CLEC3b for assessing colorectal cancer (CRC) or a precancerous condition thereof

Abstract

The present invention relates to a method for assessing colorectal cancer or a precancerous condition thereof, said method comprising (a) determining the level CLEC3B (C-type lectin domain family 3 member B) in a blood, serum or plasma sample from the subject, and assessing colorectal cancer or the precancerous condition thereof based on the level of CLEC3B determined in step a). The present invention further relates to computer-implemented methods, databases, devices, and uses related thereto.

Description

[0001] Roche Diagnostics International AG 29 September 2025

[0002] Roche Diagnostics GmbH Our Ref.: RD39557PC

[0003] CLEC3B for assessing colorectal cancer (CRC) or a precancerous condition thereof

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a method for assessing colorectal cancer or a precancerous condition thereof, said method comprising (a) determining the level of the biomarker CLEC3B (C-type lectin domain family 3 member B) in a blood, serum or plasma sample from the subject, and assessing colorectal cancer or the precancerous condition thereof based on the level of CLEC3B determined in step a). The present invention further relates to computer-implemented methods, databases, devices, kits and uses related thereto.

[0006] BACKGROUND OF THE INVENTION

[0007] Colorectal cancer (CRC) is a significant global health issue, with over 1.2 million new cases and approximately 600,000 deaths reported annually, making it the third most frequently diagnosed cancer and the fourth leading cause of cancer-related mortality worldwide (Siegel, R. L., Miller, K. D., & Jemal, A. (2020). CA: A Cancer Journal for Clinicians, 70(1), 7-30. DOI: 10.3322 / caac.21590). Because of the slow progression from detectable and curable precancerous lesions to colorectal cancer and the strong dependence of prognosis on stage at diagnosis, early detection of colorectal cancer has great potential to reduce the burden of this disease (Leslie A, Carey FA, Pratt NR, Steele RJ., Br J Surg 2002;89:845-60; Brenner H, Altenhofen L, Stock C, Hoffmeister M., Cancer Epidemiol Biomarkers Prev 2013;22: 1043-51; Brenner H, Stock C, Hoffmeister M. BMJ 2014;348:g2467). Colonoscopy is the gold standard for detecting colorectal cancer and its precursors, but its application as primary screening test is impaired by high costs, limited capacities, and typically lower adherence (Lansdorp-Vogelaar I, Knudsen AB, Brenner H., Epidemiol Rev 2011 ;33 : 88— 100., Hassan C, Giorgi Rossi P, Camilloni L, Rex DK, Jimenez- Cendales B, Ferroni E, et al ., Aliment Pharmacol Ther 2012;36:929-40). Noninvasive stool tests are an attractive alternative for colorectal cancer screening due to their low cost and suitability for home use or in a primary care setting. However, the aversion to stool handling may limit the acceptance of stool tests (Carroll MR, Seaman HE, Halloran SP. Tests and investigations for colorectal cancer screening. Clin Biochem 2014;47:921-39). Blood-based tests may be more readily accepted by patients for many medical conditions, and surveys conducted in individuals eligible for colorectal cancer screening showed a strong preference for collection of blood over feces (Osborne J, Wilson C, Moore V, Gregory T, Flight I, Young G., Open J Prev Med 2012;2:326-31). While the development of a simple and convenient blood test with diagnostic performance comparable with stool tests could contribute to improvement in the acceptance of colorectal cancer screening, the diagnostic performance of currently existing blood tests is insufficient for their routine use (Fung, K. Y. C., Tabor, B., Buckley, M. J., et al. (2015). Blood-based protein biomarker panel for the detection of colorectal cancer. PLOS ONE, 10(3), e0120425, Fung KY, Nice E, Priebe I, Belobrajdic D, Phatak A, Purins L, et al. Colorectal cancer biomarkers: to be or not to be? Cautionary tales from a road well travelled. World J Gastroenterol 2014;20:888-98). Recently, the Guardant's Shield test, a blood test, was approved by the FDA.

[0008] All guidelines recommend checking carcinoembryonic antigen concentrations at the time of diagnosis (Labianca R, Nordlinger B, Beretta GD, Mosconi Set al on behalf of the ESMO Guidelines Working Group* Early colon cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of Oncology 24 (Supplement 6): vi64-vi72, 2013.) An elevated baseline carcinoembryonic antigen (CEA) concentration is associated with worse prognosis, and concentrations that do not normalize in the postoperative phase might indicate residual disease while it has a low predictive value for diagnosis in asymptomatic patients due to its relatively low sensitivity and specificity (Thirunavukarasu P, Sukuma S el al. C-stage in Colon Cancer: Implications of Carcinoembryonic Antigen Biomarker in Staging, Prognosis, and Management. 2011. J Natl Cancer Inst 2011; 103:689- 697). However, CEA as a single marker is not recommended for screening.

[0009] Early detection of CRC, e.g. stage 1 or stage 2, significantly improves patient outcomes, as treatment is more effective at the initial stages of the disease (Siegel RL et al., Colorectal cancer statistics, 2020. CA Cancer J Clin. 2020 May;70(3): 145-164. doi: 10.3322 / caac.21601. Epub 2020 Mar 5. PMID: 32133645.19). Despite advancements in therapeutic interventions, the mortality rate remains high due to late-stage diagnosis in many patients.

[0010] Current standard screening methods for CRC include colonoscopy, sigmoidoscopy, and stool-based tests such as the fecal occult blood test (FOBT) and fecal immunochemical test (FIT). While colonoscopy is considered the gold standard due to its high sensitivity and specificity, it is invasive, costly, and requires extensive bowel preparation, which limits patient compliance (Brenner, D. E., & Rennert, G. (2005). CA: A Cancer Journal for Clinicians, 55(1), 18-35. DOI: 10.3322 / canjclin.55.1.18). Stool-based tests, though less invasive, suffer from lower sensitivity and specificity, particularly for detecting early-stage CRC (Imperiale, T. F., Ransohoff, D. F., Itzkowitz, S. H., Turnbull, B. A., & Ross, M. E. (2004). New England Journal of Medicine, 351(26), 2704-2714). Further, the aversion to stool handling may limit the acceptance of stool tests.

[0011] Given these limitations, there is a pressing need for non-invasive, reliable, and cost-effective screening methods. Blood-based biomarkers offer a promising alternative due to the minimally invasive nature of blood collection, which can lead to higher patient compliance and enable routine screening in clinical practice. Blood-based tests also have the potential to detect CRC at earlier stages, thereby improving prognosis and survival rates.

[0012] Detecting CRC at an early stage is crucial for improving patient outcomes. Equally important is the detection of adenomas, which are precancerous lesions that can develop into CRC if left untreated. Identifying and removing adenomas can prevent the progression to invasive cancer, thereby reducing CRC incidence and mortality. Current screening methods are not always effective in detecting adenomas, particularly those that are flat or located in the proximal colon. Moreover, many protein markers are more reliable for detecting advanced CRC rather than early- stage or precancerous lesions, reducing their utility for early intervention. Therefore, there is a significant need for biomarkers that can detect both early- stage CRC and adenomas.

[0013] Recent research has identified various potential blood-based biomarkers for CRC, including circulating tumor cells (CTCs), cell-free DNA (cfDNA), microRNAs (miRNAs), and proteins. Protein biomarkers, in particular, hold significant promise due to their stability in blood and the ability to reflect dynamic changes associated with tumor development and progression (Levin, B., Lieberman, D. A., McFarland, B., et al. (2018) CA: A Cancer Journal for Clinicians, 58(3), 130-160. DOL 10.3322 / CA.2007.0018). Several proteins have been proposed as CRC biomarkers, such as carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA 19-9), but these markers lack the necessary sensitivity and specificity for early- stage detection (Duffy, M. J. (2001). Clinical Chemistry, 47(4), 624-630. DOL 10.1093 / clinchem / 47.4.624). Recently, Guardant's Shield test, a blood test, received a positive FDA advisory opinion. Moreover, a study by Brenner et al. identified a panel of protein biomarkers that showed promising results in differentiating CRC patients from healthy controls with high sensitivity and specificity (Brenner, H., Tao, S., Haug, U. (2014). Blood-based biomarkers for detection of colorectal cancer: A systematic review. Clinical Epidemiology, 6, 319-331).

