about 124.435.

[0203] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B, LNC-TBP-2, CCL5 and LRRC52-AS1 has a value in the range of about 505 to 600, 510 to 595, 515 to 590, 520 to 585, 525 to 581, 526 to 581, 526.1 to 580.7, 526.104 to 580.605, 530 to 575, 540 to 565, 545 to 560, or about 550 to 555, particularly of about 526.104 to 580.605. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 505, 510, 515, 520, 525, 526, 526.1, 526.104, 527, 530, 535, 540, 545, 550, 552, 553, 553.3, 553.355, 553.5, 554, 555, 560, 565, 570, 575, 580, 585, 590, 595 or about 560, particularly of about 553.355.

[0204] In another embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for the gene or combination of genes that comprises or consists of DENND2B, LNC-TBP-2, CCL5, RNF31 and LRRC52-AS1, has a value in the range of about 400 to 470, 405 to 465, 410 to 460, 415 to 455, 420 to 450, 422 to 445, 423 to 444.5, 423.5 to 444, 423.537 to 443.992, 423.6 to 443.5, 425 to 440 or about 430 to 435, particularly of about 423.537 to 443.992. In another particular embodiment, when the gene expression level determined in step (i) of the methods is in TPM, the reference value used in step (iii) of the methods of the invention for said gene or combination of genes has a value of about 400, 405, 410, 415, 420, 422, 423, 423.5, 423.537, 423.6, 427, 430, 433, 433.5,433.765,433.8, 434, 435, 440, 443, 443.992, 444, 445, 450, 455, 460, 465 about 470, particularly of about 433.765.

[0205] In a particular embodiment, the reference value used in step (ill) of the method of the invention corresponds to the threshold value provided in table 1 below for the gene or combination of genes whose expression level is determined in step (i) of the method, particularly when the expression level(s) determined in step (i) is / are provided in TPM. In a more particular embodiment, the reference value used in step (iii) of the method, is a value corresponding to + / - 1% or + / - 2% the threshold value provided in table 1 below for the gene or combination of genes whose expression level is determined in step (i) of the method, particularly when the expression level (s) determined in step (i) is / are provided in TPM. In a more particular embodiment, the reference value used in step (iii) of the method, is a value corresponding to + / - 5%, more particularly + / - 10% the threshold value provided in table 1 below for the gene or combination of genes whose expression level is determined in step (i) of the method, particularly when the expression level(s) determined in step (i) is / are provided in TPM.

[0206] In another particular embodiment, the reference value used in step (iii) of the method of the invention is provided as a range of value, which is selected from the range of values indicated in the previous paragraphs above for the corresponding gene or combination of genes, as the range in which the reference value is comprised, particularly when the expression level(s) determined in step (i) is / are provided in TPM.

[0207] In another particular embodiment, the reference value used in step (iii) of the method of the invention is provided as a range of value, which corresponds to the range of threshold values indicated in table 1 below for the corresponding gene or combination of genes, particularly when the expression level(s) determined in step (i) is / are provided in TPM. In another particular embodiment, the reference value determined in step (iii) of the invention is provided as a range of values, wherein the lower and upper values of the range correspond to + / -1 % or + / - 2% the lower and upper values, respectively, of the range of threshold values provided in table 1 below for the gene or combination of genes whose expression level is determined in step (i) of the method, particularly when the expression level(s) determined in step (i) is / are provided in TPM. In another particular embodiment, the reference value used in step (iii) of the invention is provided as a range of values, the lower and upper values of the range correspond to +1-5%, more particularly of + / - 10% the lower and upper values, respectively, of the range of threshold values provided in table 1 below for the gene or combination of genes whose expression level is determined in step (i) of the method, particularly when the expression level(s) determined in step (i) is / are provided in TPM.

[0208] In another particular embodiment, the reference value used in step (iii) of the methods of the invention for variable S for each of the combination of genes indicated in the embodiments of any aspect of the invention is a range of values selected from those specified for the corresponding combination of genes in the embodiments above (wherein particular ranges of reference level values are specified for the corresponding combination of genes), particularly when the expression level of each gene is provided in TPM. In another particular embodiment, the reference value used in step (iii) for variable S for each of the combination of genes indicated in the embodiments of any aspect of the invention is a value selected from those specified for the corresponding combination of genes in the embodiments above (wherein particular reference level values are specified for the corresponding combination of genes), particularly when the expression level of each gene is provided in TPM.

[0209] In an embodiment, step (i) of the methods of the invention consists of determining in the isolated sample: the expression level of gene LNC-TBP-2 and the corresponding reference value is from 160 to 171, particularly from 164 to 169, or the expression level of genes LNC-TBP-2 and CCL5 and the corresponding reference value is from 300 to 310, particularly from 301 to 308, or the expression level of genes LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1, and the corresponding reference value is from 515 to 590, particularly is from 526 to 581; or the expression level of genes LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1, and RNF31, and the corresponding reference value is from 420 to 450, particularly is from 422 to 445, particularly wherein the expression level of the gene / s is provided in TPM.

[0210] In an embodiment, in those embodiments in which the expression level is determined with a method based on RT-PCR or RT-qPCR, particularly is determined by RT-qPCT, the reference value is the value of variable S calculated from the expression level determined by said method, particularly is determined by RT-qPCR, in a group of control subjects as defined above, particularly from a subject or group of subjects that do not have CRC. In another embodiment, in those embodiments in which the expression level is determined by a method based on RT-PCR or RT-qPCR, particularly is determined by RT-qPCR, the reference value is determined by any of the methods indicated above to determine the reference value.

[0211] In an embodiment, step (I) of the methods of the invention comprises, before determining the expression level of the corresponding gene or combination of genes, isolating the RNA comprised in the isolated sample, particularly the C-RNA in the sample, more particularly wherein the sample is a blood sample, yet more particularly a plasma sample. Methods to isolate the RNA from a sample are well-known by an expert in the field and include phenol-chloroform, or guanidium-thiocyanate based methods, and particularly in case of CfRNA, Plasma / Serum RNA isolation kits and following the manufacturer's instruction such as those from Norgen Biotek ®.

[0212] In another embodiment, step (I) of the method comprises, before determining the expression level of the corresponding gene or combination of genes obtaining the cDNA corresponding to the RNAs comprised in the sample, i.e. reverse-transcription of the RNA comprised in the isolated sample, particularly the C-RNA comprised in the sample. Methods for that purpose are well-known by an expert in the field and include RT- PCR or using a library preparation kit such as Lexogen® CORALL RNA-Seq Library Prep Kit and following the manufacturer's instructions.

[0213] In a further embodiment, step (I) of the method comprises, before determining the expression level of the corresponding gene or combination of genes, sequencing the cDNAs corresponding to the RNAs comprised in the sample, particularly the C-RNAs comprised in the sample. Methods for that purpose are well-known by an expert in the field and include the use of NGS sequencers, such as Illumina® NovaSeq sequencer and following the manufacturer's instructions.

[0214] In another embodiment, step (I) of the methods comprises, before determining the expression level of the corresponding gene or combination of genes, bioinformatic analysis of the data obtained from sequencing the cDNAs corresponding to the RNAs, particularly C-RNAs, comprised in the sample, to determine the expression level of the genes of interest. Bioinformatic tools for that purpose are well-known by an expert in the field and include those disclosed in the examples below.

[0215] In a particular embodiment, step (I) of the method of the invention comprises:

[0216] (I. a) isolating the RNA comprised in the sample, particularly the C-RNA in the sample, more particularly wherein the sample is a blood sample, yet more particularly a plasma sample,

[0217] (i.b) synthesizing the cDNA corresponding the RNAs, or C-RNAs, obtained from step (I. a),

[0218] (i.c) sequencing the cDNAs, or cDNA libraries, obtained from step (i.b),

[0219] (i.d) analyzing with bioinformatic tools the sequencing data obtained from step (i.c) to determine the expression level in the sample of at least one gene selected from the list consisting of: LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1, and RNF31. In a particular embodiment, the gene or group of genes whose expression level is determined in step (i.d) indicated above is any gene or combination of genes provided in any of the embodiments above of this section I. A.

[0220] In a particular embodiment, determining the expression level of the gene or group of genes in step (i) of the methods of the invention is performed by RNA sequencing, particularly wherein the expression level is provided in TPM.

[0221] In another particular embodiment, determining the expression level of the gene or group of genes in step (i) of the methods of the invention is performed by methods based on RT-PCR or RT-qPCR, particularly is performed by RT-qPCR.

[0222] As well understood by a skilled person, the term "step (i)” as used in this section I. A of the invention refers to step (i) of the method of the first aspect of the invention. The term "step (ii)" as used in this section l-A of the invention refers to step (ii) of the method of the first aspect of the invention. The term "step (iii)" as used in this section I. A of the invention refers to step (iii) of the method of the first aspect of the invention.

[0223] In an additional embodiment, the subject of study in the methods of the invention has been treated with a therapeutic intervention for the treatment of cancer, particularly for the treatment of CRC, before performing the methods of the invention. Known therapeutic interventions for that purpose include surgery, chemotherapy, targeted therapy, immunotherapy, surgery, radiation therapy and also palliative care to help relieve symptoms of cancer and side effects of treatment. In another embodiment, the subject of study in the methods of the invention has not been treated with a therapeutic intervention for the treatment of cancer, particularly for the treatment of CRC, before performing the methods of the invention.

[0224] B. Method for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer.

[0225] In a second aspect, the invention relates to an in vitro method for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer wherein the method comprises steps (i) and (ii) as defined in the first aspect of the invention, and (iii) if S calculated in step (ii) is equal or lower than the corresponding reference value, it is indicative that the individual has to start a therapeutic intervention.

[0226] In the present invention, "to initiate a therapeutic intervention” and the term "treat" are interchangeable. Thus, both are used herein to mean to intervene, or administer the subject with medicaments, to try to relieve, reduce or alleviate at least one symptom of a disease in the subject. For example, in relation to CRC disease, these terms include intervening the subject or administering medications to the subject, to try to reduce the growth or reduce the size of the tumor / s in the subject, eliminate the tumors, prevent or eliminate the spread of the tumoral cells within different tissues or organs or even help alleviate the symptoms. Accordingly, these terms also encompass trying to delay or prevent onset prior to clinical manifestation of a disease or symptom of a disease and / or reducing the risk of developing or worsening of a symptom of a disease.

[0227] In a particular embodiment, the therapeutic intervention or treatment, as used herein, is for the treatment of CRC. More particularly, when the patient has been treated with a therapeutic intervention for the treatment of cancer, particularly for the treatment of CRC, before performing the method of the second aspect, the term "to initiate a therapeutic intervention” refers to treat the patient with a treatment that is the same or different, particularly different, than that received by the patient before performing the method of the second aspect. Known therapeutic interventions for the treatment of CRC include surgery, chemotherapy, targeted therapy, immunotherapy, radiation therapy and also palliative care to help relieve symptoms of cancer and side effects of treatment.

[0228] The term "subject suspicious of suffering colon cancer”, as used herein, is a subject that has shown signs that could be assigned to colon cancer. In a particular embodiment, the subject suspicious of suffering colon cancer referred to in the method of the second aspect is as the subject indicated in the definition of "subject” in section I. A as being considered to show signs of suffering colon cancer.

[0229] As well understood by a skilled person, the reference value used in step (ill) of the method of the second aspect of the invention, is as the reference value used in step (ill) of the method of the first aspect of the invention. The definition and embodiments provided in the first aspect for defining or determining the "reference value” are equally applicable to said term in the second aspect of the invention.

[0230] The rest of terms in the context of the present aspect of the invention have the meaning defined for the first aspect of the invention. All the embodiments and definitions provided in the first aspect of the invention, particularly to define or specify step (I) of the method of the first aspect, are equally applicable to the method of the second aspect of the invention. All the embodiments and definitions provided in the first aspect of the invention, particularly to define or specify step (ii) of the method of the first aspect, are equally applicable to the method of the second aspect of the invention.

[0231] C. In vitro method for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer.

[0232] In a third aspect, the invention is addressed to an in vitro method for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer, wherein the method comprises performing steps (I) and (ii) as defined in the first aspect of the invention before and after starting the therapeutic intervention, and if the level of S calculated in step (ii) after starting the therapeutic intervention is higher than the level of S calculated in step (ii) before starting the therapeutic intervention, it is indicative that the therapeutic intervention is effective in the treatment of colon cancer.

[0233] The expression "efficacy of a therapeutic intervention”, or "therapeutic intervention is effective in the treatment of colon cancer”, as used herein, refers to the fact that a patient already diagnosed with colon cancer and that has received a therapeutic intervention for the treatment of CRC reacts favorably to the therapeutic intervention. As well understood by a skilled person, a therapy effective in the treatment of colon cancer can relieve, reduce or alleviate at least one symptom of the disease in the individual. Particularly, the effective treatment can reduce the growth or reduce the size of the tumor / s in the subject, eliminate the tumors, or even prevent or eliminate the spread of the tumoral cells within different tissues or organs. Accordingly, these terms also encompass delaying or preventing onset prior to clinical manifestation of the disease or symptom of the disease and / or reducing the risk of developing or worsening of a symptom of the disease. Known therapeutic interventions for that purpose have already been provided in the second aspect of the invention and can be applied to the third aspect of the invention.

[0234] In a particular embodiment, the therapeutic intervention or treatment, as used herein, is for the treatment of CRC, particularly as defined in the second aspect of the invention, more particularly, is the treatment of the second aspect of the invention In a particular embodiment, the method of the third aspect is performed after the method of the second aspect, wherein in step (ii) of the method of the second aspect, it was found that the individual has to start a therapeutic intervention, particularly wherein the therapeutic intervention of the third aspect is that of the second aspect.

[0235] The term "the level of S calculated in step (ii) after starting the therapeutic intervention is higher than the level of S calculated in step (ii) before starting the therapeutic intervention” as used herein, refers to the fact that the level of S calculated in step (ii) after starting the therapeutic intervention is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 90%, 95%, 100%, 125%, 150%, 175%, 200% higher than the level of S calculated in step (ii) before starting the therapeutic intervention.

[0236] The term "after starting the therapeutic intervention” refers to a time point immediately after the administration of the therapeutic intervention to the patient, particularly, to at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24h, at least 36 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, after the last administration of the therapeutic intervention to the subject.

[0237] The term "before starting the therapeutic intervention”, as used herein, refers to any time before the start of the therapeutic intervention. In a particular embodiment, said term refers to a time point immediately before starting the therapeutic intervention. In another particular embodiment, said term refers to at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24h, at least 36 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 1 .5 years, at least 2 years, at least 3 years, before starting the therapeutic intervention. In a particular embodiment, "before starting the therapeutic intervention”, refers to any time between the time when the subject was diagnosed with CRC and the start of the therapeutic intervention. In a particular embodiment, said term refers to immediately before starting the therapeutic intervention, particularly to at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24h, at least 36 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 1 .5 years, at least 2 years, at least 3 years, before starting the therapeutic intervention, provided that at said time the subject was already diagnosed with CRC. In another particular embodiment, the term refers to at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24h, at least 36 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, after being diagnosed with CRC, provided it is before starting the therapeutic intervention of study.