[0014] Many blood based markers have been evaluated as a combination of protein markers (Wild N et al A combination of serum markers for the early detection of colorectal cancer. ClinCancer Res. 2010 Dec 15; 16(24):6111-21. doi: 10.1158 / 1078-0432.CCR-10-0119) in the past. Amongst the protein panels only a few have been validated rigorously in intended use cohorts (screening, asymptomatic patients) like the panel from Wild et al. One example is the panel from Rhythm Bioscience (ColoStat). ColoSTAT sensitivity and specificity for CRC were comparable with published performance parameters for FIT, which range from 74-93% (sensitivity) and 85-96% (specificity) (ASCO poster 3529, 2023 A prospective, cross-sectional, multicentre study to evaluate the clinical performance of the ColoSTAT in vitro diagnostic for the detection of biomarkers associated with colorectal cancer). The ColoSTAT blood test and algorithm combines concentrations of 5 protein biomarkers with age and sex to provide an alternative to current CRC screening methods like the faecal immunochemical test (FIT).

[0015] Marker combinations for the assessment of CRC have described in the art (Bhardwaj M et al. 2019. Molecular Oncology 14 (2020) 8-21). For example, a three-marker signature detected early- stage CRC and advanced adenoma cases in participants of screening colonoscopy with AUCs of 0.79 (95% CI, 0.66e0.89) and 0.65 (95% CI, 0.56e0.73), respectively (Bhardwaj M et al., 2020. European Journal of Cancer 127 (2020) 30e40).

[0016] CLEC3B (C-type lectin domain family 3 member B, UniProtID P05452) is a protein that binds plasminogen and promotes the proteolytic activation of plasminogen into plasmin, which suggests that CLEC3B is involved in remodeling the extracellular matrix and cancer tissues during cancer development. CLEC3B also accumulates in the extracellular matrix in cancer tissues and co-localizes with plasminogen in an invasive front (Iram S et al Insight into the function of tetranectin in human diseases: A review and prospects for tetranectin- targeted disease treatment. 2023. Heliyon 10 (2024) e23512). CLEC3B binds plasminogen kringle domain 4. Plasminogen is a precursor of plasmin, which dissolves the fibrin of blood clots and acts as a proteolytic factor in a variety of processes including embryonic development, tissue remodeling, tumor invasion, and inflammation (Vago J et al. The Role of Plasmin / Plasminogen in Experimental Polymicrobial Sepsis. 2020. Blood (2020) 136 (Supplement 1): 16). CLEC3B is closely associated with different types of cancers. It was shown to have an important role in tissue remodeling during cancer development. Serum levels of CLEC3B are reported to be lower in CRC, breast cancer, ovarian cancer and myeloma patients compared to healthy individuals (Iram S et al Insight into the function of tetranectin in human diseases: A review and prospects for tetranectin-targeted disease treatment. 2023. Heliyon 10 (2024) e23512), but are shown to be increased in lung cancer patients compared to healthy controls (Guo Y et al. CLEC3B Identified as a Potential Lung Cancer Biomarker in Serum by Aptamer-Capture Technology. 2021. ChemistrySelect 2021, 6, 5640 -5645; Sun J et al.CLEC3B as a potential diagnostic and prognostic biomarker in lung cancer and association with the immune microenvironment.2020. Cancer Cell International volume 20, Article number: 106) using aptamer based ELISA or RT-PCR. Metastatic cancer patients showed lower serum CLEC3B levels compared to non-metastatic cancer patients (Jensen BA et al.Plasma Tetranectin Is Reduced in Cancer and Related to Metastasia. 1988. Cancer 62:869-87). In CRC, significantly shorter survival was found for patients with CLEC3B levels below a cut-off point of 7.5 mg / 1 compared to patients with levels above (Iram S et al Insight into the function of tetranectin in human diseases: A review and prospects for tetranectin-targeted disease treatment. 2023. Heliyon 10 (2024) e23512).

[0017] Immunohistochemistry (IHC) of CRC and normal colorectal mucosa show that CLEC3B is strongly expressed in the CRC stroma, with little or no expression in the normal colon mucosa (Hogdall CK et al. Serum tetranectin is an independent prognostic marker in colorectal cancer and weakly correlated with plasma suPAR, plasma PAI-1 and serum CEA. 2002. APMIS 110: 630-8, 2002). A second study showed, however, that CLEC3B gene expression is absent in CRC tissue compared to normal mucosa (Birkenkamp-Demtroder K et al. Gene Expression in Colorectal Cancer, 2002. Cancer Research 62, 4352-4363, August 1). CLEC3B accumulated in the ECM of cancer cells is suggested to derive from blood, as cancer patients show significantly reduced CLEC3B serum levels (Vago J et al. The Role of Plasmin / Plasminogen in Experimental Polymicrobial Sepsis. 2020. Blood (2020) 136 (Supplement 1): 16). Moreover, CLEC3B belongs to “epigenetic clock genes” and is involved in aging.

[0018] During carcinogenesis, the change of expression for CLEC3B in tissue followed the same pattern as in aging (Galamb O et al. Aging related methylation influences the gene expression of key control genes in colorectal cancer and adenoma. 2016. World J Gastroenterol 2016 December 21; 22(47): 10325-10340). Besides cancer, CLEC3B is also associated with other diseases, such as Sepsis, developmental disorders, cardiovascular diseases, neurological diseases, inflammation, and diabetes (Iram S et al Insight into the function of tetranectin in human diseases: A review and prospects for tetranectin-targeted disease treatment. 2023. Heliyon 10 (2024) e23512).

[0019] There is a need for biomarkers, in particular of protein biomarkers for the assessment colorectal cancer or precancerous conditions thereof. It is therefore an objective of the present invention to provide improved means and methods for colorectal cancer or precancerous conditions thereof.

[0020] The present invention, therefore, provides means and methods complying with these needs.

[0021] DESCRIPTION OF THE INVENTION

[0022] In the studies underlying the present invention, several protein biomarker were tested in plasma samples from patients with malignant findings, i.e. patients with colorectal cancer cases (CRC) or patients having a pathological tumor in situ (pTis), from patients with high grade dysplasia (HG) / advanced adenoma findings (i.e. patients with a high risk of progressing to CRC), from patients with low grade intraepithelial neoplasia (i.e. patients with a low risk of progressing to CRC, LG) and from control patients. Two different cohorts were tested (see Examples).

[0023] The protein biomarker CLEC3B was found to be second best performing single marker in cohort 1 (Potsdam CRC) in distinguishing Controls from CRC + pTis cases out of 2926 measured proteins. Additionally, for distinguishing Controls from CRC + pTis + HG / AA, CLEC3B was found as one of the top performing single markers in both cohorts. For both endpoints, CLEC3B achieved a similar AUC than the current reference marker carcinoembryonic antigen (CEA) (see Example 5) in cohort 1 (Potsdam CRC) and a nearly as high performance in cohort 2 (Blitz). CEA is so far known as the best performing single marker for CRC (Wild N et al A combination of serum markers for the early detection of colorectal cancer. ClinCancer Res. 2010 Dec 15; 16(24):6111-21. doi: 10.1158 / 1078- 0432.CCR-10-0119). Moreover, the combined determination of i) CLEC3B and ii) CEA, SOX9, anti-p53, CLEC3B, DNER, CFB, Ferritin and AREG increased the diagnostic performance. Thus, the use of the biomarker CLEC3B, optionally, in combination with one or more biomarkers selected from CEA, SOX9, anti-p53, CLEC3B, DNER, CFB, Ferritin and AREG enhances the non-invasive assessment of CRC and related conditions, thereby improving early diagnosis and overall patient management. Since the biomarker(s) can be determined in blood, serum or samples, such as in plasma samples, they are suitable for the screening of larger cohorts.