[0238] In a particular embodiment, the subject may have received other therapeutic interventions for the treatment of cancer, particularly CRC, previous to the one being studied in the third aspect of the invention. Thus, in a particular embodiment, the subject of study in the third aspect of the invention has already received a therapeutic intervention for the treatment of cancer, particularly CRC, prior to performing the method, different from that being studied in the method of the third aspect. In another particular embodiment, the therapeutic intervention of the third aspect of the invention is a second line or third line therapeutic treatment for the treatment of CRC applied to the subject.

[0239] In another particular embodiment, the subject has not received any other therapeutic intervention for the treatment of cancer, particularly CRC, previous to the one being studied in the third aspect of the invention. Thus, in a particular embodiment, the subject of study in the third aspect of the invention has not received any other therapeutic intervention for the treatment of cancer, particularly CRC, prior to performing the method, different from that being studied in the method of the third aspect. In another particular embodiment, the therapeutic intervention of the third aspect of the invention is a first line therapeutic treatment for the treatment of CRC.

[0240] The rest of terms in the context of the present aspect of the invention have the meaning defined for the first and second aspects of the invention. All the embodiments and definitions provided in the first and second aspects of the invention are equally applicable to the method of the third aspect of the invention. In a particular embodiment, the definitions and specification of step (I) of the method of the first and second aspects of the invention, are equally applicable to the method of the third aspect. In another embodiment, the definitions and specification of step (ii) of the method of the first and second aspects of the invention, are equally applicable to the method of the third aspect.

[0241] D . A method for classifying a subject into a subject cohort.

[0242] In a fourth aspect, the invention relates to a method for classifying a subject into a subject cohort, wherein the method comprises steps (I) and (ii) as defined in the first aspect of the invention and (ill) classifying the subject into:

[0243] - a cohort of subjects with colon cancer if S calculated in step (ii) is equal or lower than the corresponding reference value, or

[0244] - a cohort of subjects with no colon cancer if S calculated in step (ii) is higher than the corresponding reference value.

[0245] The term "cohort" or "patient cohort" as used herein, refers to a group of subjects affected by common diseases, environmental or temporal influences, treatments, or other traits whose progress is assessed in a research or clinical study. In the present invention the trait common for all the subjects within a cohort is that they have or do not have CRC.

[0246] In a particular embodiment, the subjects with no CRC are considered to be healthy subjects.

[0247] As well understood by a skilled person, the reference value used in step (ill) of the method of the fourth aspect of the invention, is as the reference value used in step (ill) of the method of the first aspect of the invention. The definition and embodiments provided in the first aspect for the definition or specification of "reference value” are equally applicable to said term in the fourth aspect of the invention.

[0248] The rest of terms in the context of the present aspect of the invention have the meaning defined for the first, or second aspects of the invention. All the embodiments and definitions provided in the first and second aspects of the invention, particularly to define or specify step (I) and step (ii) of the methods, are equally applicable to the method of the fourth aspect of the invention.

[0249] The in vitro methods of the invention provide diagnostic, stratifying and / or monitoring information. In one embodiment, the methods of the invention further comprise the steps of (iv) collecting the diagnostic, stratification, and / or monitoring information, and (v) saving the information in a data carrier.

[0250] In the sense of the invention a "data carrier” is to be understood as any means that contains meaningful information data for the diagnosis and / or prognosis of CRC in an individual, such as paper. The carrier may also be any entity or device capable of carrying the prognosis data. For example, the carrier may comprise a storage medium, such as a ROM, for example a CD ROM or a semiconductor ROM, or a magnetic recording medium, for example a floppy disc or hard disk. Further, the carrier may be a transmissible carrier such as an electrical or optical signal, which may be conveyed via electrical or optical cable or by radio or other means. When the diagnosis / prognosis / monitoring data are embodied in a signal that may be conveyed directly by a cable or other device or means, the carrier may be constituted by such cable or other device or means. Other carriers relate to USB devices and computer archives. Examples of suitable data carriers are paper, CDs, USB, computer archives in PCs, or sound registration with the same information.

[0251] II. Uses of the invention.

[0252] In a fifth aspect the invention relates to the use of at least one gene selected from the group consisting of LNC-TBP, CCL5, DENND2B, LRRC52-AS1, and RNF31, as a biomarker for the diagnosis of colon cancer; or as a biomarker for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer; or as a biomarker for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer; or as a biomarker for classifying a subject into a cohort of subjects with colon cancer or of subjects with no colon cancer.

[0253] In a sixth aspect, the invention relates to the use of a kit for the diagnosis of colon cancer in an individual, or for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer, or for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer, or for classifying a subject into a subject cohort of subjects with colon cancer or of subjects with no colon cancer, the kit comprising means for detecting the level of expression of at least one gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 and RNF31, and optionally a solid support.

[0254] In an embodiment of the fifth aspect, the gene or combination of genes used in the method, or used in the fifth aspect , is selected from any gene or combination of genes provided in the first aspect of the invention as those whose expression level is determined in step (I), more particularly, is the gene or combination of genes whose expression level is determined in step (I) of the method of the first aspect of the invention.

[0255] In an embodiment of the sixth aspect of the invention, the kit comprises means for detecting the level of expression of the gene or combination of genes selected from any of those provided in the first aspect of the invention as those whose expression is determined in step (I), more particularly, comprises means for detecting the level of expression of the gene or combination of genes whose expression level is determined in step (I) of the method of the first aspect of the invention.

[0256] In a particular embodiment, the fifth aspect of the invention is addressed to the use of gene LNC-TBP-2, or, alternatively a combination of gene LNC-TBP-2 with a gene selected from the group consisting of DENND2B, RNF31, LRRC52-AS1, CCL5 and combinations thereof, as a biomarker for the diagnosis of colon cancer; or as a biomarker for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer; or as a biomarker for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer; or as a biomarker for classifying a subject into a cohort of subjects with colon cancer or of subjects with no colon cancer.

[0257] In a further particular embodiment, the fifth aspect of the invention is as defined herein, wherein the gene or combination of genes used in the method is selected from the list consisting of:

[0258] - RNF31,

[0259] - DENND2B,

[0260] - LNC-TBP-2,

[0261] - LRRC52-AS1,

[0262] - CCL5,

[0263] - DENND2B and RNF31,

[0264] - LNC-TBP-2 and RNF31,

[0265] - RNF31 and LRRC52-AS1,

[0266] - CCL5 and RNF31,

[0267] - DENND2B and LNC-TBP-2,

[0268] - DENND2B and LRRC52-AS1,

[0269] - DENND2B and CCL5,

[0270] - LNC-TBP and 2-LRRC52-AS1,

[0271] - LNC-TBP-2 and CCL5,

[0272] - CCL5 and LRRC52-AS1,

[0273] - DENND2B, LNC-TBP-2 and RNF31,

[0274] - DENND2B, RNF31 and LRRC52-AS1,

[0275] - DENND2B, CCL5 and RNF31,

[0276] - LNC-TBP-2, RNF31 and LRRC52-AS1,

[0277] - LNC-TBP-2, CCL5 and RNF31,

[0278] - CCL5, RNF31 and LRRC52-AS1,

[0279] - DENND2B, LNC-TBP-2 and LRRC52-AS1,

[0280] - DENND2B, LNC-TBP-2 and CCL5,

[0281] - DENND2B, CCL5 and LRRC52-AS1,

[0282] - LNC-TBP-2, CCL5 and LRRC52-AS1,

[0283] - DENND2B, LNC-TBP-2, RNF31 and LRRC52-AS1,

[0284] - DENND2B, LNC-TBP-2, CCL5 and RNF31,

[0285] - DENND2B, CCL5, RNF31 and LRRC52-AS1,

[0286] - LNC-TBP-2, CCL5, RNF31 and LRRC52-AS1,

[0287] - DENND2B, LNC-TBP-2, CCL5 and LRRC52-AS1 and

[0288] - DENND2B, LNC-TBP-2, CCL5, RNF31 and LRRC52-AS1.

[0289] In a particular embodiment, the fifth aspect of the invention is as defined herein, wherein the gene or combination of genes used in the method is selected from the list consisting of:

[0290] LNC-TBP-2 and CCL5,

[0291] LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1,

[0292] LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 and RNF31,

[0293] LNC-TBP-2, CCL5 and DENND2B

[0294] CCL5, DENND2B and LRRC52-AS1,

[0295] LNC-TBP-2, CCL5 and LRRC52-AS1, and

[0296] LNC-TBP-2, CCL5, DENND2B and RNF31, particularly from the group consisting of: LNC-TBP-2 and CCL5, LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1, and

[0297] LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 and RNF31, more particularly from the group consisting of:

[0298] LNC-TBP-2 and CCL5,

[0299] LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1 , and

[0300] LNC-TBP-2, CCL5, DENND2B and RNF31 , yet more particularly, from the group consisting of: LNC-TBP-2 and CCL5, and LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1 .

[0301] In a particular embodiment of the sixth aspect of the invention the kit comprises means for detecting the level of expression of LNC-TBP-2, or, alternatively for detecting the expression level of a combination of gene LNC- TBP-2 with a gene selected from the group consisting of DENND2B, RNF31, LRRC52-AS1, CCL5 and combinations thereof.

[0302] In a further embodiment of the sixth aspect of the invention, the kit comprises means for detecting the level of expression of a gene or combination of genes selected from any of those provided in the fifth aspect of the invention.

[0303] In another particular embodiment of the sixth aspect of the invention, the expression level of the gene or combination of genes as defined herein corresponds to the level of RNA transcribed from the corresponding gene, particularly in a sample or volume of body fluid, as defined in the first aspect of the invention. In another particular embodiment of the sixth aspect, the expression level is provided in TPM.

[0304] In another particular embodiment, the kit is an RNA-seq, RT-PCR, RT-qPCR, PCR, or an RNA microarray kit. In another embodiment of the sixth aspect, the means for detecting the expression level of the gene or combination of genes as defined herein are primer oligonucleotides. In a particular embodiment, the primer oligonucleotides specifically hybridize or are complementary to one or more polynucleotides of the gene or combination of genes as defined herein. In a particular embodiment, the primers of the kit are non-naturally occurring oligonucleotides. In a more particular embodiment, the primers of the kit comprise artificial (i.e. human-made) modifications in the sequence or structure of the primers, as compared to the sequence in the human genome that can hybridize to their target sequence as well. Such modifications can include artificial nucleotides, or artificial nucleotide sequences, attached or directly attached to the binding sequence of the primer (particularly to the 5'- or 3'-end of said binding sequence in the primer). As well understood by a skilled person, the binding sequence of the primer is the sequence that hybridizes with the target sequence of the primer. The terms "artificial nucleotide” or "artificial nucleotide sequences” as used herein, refer to a nucleotide or nucleotide sequence, respectively, that is not attached or directly attached to the corresponding sequence in the human genome. Non-limitative examples of other artificial modifications can include labels or tags attached or directly attached to the primers, particularly, fluorescently labelled tags. More particularly, the primers are modified primers, such as LNA (locked-nucleic acid) or PNA (peptide nucleic acid) primers. In another particular embodiment, the primers comprise at least one nucleotide not found in the sequence of the human genome that may hybridize to the primer's target sequence as well.

[0305] In a particular embodiment, the kit of the sixth aspect of the invention comprises a solid support.

[0306] In the present invention, the term "solid support” includes a nitrocellulose membrane, glass or a polymer. The most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of strips, tubes, beads, discs or microplates, or any other surface suitable for conducting gene expression level analysis, particularly RNA expression level analysis.

[0307] In a particular embodiment of the sixth aspect of the invention, the means for detecting the level of expression of the selected gene(s), particularly the oligonucleotide primers, comprised in the kit comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the total amount of reagents forming the kits used in the invention, preferably of the total amount of reagents adequate for the determination of the level of expression of one or more genes forming the kit.

[0308] In another embodiment of the sixth aspect, the kit comprises instructions on how to use it, nucleotides, a polymerization enzyme (e.g. Taq polymerase) and buffer solution(s), particularly, in addition to the primers as defined herein.

[0309] The invention also contemplates a kit comprising means as defined above for detecting the level of expression of a gene or combination of genes selected from any of those provided in the fifth aspect of the invention and, optionally a solid support and / or instructions to perform any of the methods as defined in the first to fourth aspects. In a particular embodiment, the kit of the present aspect is as the kit defined in the sixth aspect of the invention. In another particular embodiment, the kit of the present aspect is as the kit defined in the previous paragraph, particularly in any embodiment above.

[0310] In an embodiment, the use of the fifth aspect or the use of the sixth aspect of the invention is for performing any of the methods of the invention. Thus, in a particular embodiment, the use as defined in any of the embodiments of the fifth aspect or in any of the embodiments of the sixth aspect, is for performing the method of the first, second, third or fourth aspect of the invention, particularly the method of the first aspect of the invention. In another particular embodiment, the use as defined in any of the embodiments of the fifth aspect or in any of the embodiments of the sixth aspect, is for performing the method of the second aspect of the invention. In another particular embodiment, the use as defined in any of the embodiments of the fifth aspect or in any of the embodiments of the sixth aspect, is for performing the method of the third aspect of the invention. In another particular embodiment, the use as defined in any of the embodiments of the fifth aspect or in any of the embodiments of the sixth aspect, is for performing the method of the fourth aspect of the invention.

[0311] All the above embodiments, related to the methods of the invention, are also embodiments of the uses provided by the present invention, too. Thus, all the definitions and embodiments provided in the first, second, third and fourth aspects of the invention are equally applicable to the use of the fifth aspect of the invention and to the use of the sixth aspect of the invention.

[0312] In another further aspect, the invention is addressed to an in vitro method for determining the probability of a subject to have colon cancer, the method comprising steps (i) and (ii) as defined in the first aspect of the invention, and (iii) determining that the subject has high probabilities to have colon cancer when S calculated in step (ii) is equal or lower than a corresponding reference value.

[0313] The term "determining the probability of a subject to have colon cancer”, as used herein, refers to the probability that a subject has already started developing, or is suffering, colon cancer at the moment of performing the method for determining the probability of a subject to have colon cancer. The term "high probability", as used herein in the method for determining the probability of a subject to have colon cancer, relates to the situation where the subject is considered to have colon cancer, although not necessarily 100% sure. In a particular embodiment, the term "high probability” as used herein refers to the situation where the subject is preliminarily considered to have colon cancer. In another particular embodiment, the term "high probability” in the context of the method for determining the probability of a subject to have colon cancer, relates to the situation where the subject shows at least 5 %, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% probabilities to have colon cancer.

[0314] In a particular embodiment of the method for determining the probability of a subject to have colon cancer, when it has been determined in step (ill) of the method that the subject has a high probability to have colon cancer, it is further recommended or determined to perform an additional colon cancer diagnostic method, particularly a colonoscopy.

[0315] Thus, an additional aspect of the invention is addressed to an in vitro method to recommend or determine whether to perform a method for the diagnosis of colon cancer in a patient, wherein the method comprises steps (I) and (II) of the first aspect of the invention, and a step (ill) consisting of recommending or determining to perform a method for the diagnosis of colon cancer when S calculated in step (II) is equal or lower than a corresponding reference value. More particularly, step (ill) of the method just defined further comprises not recommending or determining to perform a method for the diagnosis of colon cancer when S calculated in step (II) is higher than a corresponding reference value.