[0024] THE FIGURES SHOW

[0025] Figure 1: Distribution of Age in Years for the different subgroups in cohort 1 (Potsdam CRC). Overall similar age distribution between the different groups with a slight increase in age for Cases [pTis (pathological tumor in situ) and CRC],

[0026] Figure 2: Distribution of male and female subjects for the different subgroups in cohort 1 (Potsdam CRC). Overall similar distribution. Slightly more cases (CRC) and LG findings in male subjects, while more HG findings are observed in females.

[0027] Figure 3: Distribution of Age in Years for the different subgroups in cohort 2 (Blitz). Overall similar age distribution between the different groups with slightly lower values in controls and a slight increase in age for Cases (pTis and CRC). Figure 4: Distribution of male and female subjects for the different subgroups in cohort

[0028] 2 (Blitz). Overall similar distribution. More adenoma (advanced and nonadvanced) cases and slightly more CRC cases in male subjects, while slightly more controls and other findings are observed in females.

[0029] Figure 5: CLEC3B plasma samples in Cohort 1 (Potsdam CRC). Levels of CLEC3B in different subgroups consisting of control groups (Controls, other findings, low grade adenoma (LG)), high grade adenoma (HG), and colorectal cancer case groups (pTis, CRC Cases)). CLEC3B levels are decreased in pTis and slightly lower in CRC cases (A).

[0030] CLEC3B plasma samples in Cohort 2 (BLITZ CRC). Levels of CLEC3B in different subgroups consisting of control groups (controls, other findings, low grade adenoma (LG)), high grade adenoma (HG), and colorectal cancer case groups (pTis, CRC Cases)). CLEC3B levels are decreased in CRC cases (B).

[0031] Figure 6: Plasma CLEC3B levels slightly decrease with increasing clinical stages in cases with colorectal cancer compared to control groups in cohort 1 (Potsdam CRC). LG = low grade adenoma, HG = high grade (advanced) adenoma, stage 0 ( = pTis) to stage IV = colorectal cancer stage 0 to stage IV (A).

[0032] Plasma CLEC3B levels slightly decrease with increasing clinical stages in cases with colorectal cancer compared to control groups in cohort 2 (BLITZ cohort). NAA = non advanced adenoma (= low grade adenoma), AA = advanced adenoma ( = high grade adenoma, stage 0 ( = pTis) to stage IV = colorectal cancer stage 0 to stage IV (B).

[0033] Figure 7: Plasma CLEC3B levels are decreased in both colon, rectum and rectosigmoid junction colorectal cancer cases with no significant difference in location in cohort 1 (A).

[0034] Plasma CLEC3B levels are decreased in both colon, rectum, cecum and rectosigmoid junction colorectal cancer cases with no significant difference in location in cohort 2 (B).

[0035] Figure 8: CLEC3B is decreased in plasma samples of subjects with colorectal cancer cases (CRC) stage I - IV, and pTis (carcinoma in situ) (CRC+pTis) compared to control subject with non- findings, other findings and low grade adenoma (Controls) (Cohort 1). Figure 9: CLEC3B is decreased in plasma samples of subjects with colorectal cancer cases (CRC) stage I - IV, pTis (carcinoma in situ) and high grade adenoma (CRC+pTis+HG) compared to control subject with non-findings, other findings and low grade adenoma (Controls) (Cohort 1).

[0036] Figure 10: CLEC3B is decreased in plasma samples of subjects with colorectal cancer cases (CRC) stage I - IV, and pTis (carcinoma in situ) (CRC+pTis) compared to control subject with non-findings, other findings and non-advanced adenoma (Cntrols) (Cohort 2).

[0037] Figure 11: CLEC3B is decreased in plasma samples of subjects with colorectal cancer cases (CRC) stage I - IV, pTis (carcinoma in situ) and advanced adenoma (CRC+pTis+AA) compared to control subject with non-findings, other findings and non-advanced adenoma (Controls) (Cohort 2).

[0038] Figure 12: CEA in plasma samples of subjects with CRC Cases and pTis compared to controls, with other findings, and low grade adenoma (Cohort 1).

[0039] Figure 13: CEA in plasma samples of subjects with CRC Cases, pTis and high grade or advanced adenoma compared to controls, with other findings, and low grade adenoma (Cohort 1).

[0040] Figure 14: CEA in plasma samples of subjects with CRC Cases and pTis compared to controls, with other findings, and low grade adenoma (Cohort 2).

[0041] Figure 15: CEA in plasma samples of subjects with CRC Cases, pTis and high grade or advanced adenoma compared to controls, with other findings, and low grade adenoma (Cohort 2).

[0042] BRIEF SUMMARY OF THE PRESENT INVENTION

[0043] The present invention relates to a method for assessing colorectal cancer or a precancerous condition thereof (such as advanced adenoma) in a subject, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) in a sample, such as a blood, serum or plasma sample, from the subject, and b) assessing colorectal cancer or the precancerous condition thereof. The assessment made in step b) of the present invention is, typically, based on the level of CLEC3B determined in step a). For example, colorectal cancer or a precancerous condition thereof is assessed by comparing the determined level of CLEC3B to a reference level. Alternatively, the colorectal cancer or a precancerous condition thereof is assessed by calculating a score for assessing colorectal cancer or the precancerous condition thereof based on the determined level of CLEC3B.

[0044] Accordingly, the present invention relates to a method for assessing colorectal cancer or a precancerous condition thereof in a subject, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) in a sample from the subject, and b) comparing the level of CLEC3B to a reference level and / or calculating a score for assessing colorectal cancer or the precancerous condition thereof, thereby assessing colorectal cancer or the precancerous condition thereof.

[0045] In a preferred embodiment of the method of the present invention, the test subject is a subject suspected to suffer from colorectal cancer or a precancerous condition thereof.

[0046] Preferably, the reference level is a level of CLEC3B which allows for assessing colorectal cancer or a precancerous condition thereof (e.g. for diagnosing colorectal cancer or a precancerous condition thereof). In a preferred embodiment, the reference level is a predetermined level of CLEC3B, such as a predetermined level of CLEC3B, which allows for assessing colorectal cancer or a precancerous condition thereof. Also preferably, the calculated score is a score, which allows for assessing colorectal cancer or a precancerous condition thereof.

[0047] In a preferred embodiment of the method of the present invention, the precancerous condition of colorectal cancer is advanced adenoma. Thus, the assessment is the assessment of colorectal cancer or advanced adenoma.

[0048] In a first aspect of the method of the present invention, the assessment of colorectal cancer or the precancerous condition thereof is the diagnosis of colorectal cancer or the precancerous condition thereof. Thus, it is diagnosed, whether a subject suffers from colorectal cancer or a precancerous condition thereof, or not.

[0049] Accordingly, the present invention relates to a method for diagnosing colorectal cancer or a precancerous condition thereof in a subject, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) in a sample from the subject, and b) comparing the level of CLEC3B to a reference level and / or calculating a score for diagnosing colorectal cancer or the precancerous condition thereof, thereby diagnosing colorectal cancer or the precancerous condition thereof.

[0050] In a preferred embodiment of the above diagnostic method, it is diagnosed whether the subject suffers from CRC (colorectal cancer), or not.

[0051] In an embodiment of the above diagnostic method, the diagnosis is made in order to exclude, i.e. rule-out colorectal cancer or a precancerous condition thereof in a subject.