[0316] In a further aspect, the invention is addressed to an in vitro screening method to identify subjects to whom a diagnostic method of CRC is to be performed, wherein the method comprises steps (I) and (II) as defined in the first aspect of the invention and (ill) identifying the subject as a subject to whom a diagnostic method of CRC is to be performed, if S calculated in step (ii) is equal or lower than the corresponding reference value, or as a subject to whom a diagnostic method of CRC is not to be performed if S calculated in step (ii) is higher than the corresponding reference value.

[0317] Different methods for the diagnosis of colon cancer in a subject are well-known by an expert in the field. Non- limitative examples of such methods include a colonoscopy, analysis of a biopsy sample, Fecal Immunochemical Test (FIT), well-known imaging tests, or combinations thereof. In a particular embodiment, the method for the diagnosis of colon cancer in the context of the three previous aspects of the invention is a colonoscopy. In a more particular embodiment, the method for diagnosis of CRC in the context of the three previous aspects of the invention is any CRC diagnostic method, provided it is different from the diagnostic method of the first aspect of the invention. More particularly, the method for the diagnosis of colon cancer in the context of the three previous aspects of the invention is as indicated in the definition of the term "different diagnostic method of CRC” in the first aspect.

[0318] In a particular embodiment, the reference value referred in the three previous aspects just defined is as defined in the first aspect of the invention, and thus the definitions and embodiments of the first aspect that define or determine the reference value of the first aspect are equally applicable to the reference value referred in the three previous aspects.

[0319] All the definitions and embodiments provided in the first, second, third and fourth aspects of the invention are equally applicable to the in vitro method for determining the probability of a subject to have colon cancer, to the in vitro method to recommend or determine to perform a method for the diagnosis of colon cancer in a patient, and to the in vitro screening method to identify subjects to whom a diagnostic method of CRC is to be performed.

[0320] In a further aspect, the invention provides a method for diagnosing and treating CRC, said method comprising steps (i) and (ii) as defined in the first aspect of the invention, (iii) identifying the subject having colon cancer when S calculated in step (ii) is equal or lower than a corresponding reference value, and (iv) administering a treatment to the patient diagnosed as having CRC. In a particular embodiment, the treatment is for CRC, particularly it is selected from surgery to remove the CRC tumor, chemotherapy, targeted therapy, immunotherapy, radiation therapy, palliative cares and combinations thereof.

[0321] In a further aspect, the invention provides a method for diagnosing and treating CRC, said method comprising steps (I), (ii) and (iii) as defined in the method to recommend or determine to perform a method for the diagnosis of colon cancer in a patient, (iv) performing a diagnostic method when recommended or determined in step (iii), (v) identifying the subject as having colon cancer when the diagnostic method performed in step (iv) diagnoses the patient as suffering colon cancer, and (vi) administering a treatment to the patient diagnosed as having CRC. In a particular embodiment of the aspect defined in the present paragraph, the treatment is a treatment for CRC, particularly, it is selected from surgery to remove the CRC tumor, chemotherapy, targeted therapy, immunotherapy, radiation therapy, palliative cares and combinations thereof.

[0322] Finally, in another aspect, the invention relates to an algorithm for carrying out any of the methods of the invention as defined in the above aspects. In the sense of the invention, the term "algorithm” is also synonymous with a panel or decision diagrams, predictors and combinations of data to correctly categorize an individual sample.

[0323] According to aspects and embodiments of the invention, the methods of the invention can be performed using a mathematical algorithm that assesses a detectable level of RNA corresponding to the gene or combination of genes as described herein in any of the methods of the invention, either in conjunction with or independent of other clinical parameters, to correctly categorize an individual sample as originating from a healthy patient, or from a patient having CRC.

[0324] The classification algorithm may be as simple as determining whether or not the amount of a specific biomarker or subset of biomarkers measured are above or below the reference level as defined in the first aspect of the invention. When multiple biomarkers are used, the classification algorithm may be a linear regression formula. Alternatively, the classification algorithm may be the product of any of a number of learning algorithms. In the case of complex classification algorithms, it may be necessary to perform the algorithm on the data, thereby determining the classification, using a computer, e.g., a programmable digital computer. In either case, one can then record the status on tangible medium, for example, in computer- readable format such as a memory drive or disk or simply printed on paper. The result also could be reported on a computer screen. This algorithm is used as a CRC diagnostic method or as a method as described in any of the aspects of the invention, and is in particular part of the kits used in the sixth aspect of the invention, particularly for carrying out the methods disclosed in former aspects.

[0325] Throughout the description and claims the word "comprise” and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise” encompasses the case of "consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. Further aspects / embodiments of the present invention can be found in the following clauses:

[0326] 1- Method for the diagnosis in vitro of colon cancer in a subject that comprises:

[0327] (I) Determining in a sample isolated from the subject the expression level of at least one gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31 ,

[0328] (II) Calculating a variable S, wherein:

[0329] S= A +B + C - D - E, wherein:

[0330] - A is the expression level of gene LNC-TBP-2 when the expression level of gene LNC-TBP-2 is determined in step (I), or is 0 when the expression level of gene LNC-TBP-2 is not determined in step (I),

[0331] - B is the expression level of gene CCL5 when the expression level of gene CCL5 is determined in step (I), or is 0 when the expression level of gene CCL5 is not determined in step (I)

[0332] - C is the expression level of gene DENND2B when the expression level of gene DENND2B is determined in step (I), or is 0 when the expression level of gene DENND2B is not determined in step (I).

[0333] - D is the expression level of gene LRRC52-AS1 when the expression level of gene LRRC52-AS1 is determined in step (I), or 0 when the expression level of gene LRRC52-AS1 is not determined in step (I),

[0334] - E is the expression level of gene RNF31 when the expression level of gene RNF31 is determined in step (I), or is 0 when the expression level of gene RNF31 is not determined in step (I) and

[0335] (ill) Diagnosing the subject as having colon cancer if S calculated in step (II) is equal or lower than a corresponding reference value and as not having colon cancer if S calculated in step (II) is higher than said corresponding reference value.

[0336] 2- The method according to clause 1, wherein step (I) further comprises determining in the sample isolated from the patient, the expression level of at least one additional gene (i.e. further comprises determining the expression level of at least one additional gene not selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31) and the variable S in step (II) is:

[0337] N

[0338] S= A +B + C - D - E + a Fj , wherein i=i

[0339] F is the expression level of the at least one additional gene, N is the number of additional genes whose expression level is determined in step (I) and a is (-1) if an increased expression level of the corresponding gene is associated to having CRC or is (1) if a decreased expression level of the corresponding gene is associated to having CRC.

[0340] 3- The method according to any one of clauses 1 or 2, wherein S= A +B + C - D - E + G, wherein:

[0341] - A, B, C, D and E are as defined in clause 1 , and:

[0342] N

[0343] - G is obtained applying formula G= F; , when step (I) further comprises determining the expression i=i level of at least one additional gene (i.e. further comprises determining the expression level of at least one additional gene not selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52- AS1 , and RNF31); wherein F, N, and "a” are as defined in clause 2; or

[0344] - G is 0 when step (I) does not further comprise determining the expression level of at least one additional gene (i.e. does not further comprise determining the expression level of a gene not selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31).

[0345] 4- The method according to any one of clauses 1-3, wherein step (I) comprises determining in the isolated sample the expression level of gene LNC-TBP-2, particularly of gene LNC-TBP-2 and of at least one additional gene selected from the list consisting of CCL5, DENND2B, LRRC52-AS1 , and RNF31. 5- The method according to any one of clauses 1-4, wherein step (i) comprises determining in the isolated sample the expression level of gene CCL5, particularly of gene CCL5 and of at least one additional gene selected from the list consisting of LNC-TBP-2, DENND2B, LRRC52-AS1, and RNF31,

[0346] 6- The method according to any one of clauses 1-5, wherein step (I) comprises determining in the isolated sample the expression level of gene DENND2B, particularly of gene DENND2B and of at least one additional gene selected from the list consisting of LNC-TBP-2, CCL5, LRRC52-AS1, and RNF31,

[0347] 7- The method according to any one of clauses 1-6, wherein step (I) comprises determining in the isolated sample the expression level of gene LRRC52-AS1, particularly of gene LRRC52-AS1 and of at least one additional gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, and RNF31,

[0348] 8- The method according to any one of clauses 1-7, wherein step (I) comprises determining in the isolated sample the expression level of gene RNF31, particularly of gene RNF31 and of at least one additional gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1.

[0349] 9- The method according to any one of clauses 1-8 wherein "at least one” consists of 1.

[0350] 10- The method according to any one of clauses 1-8 wherein "at least one” consists of 2.

[0351] 11- The method according to any one of clauses 1-8 wherein "at least one” consists of 3.

[0352] 12- The method according to any one of clauses 1-8 wherein "at least one” consists of 4.

[0353] 13- The method according to any one of clauses 1-8 wherein "at least one” consists of 5.

[0354] 14- The method according to any one of clauses 1-13, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the genes and combination of genes selected from the list disclosed in table 1.

[0355] 15- The method according to any one of clauses 1-14, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.7, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.7.

[0356] 16- The method according to any one of clauses 1-15, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.75, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.75.

[0357] 17- The method according to any one of clauses 1-16, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.76, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.76.

[0358] 18- The method according to any one of clauses 1-17, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.77, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.77.

[0359] 19- The method according to any one of clauses 1-18, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.78, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.78.

[0360] 20- The method according to any one of clauses 1-19, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.79, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.79.

[0361] 21- The method according to any one of clauses 1-20, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.8, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.8. 22- The method according to any one of clauses 1-21, wherein step (i) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.81, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.81.

[0362] 23- The method according to any one of clauses 1-22, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.82, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.82.

[0363] 24- The method according to any one of clauses 1-23, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.83, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.83.

[0364] 25- The method according to any one of clauses 1-24, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.84, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.84.

[0365] 26- The method according to any one of clauses 1-25, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.85, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.85.

[0366] 27- The method according to any one of clauses 1-26, wherein step (I) comprises determining in the isolated sample the expression level of one or more of the combination of genes shown in table 1 to provide an AUG equal to or higher than 0.86, particularly shown in table 2 to provide an AUG in expressed samples equal to or higher than 0.86.

[0367] 28- The method according to any one of clauses 15-27, wherein "one or more”, consists of one.

[0368] 29- The method according to any one of clauses 15-27, wherein "one or more”, consists of two.

[0369] 30- The method according to any one of clauses 15-27, wherein "one or more”, consists of three.

[0370] 31- The method according to any one of clauses 15-27, wherein "one or more”, consists of four.

[0371] 32- The method according to any one of clauses 15-27, wherein "one or more”, consists of five.

[0372] 33- The method according to any one of clauses 15-27, wherein "one or more”, consists of six.

[0373] 34- The method according to any one of clauses 15-27, wherein "one or more”, consists of "all”.

[0374] 35- The method according to any one of clauses 1-34, wherein step (I) comprises determining in the isolated sample the expression level of gene(s):

[0375] LNC-TBP-2 and CCL5,

[0376] LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1,

[0377] LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 and RNF31,

[0378] LNC-TBP-2, CCL5 and DENND2B

[0379] CCL5, DENND2B and LRRC52-AS1,

[0380] LNC-TBP-2, CCL5 and LRRC52-AS1,

[0381] LNC-TBP-2, CCL5, DENND2B and RNF31,

[0382] LNCTBP2, CCL5, and RNF31

[0383] DENND2B, CCL5, and RNF31,

[0384] CCL5 and LRRC52-AS1

[0385] CCL5 and DENND2B

[0386] CCL5 and LRRC52-AS1,

[0387] CCL5 and RNF31, or

[0388] CCL5.

[0389] 36- The method according to any one of clauses 1-35, wherein step (I) comprises determining in the isolated sample the expression level of genes:

[0390] LNC-TBP-2 and CCL5,

[0391] LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1 ,

[0392] LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 and RNF31 ,

[0393] LNC-TBP-2, CCL5 and DENND2B

[0394] CCL5, DENND2B and LRRC52-AS1 ,

[0395] LNC-TBP-2, CCL5 and LRRC52-AS1 , and

[0396] LNC-TBP-2, CCL5, DENND2B and RNF31 ,

[0397] 37- The method according to any one of clauses 1-36, wherein step (I) comprises determining in the isolated sample the expression level of genes:

[0398] LNC-TBP-2 and CCL5,

[0399] LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1 , and

[0400] LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 and RNF31 ,

[0401] 38- The method according to any one of clauses 1-37, wherein step (i) comprises determining in the isolated sample the expression level of:

[0402] LNC-TBP-2 and CCL5,

[0403] LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1 , and

[0404] LNC-TBP-2, CCL5, DENND2B and RNF31 .

[0405] 39- The method according to any one of clauses 1-38, wherein step (i) comprises determining in the isolated sample the expression level of:

[0406] LNC-TBP-2 and CCL5, and

[0407] LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1 .

[0408] 40- The method according to any one of clauses 1-39, wherein step (i) comprises determining in the isolated sample the expression level of genes LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31.

[0409] 41- The method according to any one of clauses 1-40 wherein the method comprises an additional step (iv) in which the subject is selected to additionally perform a different diagnostic method of CRC, if S calculated in step (ii) is equal or lower than the corresponding reference value, particularly wherein said different diagnostic method of CRC comprises or consists of a colonoscopy, a FIT, or the combination of both, and more particularly, not selecting the subject to additionally perform a different diagnostic method of CRC as just defined, if S calculated in step (ii) is higher than the corresponding reference value.

[0410] 42- An in vitro method for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer wherein the method comprises steps (i) and (ii) as defined in any one of clauses 1-41, and (iii) if S calculated in step (ii) is equal or lower than the corresponding reference value, it is indicative that the individual has to start a therapeutic intervention.

[0411] 43- An in vitro method for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer, wherein the method comprises performing steps (i) and (ii) as defined in any one of clauses 1-41 before and after starting the therapeutic intervention, and if the level of S calculated in step (ii) after starting the therapeutic intervention is higher than the level of S calculated in step (ii) before starting the therapeutic intervention, it is indicative that the therapeutic intervention is effective in the treatment of colon cancer.

[0412] 44- A method for classifying a subject into a subject cohort, wherein the method comprises steps (i) and (ii) as defined in any one of clauses 1-41 and (iii) classifying the subject into:

[0413] - a cohort of subjects with colon cancer if S calculated in step (ii) is equal or lower than the corresponding reference value, or

[0414] - a cohort of subjects with no colon cancer if S calculated in step (ii) is higher than the corresponding reference value.

[0415] 45- An in vitro screening method to identify subjects to whom a diagnostic method of CRC is to be performed, wherein the method comprises steps (i) and (ii) as defined in any one of clauses 1-41 and (iii) identifying the subject as a subject to whom a diagnostic method of CRC is to be performed, if S calculated in step (ii) is equal or lower than the corresponding reference value, or as a subject to whom a diagnostic method of CRC is not to be performed if S calculated in step (ii) is higher than the corresponding reference value.

[0416] 46- The in vitro screening method according to clause 45, wherein the diagnostic method of CRC is different from the in vitro method for the diagnosis of CRC defined in any one of clauses 1-41 .