[0052] Accordingly, the present invention relates to a method for ruling out colorectal cancer or a precancerous condition thereof in a subject, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) in a sample from the subject, and b) comparing the level of CLEC3B to a reference level and / or calculating a score for ruling out colorectal cancer or the precancerous condition thereof, thereby ruling out colorectal cancer or the precancerous condition thereof.

[0053] In an alternative embodiment of the above diagnostic method, the diagnosis is made in order to rule in colorectal cancer or a precancerous condition thereof in a subject.

[0054] Accordingly, the present invention relates to a method for ruling in colorectal cancer or a precancerous condition thereof in a subject, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) in a sample from the subject, and b) comparing the level of CLEC3B to a reference level and / or calculating a score for ruling in colorectal cancer or the precancerous condition thereof, thereby ruling in colorectal cancer or the precancerous condition thereof.

[0055] In a second aspect of the method of the present invention, the assessment of colorectal cancer or the precancerous condition thereof is

[0056] A. the differentiation whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, or

[0057] B. the differentiation whether a subject i) suffers from colorectal cancer or ii) does not suffer from colorectal cancer.

[0058] Accordingly, the present invention relates to a method for differentiating whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) in a sample from the subject, and b) comparing the level of CLEC3B to a reference level and / or calculating a score, thereby differentiating whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma.

[0059] Also, the present invention relates to a method for differentiating whether a subject i) suffers from CRC or ii) does not suffer from CRC, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) in a sample from the subject, and b) comparing the level of CLEC3B to a reference level and / or calculating a score, thereby differentiating whether a subject i) suffers from CRC or ii) does not suffer from CRC.

[0060] In a third aspect of the method of the present invention, the assessment of colorectal cancer or the precancerous condition thereof is the identification of a subject who is eligible to one or more (further) diagnostic measures for diagnosing colorectal cancer or a precancerous condition thereof. Thus, it can be decided whether the patient shall be subjected to further diagnostic measures, or not.

[0061] Accordingly, the present invention relates to a method for identifying a subject who is eligible to one or more diagnostic measures for diagnosing colorectal cancer or a precancerous condition thereof, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) in a sample from the subject, and b) comparing the level of CLEC3B to a reference level and / or calculating a score, thereby identifying a subject who is eligible to said one or more diagnostic measures.

[0062] In a preferred embodiment of the above method for identifying a subject, the one or more further diagnostic measures are selected from Colonoscopy, Sigmoidoscopy, CT CoIonography (Virtual Colonoscopy), Double-Contrast Barium Enema, Capsule Endoscopy, a fecal immunochemical test and a stool DNA test. In case, the subject has been identified to be eligible to said one or more further diagnostic measures, the method may further comprise recommending or subjecting the identified patient to said one or more further diagnostic measures.

[0063] Accordingly, the method of present invention may comprise the further step of subjecting a subject who has been identified to be eligible to said one or more further diagnostic methods in order to diagnose colorectal cancer or a precancerous condition thereof. In accordance with the present invention, the assessment of colorectal cancer or the precancerous condition thereof (such as the diagnosis, the differentiation, the identification,) may be further based on the level of at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9, DNER , CFB, Ferritin and AREG.

[0064] Accordingly, the method of the present invention may comprise the following steps, a) determining the level of CLEC3B (C-type lectin domain family 3 member B) and the level(s) of at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9 SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin) in a sample from the subject, and b) comparing the level of CLEC3B and the level of the at least one further biomarker to reference levels, and / or calculating a score for assessing colorectal cancer or the precancerous condition thereof, wherein the score is calculated based on the level of CLEC3B and the level(s) of the at least one further biomarker, whereby colorectal cancer or the precancerous condition thereof is assessed.

[0065] In an embodiment, the levels of CLEC3B and SOX9 are determined.

[0066] In another embodiment, the levels of CLEC3B and Ferritin are determined.

[0067] In yet another embodiment, the levels of CLEC3B and CFB are determined.

[0068] In yet another embodiment, the levels of CLEC3B and AREG are determined.

[0069] In yet another embodiment, the levels of CLEC3B and anti-p53 are determined.

[0070] In yet another embodiment, the levels of CLEC3B and DNER are determined.

[0071] In yet another embodiment, the levels of CLEC3B and CEA or are determined.

[0072] In yet another embodiment, the levels of CLEC3B, CEA and anti-p53 are determined.

[0073] The method of the present invention can be also carried out as computer implemented method. Accordingly, the present invention further relates to a computer-implemented method for assessing colorectal cancer or a precancerous condition thereof, comprising a) receiving, at a processing unit, a value for the level of CLEC3B in a blood, serum or plasma sample from a subject, and, optionally at least one further value for the level of at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9, DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin) in said blood, serum or plasma sample, b) comparing, by said processing unit, the value or values received in step a) to a reference or to references and / or calculating, by said processing unit, a score for assessing colorectal cancer or the precancerous condition thereof, wherein the score is based on the value or values received in step a), and c) assessing colorectal cancer or the precancerous condition thereof based on the result of step b).

[0074] Moreover, the present invention relates to the use of i. the biomarker CLEC3B, or at least one detection agent thereof, and optionally ii. at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9, DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), or at least one detection agent for said at least one further biomarker, in a sample (such as blood, serum or plasma) from a subject for assessing colorectal cancer or a precancerous condition thereof.

[0075] Moreover, the present invention relates to the use of the biomarker CLEC3B, or one or more detection agents for said biomarker, in a sample from a subject as referred to herein, such as in a blood, serum or plasma sample for improving the diagnostic performance of a biomarker (or a panel of biomarkers) for colorectal cancer or a precancerous condition thereof in the assessment of colorectal cancer or a precancerous condition thereof. Preferably, the assessment is the diagnosis or identification as described herein. Preferably, the biomarker whose performance is increased is CEA, anti-p53, DNER, CFB, SOX9, Ferritin or AREG, or a panel thereof. In an embodiment, the performance increase is an increased sensitivity. In another embodiment, the performance increase is an increased specificity.

[0076] Moreover, the present invention relates to a kit for assessing colorectal cancer or a precancerous condition thereof, said kit comprising at least one detection agent for the biomarker CLEC3B and, optionally, at least one detection agent for at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9, DNER , CFB, Ferritin and AREG.

[0077] Moreover, the present invention relates to a database comprising one or more stored references for the biomarker CLEC3B and, optionally one or more stored references for at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9, DNER , CFB, Ferritin and AREG. Preferably, the one or more stored references allow for assessing colorectal cancer or a precancerous condition thereof. Further, the present invention relates to a computer program including computer-executable instructions for performing the computer-implemented method of the present invention when the program is executed on a computer or computer network.

[0078] Moreover, the present invention relates to a device for assessing colorectal cancer or a precancerous condition thereof, said device comprising: a) at least one measuring unit for determining a level of the biomarker CLEC3B and, optionally, a level of at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9, DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), in a sample, such as a blood, serum or plasma sample, from a subject, said at least one measuring unit comprising at least one detection agent for the biomarker CLEC3B and, optionally, at least one detection agent for the biomarker CEA, at least one detection agent for the biomarker anti-p53, at least one detection agent for the biomarker CLEC3B, at least one detection agent for the biomarker DNER, at least one detection agent for the biomarker Ferritin, at least one detection agent for the biomarker CFB and / or at least one detection agent for the biomarker AREG, and b) an evaluation unit operably linked to the measuring unit, said evaluation unit comprising a data processor comprising instructions for i) carrying out a comparison of the level of the biomarker CLEC3B and, optionally, of the level of said at least one further biomarker to a reference or references and / or for ii) carrying out a calculation of a score for assessing colorectal cancer or a precancerous condition thereof, wherein the score is calculated based on the level of CLEC3B and, optionally, on the level(s) of the at least one further biomarker.