[0417] 47- The in vitro screening method according to any one of clauses 45-46, wherein the diagnostic method of CRC comprises or consists of a colonoscopy, analysis of a biopsy sample, Fecal Immunochemical Test (FIT), well-known imaging tests, or combinations thereof.

[0418] 48- The method according to any one of clauses 1-47 wherein the sample is a blood sample, in particular, a plasma sample.

[0419] 49- The method according to any one of the previous clauses, wherein the expression level determined in step (i) is provided in counts per million (CPM).

[0420] 50- The method according to any one of the previous clauses wherein determining the expression level in step (i) is performed by RNA sequencing.

[0421] 51- The method according to any one clauses 1-50, wherein the reference value used in step (iii) corresponds to the threshold value provided in table 1 for the gene or combination of genes whose expression level is determined in step (i), particularly when the expression level (s) determined in step (i) is / are provided in counts per million.

[0422] 52- The method according to any one of clauses 1- 51, wherein the reference value used in step (iii) corresponds to + / - 1% , 2%, 3%, 4%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, or 20%, the threshold value provided in table 1 for the gene or combination of genes whose expression level is determined in step (i), particularly when the expression level (s) determined in step (i) is / are provided in counts per million.

[0423] 53- The method according to any one of clauses 1-52, wherein the reference value used in step (iii) is provided as a range of values that corresponds to the range of threshold values indicated in table 1 for the gene or combination of genes whose expression level is determined in step (i), particularly when the expression level(s) determined in step (i) is / are provided in CPM.

[0424] 54- The method according to any one of clauses 1-53, wherein the reference value used in step (iii) is provided as a range of values, wherein the lower and upper values of the range correspond to + / -1%, 2%, 3%, 4%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, or 20% the lower and upper values, respectively, of the range of threshold values provided in table 1 for the gene or combination of genes whose expression level is determined in step (i), particularly when the expression level(s) determined in step (i) is / are provided in CPM.

[0425] 55- The method according to any one of the previous clauses, particularly wherein the expression level(s) determined in step (i) is / are provided in CPM, wherein:

[0426] - step (i) consists of determining in the isolated sample, the expression level of gene LNC-TBP-2 and the corresponding reference value is from 160 to 171, particularly from 164 to 169, or

[0427] - step (i) consists of determining in the isolated sample the expression level of genes LNC-TBP-2 and CCL5 and the corresponding reference value is from 300 to 310, particularly from 301 to 308, or

[0428] - step (i) consists of determining in the isolated sample the expression level of genes LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1, and the corresponding reference value is from 515 to 590, particularly from 526 to 581, or

[0429] - step (i) consists of determining in the isolated sample the expression level of genes LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1, and RNF31, and the corresponding reference value is from 420 to 450, particularly from 422 to 445;

[0430] 56- Use of at least one gene selected from the group consisting of LNC-TBP-2, DENND2B, RNF31, LRRC52- AS1, CCL5 and combinations thereof, as a biomarker for the diagnosis of colon cancer; or as a biomarker for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer; or as a biomarker for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer; or as a biomarker for classifying a subject into a cohort of subjects with colon cancer or of subjects with no colon cancer; or as a biomarker to identify subjects to whom a diagnostic method of CRC is to be performed.

[0431] 57- Use of a kit for the diagnosis of colon cancer in an individual, or for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer; or for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer; or for classifying a subject into a subject cohort of subjects with colon cancer or of subjects with no colon cancer; or for identifying subjects to whom a diagnostic method of CRC is to be performed; the kit comprising means for detecting the level of expression of at least one gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52- AS1 and RNF31 , and optionally a solid support.

[0432] 58- The use according to clause 57, wherein the means for detecting the expression level of the gene / s are primers.

[0433] 59- The use according to any one of clauses 56-58, wherein the at least one gene comprises LNC-TBP-2.

[0434] 60- The use according to any one of clauses 56-59, wherein the at least one gene comprises CCL5.

[0435] 61- The use according to any one of clauses 56-60, wherein the at least one gene comprises DENND2B

[0436] 62- The use according to any one of clauses 56-61 , wherein the at least one gene comprises LRRC52-AS1 .

[0437] 63- The use according to any one of clauses 56-61 , wherein the at least one gene comprises RNF31 .

[0438] 64- The use according to any one of clauses 56-62, wherein the at least one gene consists of the gene(s) whose expression level is determined in step (I) of the method as defined in any one of clauses 1-55.

[0439] The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

[0440] Examples

[0441] Example 1 : Materials and methods

[0442] Intravenous blood was obtained from 30 CRC patients and 14 controls using Streck RNA Complete BCT tubes (https: / / www.streck.com / products / stabilization / rna-complete-bct / ). After blood draw, the tubes were inverted 8-10 times and plasma was isolated following the manufacturer's instructions within 5 days from extraction. Hemolysis was measured using NanoDrop™ and determining the absorbance of the plasma (2ul) at 414 nm, with water as blank. Only samples with absorbance levels below 0.2AU were used.

[0443] To obtain the cfRNA, we started from 1 ml of plasma per patient and used Norgen Plasma / Serum RNA isolation kit (https: / / norgenbiotek.com / product / plasmaserum-rna-purification-kits) without modifications of the protocol. The size profile of the isolated RNA was determined using Bioanalyzer and in all cases, samples showed an expected peak at 50-150 nt, classically found in all cfRNA samples.

[0444] To measure the relative abundance of each molecule in the sample, the cfRNA was transformed into sequenceable cDNA. For that purpose the samples were prepared using the kit CORALL RNA-Seq Library Prep Kit (https: / / www.lexogen.com / corall-rna-seq / ) starting with the maximum volume possible. After library preparation, the samples were quantified using Qubit™ to measure the DNA yield of the library . To measure the size profiles of the library, a second round of bioanalyzers was performed to determine the number of peaks and in all cases, samples showed the expected peak at 250-350 nt. After that, libraries were sequenced to obtain between 12 and 17 million reads 150bp x PE in a NovaSeq sequencer (Illumina®; the obtained mean was of 15.7M and the standard deviation of 1.89M).

[0445] Trim Galore (Krueger F. 2023) was used to remove adapters, remove read pairs with a quality score below 20, and to perform quality control of the samples. The trimmed reads were mapped to the human genome (UCSC genome build hg38) using STAR (Dobin A. et al., 2013) and the gene annotations from GENCODE v39 with the following parameters: -readFilesCommand zcat -twopassMode Basic -outSAMprimaryFlag AIIBestScore -outFilterMismatchNmax 999 — alignlntronMin 20 — alignlntronMax 1000000 -- alignMatesGapMax 1000000 — outFilterType BySJout -alignSJDBoverhangMin 3 -alignSJoverhangMin 8 -- peOverlapNbasesMin 40 -peOverlapMMp 0.8 -quantMode TranscriptomeSAM -quantTranscriptomeBan Singleend. UMI-tools was used to remove the PGR duplicates (Smith T. et al., 2017). Salmon was used to perform the gene quantification of the genome alignment and to create the gene count matrix (Patro R et al., 2017).

[0446] The gene expression values were used by the Least Absolute Shrinkage and Selection Operator (LASSO) machine learning algorithm to select the genes that could discriminate between ORC and controls.