[0079] The present invention further relates to a method of treating a subject, said method comprising a) determining the level of CLEC3B (C-type lectin domain family 3 member B) and, optionally, the level(s) of at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, Ferritin and AREG in a sample from the subject, and b) diagnosing colorectal cancer or the precancerous condition thereof based on the level of CLEC3B and, optionally, the level(s) of at least one further biomarker, and c) treating a subject diagnosed to suffer from colorectal cancer or the precancerous condition thereof with a suitable therapeutic measure that aims to treat colorectal cancer or a precancerous condition thereof.

[0080] In an embodiment of the above method of treatment, the diagnosis of colorectal cancer or a precancerous condition thereof may be based on the biomarker level(s) only. In an alternative embodiment, the diagnosis may be based on the biomarker level(s) and on one or more further diagnostic measures selected from Colonoscopy, Sigmoidoscopy, CT CoIonography (Virtual Colonoscopy), Double-Contrast Barium Enema, Capsule Endoscopy, a fecal immunochemical test and a stool DNA test.

[0081] Moreover, the present invention relates to a method for determining the level of a first biomarker, said first biomarker being CLEC3B, the level of a second biomarker and, optionally, the level of a third biomarker in a sample blood, serum or plasma from a subject suspected to suffer from colorectal cancer or a precancerous condition thereof comprising i) contacting a portion of said blood, serum or plasma sample with one or more detection agents that specifically bind to the first biomarker CLEC3B present in the sample, thereby allowing the formation of first complex comprising said biomarker and said one or more detection agents and ii) contacting the same or a different portion of said blood, serum or plasma sample with one or more detection agents that specifically bind a second biomarker selected from the group consisting of SOX9 (SRY-box transcription factor 9), carcinoembryonic antigen (CEA), anti-p53 (ap53), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), and thereby allowing the formation of a second complex comprising said second biomarker and said one or more detection agents, and optionally iii) contacting the same portion as in i) or ii), or a different portion of said blood, serum or plasma sample with one or more detection agents that specifically bind a third biomarker selected from the group consisting of SOX9 (SRY-box transcription factor 9), carcinoembryonic antigen (CEA), anti-p53 (ap53), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), and thereby allowing the formation of a third complex comprising said third biomarker and said one or more detection agents, and iv) determining the level of the first biomarker, the second biomarker and, optionally, the third biomarker by determining the level of the first complex, the second complex, and optionally the third complex.

[0082] In preferred embodiment of the above method, the sample is from a subject suffering from colorectal cancer or a precancerous condition. For example, the sample is from a subject suffering from CRC stage 0 (which is also referred to as pTis (pathological tumor in situ)) or from advanced adenoma.

[0083] DETAILED SUMMARY OF THE PRESENT INVENTION / DEFINITIONS

[0084] It is to be understood that as used in the specification and in the claims, “a” or “an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “an” item can mean that at least one item can be utilized.

[0085] As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. The term “comprising” also encompasses embodiments where only the items referred to are present, i.e. it has a limiting meaning in the sense of “consisting of’.

[0086] Further, it will be understood that the term “at least one” as used herein means that one or more of the items referred to following the term may be used in accordance with the invention. For example, if the term indicates that at least one sampling unit shall be used this may be understood as one sampling unit or more than one sampling units, i.e. two, three, four, five or any other number. Depending on the item the term refers to, the skilled person understands as to what upper limit the term may refer, if any. For example, the term at least one further biomarker may mean, one, two, three, four or five biomarkers.

[0087] The term “about” as used herein means that with respect to any number recited after said term an interval accuracy exists within in which a technical effect can be achieved. Accordingly, "about" as referred to herein, preferably, refers to the precise numerical value or a range around said precise numerical value of ±20 %, preferably ±15 %, more preferably ±10 %, or even more preferably ±5 %. As used herein, the term “level” includes any and all measure of quantity deemed suitable by the skilled person, and in particular includes an absolute amount of a compound referred to herein, a relative level, or a concentration of the compound, as well as any value or parameter which correlates thereto or can be derived therefrom, in an embodiment by standard mathematical operations. Such values or parameters comprise intensity signal values from all specific physical or chemical properties obtained from the said compounds by direct measurements, e.g., intensity values in mass spectra or NMR spectra. Moreover, encompassed are all values or parameters which are obtained by indirect measurements specified elsewhere in this description, e.g., response levels determined from biological read out systems in response to the compounds or intensity signals obtained from specifically bound ligands, such as detection compounds.

[0088] The term “determining” as used herein refers to semiquantitative or quantitative determination of a biomarker referred to herein. Determining the level of a biomarker may be carried out by any technique which allows for establishing a measure of quantity of a biomarker in a semiquantitative or quantitative manner. Suitable techniques depend on the molecular nature and the properties of the biomarkers and are discussed elsewhere herein in more detail.

[0089] The term “comparing” as used herein encompasses comparing the determined level for a biomarker as referred to herein to a reference. It is to be understood that comparing as used herein refers to any kind of comparison made between the value for the level with the reference. However, it is to be understood that, in an embodiment, identical types of values are compared with each other, e.g., if an absolute amount is determined, the reference shall also be an absolute amount, if a relative amount is determined, the reference shall also be a relative amount, etc. The term comparing also encompasses comparing a calculated score with a suitable reference core. The comparison may be carried out manually or computer assisted. The value of the level and the reference can be, e.g., compared to each other and the said comparison can be automatically carried out by a computer program executing an algorithm for the comparison. The computer program carrying out the said evaluation will provide the desired assessment in a suitable output format.

[0090] The term “kit” as used herein refers to a collection of the aforementioned components, typically, provided in separate compound(s) or as a mixture of compounds. The means are, in an embodiment, provided in a single container (i.e. a housing), in a further embodiment enabling common translocation, e.g. transport, of the components. The container also typically comprises instructions for carrying out the method of the present invention. These instructions may be in the form of a manual or may be provided by a computer program code which is capable of carrying out or supports the determination of the biomarkers referred to in the methods of the present invention when implemented on a computer or a data processing device. The computer program code may be provided on a data storage medium or device such as an optical storage medium (e.g., a Compact Disc) or directly on a computer or data processing device or may be provided in a download format such as a link to an accessible server or cloud. Moreover, the kit may, usually, comprise standards for reference levels of biomarkers for calibration purposes. The kit may also comprise further components which are necessary for carrying one of the methods described herein, may assist in doing so, or may provide further functions; in an embodiment, the further component is a solvent, a buffer, a diluent, a washing solution and / or one or more reagent(s) required for detection of the biomarkers. Further, the kit may comprise the device of the invention either in parts or in its entirety.

[0091] The term “device” as used herein relates to a combination of means comprising the aforementioned units operatively linked to each other as to allow the determination of the levels of biomarkers and evaluation thereof according to a method as specified herein such that an assessment can be provided. The device comprises at least one measuring unit and at least one evaluation unit.

[0092] The term “detection agent”, for which also "detection compound" may be used, as used herein, refers to any agent which allows determination, in an embodiment specific determination, of a level of at least one biomarker. Thus, the detection agent may in particular be a reaction substrate, e.g. in case the biomarker has catalytic activity, e.g. is an enzyme; or the detection agent may be an agent binding, in an embodiment specifically, to a biomarker or analyte thereof. The skilled person selects suitable detection substrates in dependence on the biomarker, i.e. catalytic activity, to be detected, based on information available in the art. For specific biomarkers, example substrates are provided herein above. Also, binding agents for specific antigens, as well as methods for providing them, are known in the art. As indicated herein above, the detection agent being a binding agent in an embodiment specifically binds to a biomarker, i.e. does not cross-react with other components present in the sample. Typically, a detection agent specifically binding a biomarker as referred to herein may be an antibody, an antibody fragment or derivative, an aptamer, a ligand for the biomarker, a receptor for the biomarker, an enzyme known to bind and / or convert the biomarker, or a small molecule known to specifically bind to the biomarker. For example, antibodies as referred to herein as detection agents include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F(ab)2 fragments that are capable of binding antigen or hapten. Aptamer detection agents, e.g., may be nucleic acid or peptide aptamers. Methods to prepare such aptamers are well- known in the art. The detection agent may be fused or linked permanently or reversibly to a detectable label. Suitable labels are well known to the skilled artisan. Suitable detectable labels are any labels detectable by an appropriate detection method. Typical labels include gold particles, latex beads, acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels ("e.g. magnetic beads", including paramagnetic and superparamagnetic labels), and fluorescent labels.