[0447] To determine the patient dependent score, the TPM normalized expression of the genes was combined through addition or subtraction (determined by sign in front of the gene - / + in table 2 below). The optimal threshold, the ROC curves and the AUCs were calculated by applying the roc function of the pROC package (Robin X. et al., 2011) to the patient dependent scores in ORC and control samples (see table 1 below). If the patient dependent score value was higher than the threshold, then the sample was considered to be from a non-cancer patient while if the value was below said threshold then the sample was considered to be from a cancer patient. The threshold range was defined as the range between the two threshold-defining samples, being the sample classified as ORC with the highest score and the sample classified as control with the lowest score. The ROC curves were produced and the AUCs calculated by two different methods: using all of the samples; and excluding the samples where there was no expression of the gene or combination of the genes. This second method avoids the random classification of samples in which the expression level of the gene of interest could not be detected. This procedure is repeated for each gene or combination of genes.

[0448] Example 2. Results

[0449] From the LASSO machine learning algorithm a total of 5 genes were found that could be used to discriminate between ORC and controls.

[0450] Using the genes selected by the LASSO model, several gene combinations were obtained with high discriminative value, evidenced by the high AUG in several of the gene combinations in Table 1. Between them, there is one RNA, CCL5, which has a high AUG by itself but an even increased AUG when combined with other genes. Moreover, some of the genes are not found in many of the samples, which lowers their AUG. When computing the AUCs only for the samples where the expression of the genes could be detected, the AUCs increase, as reported in table 2.

[0451] Table 1. AUCs, thresholds and threshold ranges of each of the gene combinations.

[0452]

[0453] Table 2. AUG values for each gene combination, including the AUG calculated exclusively for samples expressing the genes within the respective gene combinations, as well as the percentage of samples expressing said genes in each combination.

[0454] Citation List

[0455] Burtis C. A. et al., 2008, Chapter 14, section "Statistical Treatment of Reference Values” Krueger F. Trim Galore [Internet], 2023 [cited 2023 Apr 25], Available from: https: / / Qithub.com / FelixKrueqer / TnmGalore

[0456] Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinforma Oxf Engl. 2013 Jan 1 ;29(1): 15-21.

[0457] Patro R, Duggal G, Love Ml, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017 Apr; 14(4):417- 9.

[0458] Robin X, Turck N, Hainard A, Tiberti N, Lisacek F, Sanchez JC, et al. pROC: an open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinformatics. 2011 Mar 17;12(1):77.

[0459] Smith T, Heger A, Sudbery I. UMI-tools: Modelling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy. Genome Res. 2017 Jan 18;gr.209601 .116.

Claims

Claims1- Method for the diagnosis in vitro of colon cancer in a subject that comprises:(i) Determining in a sample isolated from the subject the expression level of at least one gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31 ,(II) Calculating a variable S, wherein:S= A +B + C - D - E, wherein:- A is the expression level of gene LNC-TBP-2 when the expression level of gene LNC-TBP-2 is determined in step (I), or is 0 when the expression level of gene LNC-TBP-2 is not determined in step (I),- B is the expression level of gene CCL5 when the expression level of gene CCL5 is determined in step (I), or is 0 when the expression level of gene CCL5 is not determined in step (I)- C is the expression level of gene DENND2B when the expression level of gene DENND2B is determined in step (I), or is 0 when the expression level of gene DENND2B is not determined in step (I).- D is the expression level of gene LRRC52-AS1 when the expression level of gene LRRC52-AS1 is determined in step (I), or 0 when the expression level of gene LRRC52-AS1 is not determined in step (I),- E is the expression level of gene RNF31 when the expression level of gene RNF31 is determined in step (I), or is 0 when the expression level of gene RNF31 is not determined in step (I) and(ill) Diagnosing the subject as having colon cancer if S calculated in step (II) is equal or lower than a corresponding reference value and as not having colon cancer if S calculated in step (II) is higher than said corresponding reference value.2- The method according to claim 1 , wherein step (I) further comprises determining in the sample isolated from the patient, the expression level of at least one additional gene and the variable S in step (II) is:NS= A +B + C - D - E + a Fbwherein i=iF is the expression level of the at least one additional gene, N is the number of additional genes whose expression level is determined in step (I) and a is (-1) if an increased expression level of the corresponding gene is associated to having CRC or is (1) if a decreased expression level of the corresponding gene is associated to having CRC.3- The method according to any one of claims 1-2, wherein step (I) comprises determining in the isolated sample the expression level of gene LNC-TBP-2.4- The method according to any one of claims 1-3, wherein step (I) comprises determining in the isolated sample the expression level of: genes LNC-TBP-2 and CCL5, genes LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1 , or genesLNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31 .5- An in vitro method for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer wherein the method comprises steps (I) and (II) as defined in any one of claims 1-4, and (ill) if S calculated in step (II) is equal or lower than the corresponding reference value, it is indicative that the individual has to start a therapeutic intervention.6- An in vitro method for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer, wherein the method comprises performing steps (I) and (ii) as defined in any one of claims 1- 4 before and after starting the therapeutic intervention, and if the level of S calculated in step (ii) after starting the therapeutic intervention is higher than the level of S calculated in step (ii) before starting the therapeutic intervention, it is indicative that the therapeutic intervention is effective in the treatment of colon cancer.7- A method for classifying a subject into a subject cohort, wherein the method comprises steps (I) and (ii) as defined in any one of claims 1- 4 and (ill) classifying the subject into:- a cohort of subjects with colon cancer if S calculated in step (ii) is equal or lower than the corresponding reference value, or- a cohort of subjects with no colon cancer if S calculated in step (ii) is higher than the corresponding reference value.8 - The method according to any one of the previous claims wherein the sample is a blood sample, in particular, a plasma sample.9- The method according to any one of the previous claims, wherein: step (I) consists of determining in the isolated sample, the expression level of gene LNC-TBP-2 and the corresponding reference value is from 160 to 171 , particularly from 164 to 169, or step (I) consists of determining in the isolated sample the expression level of genes LNC-TBP-2 and CCL5 and the corresponding reference value is from 300 to 310, particularly from 301 to 308, or step (I) consists of determining in the isolated sample the expression level of genes LNC-TBP-2, CCL5, DENND2B, and LRRC52-AS1 , and the corresponding reference value is from 515 to 590, particularly from 526 to 581 , or step (I) consists of determining in the isolated sample the expression level of genes LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 , and RNF31 , and the corresponding reference value is from 420 to 450, particularly from 422 to 445.10- The method according to any one of the previous claims, wherein the expression level of the gene or combination of genes determined in step (I) is provided in transcripts per million (TPM).11- The method according to any one of the previous claims wherein determining the expression level of the gene or group of genes in step (I) is performed by RNA sequencing.12- Use of at least one gene selected from the group consisting of LNC-TBP-2, DENND2B, RNF31 , LRRC52- AS1 , CCL5 and combinations thereof, as a biomarker for the diagnosis of colon cancer; or as a biomarker for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer; or as a biomarker for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer; or as a biomarker for classifying a subject into a cohort of subjects with colon cancer or of subjects with no colon cancer.13- The use according to claim 12, wherein the gene or combination of genes is one selected from the group consisting of: gene LNC-TBP2 gene LNC-TBP-2 with gene CCL5, gene LNC-TBP-2 with genes CCL5, DENND2B, and LRRC52-AS1 , and gene LNC-TBP-2 with genes CCL5, DENND2B, LRRC52-AS1 and RNF31 .14- Use of a kit for the diagnosis of colon cancer in an individual, or for deciding or recommending whether to initiate a therapeutic intervention in an individual suspicious of suffering colon cancer, or for determining the efficacy of a therapeutic intervention in a patient already diagnosed with colon cancer, or for classifying a subject into a subject cohort of subjects with colon cancer or of subjects with no colon cancer, the kit comprising means for detecting the level of expression of at least one gene selected from the list consisting of LNC-TBP-2, CCL5, DENND2B, LRRC52-AS1 and RNF31 , and optionally a solid support.15- The use according to claim 14, wherein the means for detecting the expression level of the gene / s are primers.

Images