[0093] “Specific binding” of a detection agent means that it should not bind substantially to, i.e. cross-react with, another peptide, polypeptide or substance present in the sample to be analyzed. Preferably, the specifically bound biomarker should be bound with at least 3 times higher, more preferably at least 10 times higher and even more preferably at least 50 times higher affinity than any other components of the sample. Non-specific binding may be tolerable, if it can still be distinguished and measured unequivocally, e.g. according to its size on a Western Blot, or by its relatively higher abundance in the sample.

[0094] The term “computer-implemented” as used herein means that the method is carried out in an automated fashion on a data processing unit which is, typically, comprised in a computer or similar data processing device. The data processing unit shall receive values for the level of the biomarkers. Such values can be the levels, relative levels or any other calculated value reflecting the level as described elsewhere herein in detail. Accordingly, it is to be understood that the aforementioned method does not require the determination of levels for the biomarkers but rather uses values for already predetermined levels.

[0095] The term "receiving", as used herein, relates to acquiring the indicated information, in particular a value of a parameter such as a level of a biomarker, in a manner enabling basing the assessment on said information. Thus, in an embodiment, receiving is reading the information from a data carrier, e.g. in the form of a data sheet, analysis device output, e.g. a result of an immunoassay, a mass spectrum, or the like; or from a database comprising at least the relevant information. In an embodiment, the information obtained comprises value(s) for level(s) of biomarkers determined as specified herein above. The data carrier and / or the database may be local, i.e. physically connected to a device used to perform steps of the method; they may, however, also be remote, accessible via a network connection or via the internet; thus, the data carrier and / or the data carrier may e.g. be cloud storage. Also, the method may be implemented as a service provided by means of a network connection, e.g. via internet, in which values of the biomarkers, or one or more score(s) derived therefrom, are obtained from e.g. a device performing the determination or medical practitioner, and the result of the comparison it output.

[0096] The term “database”, as used herein, refers to a collection of data which may be physically and / or logically grouped together. Accordingly, the database in an embodiment comprises an allocation of references to assessment results. As the skilled person understands from the description herein above, "stored references for the biomarker. ..." may also be one or more scores derived from said references. The database, in an embodiment, comprises further data, such as upper and / or lower detection limits, references for further biomarkers, in particular those described herein above, data relevant for plausibility checks, and the like. In a further embodiment, the database comprises data on one or more assay methods to use, lot-specific data, e.g. for calibrator samples, and the like. In an embodiment, the database may be implemented in a single data storage medium or in physically separated data storage media being operatively linked to each other. In an embodiment, the database comprises a data collection on a suitable storage medium, in an embodiment tangibly embedded thereon. Moreover, the database, in an embodiment, further comprises a database management system. The database management system is, in an embodiment, a network-based, hierarchical or object-oriented database management system. Furthermore, the database may be a federal or integrated database. In a further embodiment, the database will be implemented as a distributed (federal) system, e.g. as a Client-Server-System. In a further embodiment, the database is structured as to allow a search algorithm to compare a test data set with the data sets, in particular the references, comprised by the data collection. Specifically, by using such an algorithm, the database can be searched for similar or identical data sets being indicative for a medical condition or effect as set forth above (e.g. a query search). Thus, in an embodiment, if a data set fulfilling the comparison criteria as detailed elsewhere herein can be identified in the database, the test data set will be associated with the said medical condition or effect. Consequently, the information obtained from the database can be used, e.g., as a reference for the methods described elsewhere herein.

[0097] As set forth above, the present invention relates to method for assessing colorectal cancer or a precancerous condition thereof in a subject. The method will be described herein below in more detail. The present invention further relates to computer-implemented methods, databases, devices, kits and uses related thereto. The definition and explanations given in connection with the method for assessing colorectal cancer or a precancerous condition thereof in a subject, preferably, apply mutatis mutandis to computer-implemented methods, databases, devices, kits and uses related thereto.

[0098] The term "colorectal cancer", typically, includes the well-accepted medical definition that defines colorectal cancer as a medical condition characterized by cancer of cells of the intestinal tract below the small intestine (i.e., the large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum). "Cancer cells" refer to any cells that exhibit uncontrolled growth in a tissue or organ of a multicellular organism.

[0099] In a preferred embodiment, the term “colorectal cancer” includes stage 0, stage I, stage II, stage III, and stage IV cancer (preferably according to the UICC Classification of Malignant Tumours, see Virchows Arch. 2018 Apr;472(4):519-531. doi: 10.1007 / s00428-017-2276-y. Epub 2017 Dec 5. PMID: 29209757). Thus, the cancer to be diagnosed may be stage 0 (pTis), stage I, stage II, stage III or stage IV colorectal cancer. A higher number indicates a more advanced cancer and likely a worse outcome. CRC stage 0 is typically also referred to as pTis (pathological tumor in situ). This stage refers to a very early stage of cancer where malignant cells are present only in the innermost lining of the colon or rectum and have not spread to deeper layers or other tissues. It represents a pre-invasive stage of cancer. In CRC stage I, the cancer has grown into deeper layers of the colon or rectum but hasn’t spread to nearby lymph nodes or distant sites. In CRC stage II, the cancer has penetrated through the wall of the colon or rectum and may involve nearby tissues. In CRC Stage III has reached nearby lymph nodes. CRC stage IV is an advantaged stage, the cancer has spread to distant organs.

[0100] The precancerous condition in accordance with the present invention is preferably adenoma, and in particular advanced adenoma. Adenomas are benign tumors found in the colon and rectum. In particular, they are noninvasive neoplastic lesions of the columnar epithelium. Adenomas are often discovered during routine screenings such as colonoscopies. They are considered precancerous because they have the potential to develop into colorectal cancer over time. Adenomas are generally classified based on their histological features and size, which helps determine their potential risk of becoming malignant. The term "advanced adenoma" is used to describe those adenomas that carry a higher risk of progressing to cancer. This classification includes adenomas that are 1 cm or larger in size, adenomas with a villous or tubulovillous histology, and adenomas exhibiting high grade dysplasia (HGD). HGD indicates significant dysplasia, or abnormal cell growth, which is a strong predictor of future cancer development if left untreated. In contrast, non-advanced adenomas (adenomas that less than 1 cm in size) have a lower risk of becoming invasive cancer compared to advanced adenomas. They are considered early precancerous lesions but with less severe cellular abnormalities. In the studies underlying the present invention, they were considered as benign findings.

[0101] In a preferred embodiment, the assessment of the present invention is the assessment of CRC or advanced adenoma (such as the differentiation or diagnosis as referred to herein). In a particular preferred embodiment, the assessment is the assessment of CRC (such as the diagnosis).

[0102] The assessment made in accordance with the method of the present invention is preferably based on the biomarker CLEC3B. Accordingly, the method of the present invention comprises step a) of determining the level of CLEC3B (C-type lectin domain family 3 member B) in a sample, such as a blood, serum or plasma sample, from the subject. In a preferred embodiment, the method of the present invention, the method further comprises in step a) the determination of the level(s) of at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9, DNER , CFB, Ferritin and AREG.

[0103] Thus, the present invention relates to a method for assessing colorectal cancer or a precancerous condition thereof (in particular high grade advanced adenoma) in a subject, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) and optionally, the level of at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, Ferritin and AREG, in a sample, such as a blood, serum or plasma sample, from the subject, and b) assessing colorectal cancer or the precancerous condition thereof based on the level of the biomarker CLEC3B and optionally the level of the at least one further biomarker as determined in step a).

[0104] In an embodiment, step b) of the above method comprises comparing the level of CLEC3B to a reference level for CLEC3B and, optionally, comparing the level of the at least one further biomarker to a reference level for said at least one further biomarker. Thereby, colorectal cancer or the precancerous condition thereof is assessed (i.e. based on the comparison step).

[0105] In an alternative embodiment, step b) of the above method comprises calculating a score for assessing colorectal cancer or the precancerous condition thereof, wherein the score is calculated based on the level of CLEC3B and, optionally, the level(s) of the at least one further biomarker, Thereby, colorectal cancer or the precancerous condition thereof is assessed (i.e. based on the calculated score).

[0106] The method as referred to in accordance with the present invention preferably, is an ex vivo and more preferably an in vitro method. Moreover, the method may be a method which essentially consists of the aforementioned steps or a method which includes further steps. Thus, it may comprise steps in addition to those explicitly mentioned above. For example, the method may comprise prior to step a) the step of selecting a sample from a subject as referred to herein, e.g. a patient suspected to suffer from colorectal cancer or a precancerous condition thereof. For example, further steps may relate to the determination of further markers and / or evaluation of the results obtained by the method. The method may be carried out manually or assisted by automation. Preferably, the method may in total or in part be assisted by automation, e.g., by a suitable robotic and sensory equipment for the determination in step (a) or a computer-implemented calculation step or comparison step. In accordance with the present invention, colorectal cancer or a precancerous condition thereof shall be assessed. Preferably, the term “assessing colorectal cancer or a precancerous condition thereof’, relates to

[0107] • the diagnosis whether a subject is likely, or is not likely, to suffer from colorectal cancer or a precancerous condition thereof (such as the rule-in or rule-out of colorectal cancer or a precancerous condition thereof),

[0108] • the diagnosis of colorectal cancer (such as the rule-in or rule-out of colorectal cancer),

[0109] • the differentiation whether a subject i) suffers from colorectal cancer or ii) does not suffer from colorectal cancer,

[0110] • the differentiation whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, or

[0111] • the identification of a subject who is eligible to one or more further diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof.

[0112] The various assessments of the present invention are described herein below in more detail. As will be understood by those skilled in the art, the assessment of the present invention is usually not intended to be correct for 100% of the subjects to be tested. The term, typically, requires that a correct assessment (such as the diagnosis, differentiation, or identification of a subject as referred as referred to herein) can be made for a statistically significant portion of subjects. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Typically envisaged confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-values are, typically, 0.2, 0.1, 0.05. Also typically, the assessment may be done with a preselected sensitivity and specificity.

[0113] Establishing an assessment as referred to herein may be based on the aforesaid assessment. For example, the diagnosis of colorectal cancer or a precancerous condition thereof may be based on the determined level of the biomarker CLEC3B and optionally on the determined level of the at least one further biomarker. However, in a further embodiment, the assessment based on the aforesaid assessment is based on the determined level(s) of the biomarker in combination with further diagnostic information, such as the results from one or more further diagnostic measures for assessing colorectal cancer or a precancerous condition thereof. In embodiments, the further diagnostic information taken into account for the assessment (e.g. by including them into a calculated score) may comprise or consist of age and / or sex. Preferred further diagnostic measures for assessing colorectal cancer or a precancerous condition thereof are Colonoscopy, Sigmoidoscopy, CT CoIonography (Virtual Colonoscopy), Double-Contrast Barium Enema, Capsule Endoscopy, a fecal immunochemical test and a stool DNA test.

[0114] Alternatively, the biomarker CLEC3B or the biomarker combinations of the present invention is (are) combined with one or more of the biomarkers for colorectal cancer as disclosed in EP 2 829 881 Bl : DKK-3 (dickkopf homolog 3), M2PK (pyruvate kinase muscle 2), IGFBP2 (insulin-like growth factor binding protein-2), EpCAM (epithelial cell adhesion molecule), IL- 13 (interleukin- 13), and IL-8 (interleukin-8). Thus, the marker levels are determined. For the assessment as referred to herein, the determined levels may be compared to reference levels. Alternatively, a score may be calculated based on the levels of the markers.

[0115] In an embodiment, the biomarker CLEC3B is combined with DKK-3 (dickkopf homolog 3), M2PK (pyruvate kinase muscle 2), and IGFBP2 (insulin-like growth factor binding protein- 2).

[0116] In another embodiment, the biomarker CLEC3B is combined with M2PK, IGFBP2, and EpCAM (epithelial cell adhesion molecule).

[0117] In yet another embodiment, the biomarker CLEC3B is combined with M2PK, IGFBP2 and IL- 13 (interleukin- 13).

[0118] In yet another embodiment, the biomarker CLEC3B is combined with M2PK, IGFBP2 and IL-8 (interleukin-8).

[0119] Diagnosing colorectal cancer or a precancerous condition thereof

[0120] In a preferred embodiment of the present invention, the assessment of colorectal cancer or a precancerous condition thereof is the diagnosis of colorectal cancer or a precancerous condition thereof, i.e. it is diagnosed whether a test subject is likely to suffer from colorectal cancer or a precancerous condition thereof (in particular advanced adenomas), or is not likely to suffer from colorectal cancer or a precancerous condition thereof. In a particularly preferred embodiment, the assessment is the diagnosis of CRC. Accordingly, it is diagnosed, whether a subject is likely to suffer from colorectal cancer, or not. In case a subject is diagnosed to be likely to suffer from colorectal cancer or a precancerous condition thereof, a further diagnostic measure such as colonoscopy could be recommended or initiated.

[0121] Accordingly, the present invention envisages a method for diagnosing colorectal cancer or a precancerous condition thereof in a subject, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) and, optionally, the level of at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, Ferritin and AREG in a sample from the subject, and b) diagnosing colorectal cancer or the precancerous condition thereof based on the level of CLEC3B and, optionally, the level of the at least one further biomarker determined in step a).

[0122] In a preferred embodiment, step b) of the above diagnostic method comprises

[0123] (b) comparing the level of the biomarker CLEC3B to a reference level for CLEC3B and, optionally, comparing the level of the at least one further biomarker to a reference level for said at least one further biomarker, whereby colorectal cancer or a precancerous condition thereof is to be diagnosed.

[0124] Alternatively, a score may be calculated for the diagnosis of colorectal cancer or a precancerous condition thereof. Thus, the diagnosis is based on the score. Accordingly, in an another preferred embodiment, step b) of the above diagnostic method comprises (b) calculating a score for diagnosing colorectal cancer or the precancerous condition thereof, wherein the score is calculated based on the level of CLEC3B and, optionally, the level(s) of the at least one further biomarker, whereby colorectal cancer or a precancerous condition thereof is to be diagnosed.

[0125] In accordance with the above method, it shall be diagnosed whether a test subject is likely to suffer from colorectal cancer or a precancerous condition thereof (in particular advanced adenomas), or is not likely to suffer from colorectal cancer or a precancerous condition thereof. Accordingly, the term “diagnosing” typically refers to assessing the probability of a subject to suffer from the disease or not (at the time of the testing).

[0126] A subject who is likely to suffer from a disease or condition has higher probability of suffering from the disease or condition as compared to the average risk in a cohort of subjects (i.e. a group of subjects). The probability depends on the preselected sensitivity of the assay. A subject who is not likely to suffer from a disease or condition has lower probability of suffering from the disease or condition as compared to the average risk in a cohort of subjects (i.e. a group of subjects).

[0127] Preferably, the subject to be tested in connection with the method for diagnosing of colorectal cancer or a precancerous condition thereof is a subject who is suspected to suffer from colorectal cancer or a precancerous condition thereof (as described elsewhere herein). However, it is also contemplated that the subject already has been diagnosed previously to suffer from colorectal cancer or a precancerous condition thereof and that the previous diagnosis is confirmed by carrying out the method of the present invention. In a preferred embodiment of the diagnostic method, colorectal cancer or a precancerous condition thereof is ruled in. A patient in which the diagnosis is ruled in has high likelihood to suffer from colorectal cancer or a precancerous condition thereof. Typically, such a patient requires further diagnostic measures to confirm the diagnosis and / or suitable therapeutic measures to confirm the diagnosis (as described elsewhere herein). A rule in of a disease requires a reference level which allows for a high specificity.

[0128] In another preferred embodiment of the diagnostic method, colorectal cancer or a precancerous condition thereof is ruled out. Thus, a diagnosis of colorectal cancer or a precancerous condition thereof is excluded. A patient in which the diagnosis is ruled out has a low likelihood to suffer from colorectal cancer or a precancerous condition thereof. Typically, such a patient does not require further diagnostic or therapeutic measures with respect to colorectal cancer or a precancerous condition thereof. A rule out of a disease requires a reference level which allows for a high sensitivity. Ruling-out out colorectal cancer or a precancerous condition thereof is of particular interest since further diagnostic measures can be avoided.

[0129] In an embodiment of the method of diagnosing colorectal cancer or a precancerous condition thereof, said method further comprises a step of recommending and / or initiating one or more therapeutic methods that aim to treat colorectal cancer or a precancerous condition thereof. Preferably, said one or more therapeutic measures are recommended or initiated if it is diagnosed that the subject suffers from colorectal cancer or a precancerous condition thereof. Preferred therapeutic measures are described elsewhere herein. In an embodiment, the therapeutic measure is the resection of the tumor or the precancerous condition, e.g. of the advanced adenoma(s).

[0130] The direction of the individual markers for the diagnosis of colorectal cancer or a precancerous condition thereof, i.e. whether an increased or decreased level of the markers (as compared to the reference) is indicative for the diagnosis is disclosed elsewhere herein.

[0131] Method for differentiation

[0132] The term “differentiating” as used herein means, in a first embodiment, differentiating whether i) a subject suffers from CRC or advanced adenoma or ii) whether the subject does not suffer from CRC or advanced adenoma. A subject who does not suffer from CRC or advanced adenoma may be a subject who has no findings (non- findings), other findings (e.g. inflammatory bowel disease) or only benign findings (non-advanced adenoma or low grade adenoma) when undergoing colonoscopy. In a second embodiment, the term “differentiating” means differentiating whether i) a subject suffers from CRC or ii) whether the subject does not suffer from CRC. A subject who does not suffer from CRC may be a subject who has no findings (non-findings), other findings (e.g. inflammatory bowel disease), benign findings (non-advanced adenoma or low grade adenoma) or advanced adenoma when undergoing colonoscopy. Thus, it can differentiated whether a subject suffers from CRC or ii) whether the subject from inflammatory bowel disease.

[0133] Accordingly, the present invention relates to a method for differentiating whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) and, optionally, the level of at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, Ferritin and AREG in a sample from the subject, and b) comparing the level of the biomarker CLEC3B to a reference level for CLEC3B and, optionally, comparing the level of the at least one further biomarker to a reference level for said at least one further biomarker, whereby it differentiated whether the subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma.

[0134] Alternatively, step b) may comprise calculating a score for differentiating between i) and ii), wherein the score is calculated based on the level of CLEC3B and, optionally, the level(s) of the at least one further biomarker.

[0135] Alternatively, the present invention relates to a method for differentiating whether a subject i) suffers from CRC or ii) does not suffer from CRC, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) and, optionally, the level of at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, Ferritin and AREG in a sample from the subject, and b) comparing the level of the biomarker CLEC3B to a reference level for CLEC3B and, optionally, comparing the level of the at least one further biomarker to a reference level for said at least one further biomarker, whereby it is differentiated whether the subject i) suffers from CRC or ii) does not suffer from CRC.

[0136] Alternatively, step b) may comprise calculating a score for differentiating between i) and ii), wherein the score is calculated based on the level of CLEC3B and, optionally, the level(s) of the at least one further biomarker. In some embodiments, the differentiation may comprise further steps such as the confirmation of the differentiation (by further therapeutic measures described herein below). Thus, the term “differentiation” in the context of the present invention also encompasses aiding a physician in the differentiation as referred to herein. In some embodiments, the differentiation may comprise further therapeutic measures.

[0137] Method for identifying a subject who is eligible to further diagnostic measures

[0138] In accordance with this embodiment of method of the present invention, it shall be assessed whether a subject is eligible to further diagnostic measures for diagnosing colorectal cancer or a precancerous condition thereof. Thus, the biomarker can be used for the screening of larger cohorts. The term “identifying a subject” as used herein preferably refers to using the information or data generated relating to the level of CLEC3B and optionally relating to the level of the at least one further biomarker as referred to herein in a sample to identify a subject who is eligible to such further diagnostic measures. Thus, it is assessed whether the subject should be subjected to such further diagnostic methods. In a preferred embodiment, a subject who has been identified as a subject who is likely to suffer from colorectal cancer or a precancerous condition thereof (based on the level of the biomarker(s)) is eligible to such further diagnostic measures. Typically, the further measures are applied in order to confirm the diagnosis, or not. Accordingly, said further measure shall diagnose the presence of absence of colorectal cancer or a precancerous condition thereof, in said subject.

[0139] Accordingly, the present invention relates to a method for identifying a subject who is eligible to one or more diagnostic measures for diagnosing colorectal cancer or a precancerous condition thereof, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B) and, optionally, the level of at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, CLEC3B, DNER, CFB, Ferritin and AREG in a sample from the subject, and b) comparing the level of the biomarker CLEC3B to a reference level for CLEC3B and, optionally, comparing the level of the at least one further biomarker to a reference level for said at least one further biomarker, whereby it is assessed whether a subject is eligible to one or more diagnostic measures for diagnosing colorectal cancer or a precancerous condition thereof

[0140] Alternatively, step b) may comprise calculating a score for differentiating between i) and ii), wherein the score is calculated based on the level of CLEC3B and, optionally, the level(s) of the at least one further biomarker, whereby it is assessed whether a subject is eligible to one or more diagnostic measures for diagnosing colorectal cancer or a precancerous condition thereof.

[0141] In another preferred embodiment of the method of identification, the one or more further diagnostic measure is ruled out. Thus, the patient is not eligible to said one or more diagnostic measures, i.e. does require a further assessment. A patient who is not eligible to said one or more diagnostic measures is a patient who is unlikely to suffer from colorectal cancer or a precancerous condition thereof (based on the level of the biomarkers).

[0142] Specifically, the measure is ruled in, if the patient is likely to suffer from colorectal cancer or a precancerous condition thereof (based on the level of the biomarkers). Thus, the patient is eligible to said one or more diagnostic measures.

[0143] The further diagnostic measure for diagnosing colorectal cancer or a precancerous condition thereof can be any measure which allows for diagnosing colorectal cancer or a precancerous condition thereof. In a preferred embodiment, the diagnostic measures is selected from Colonoscopy, Sigmoidoscopy, CT CoIonography (Virtual Colonoscopy), Double-Contrast Barium Enema, Capsule Endoscopy, a fecal immunochemical test and a stool DNA test. Such measures are known in the art and e.g. described in Labianca R, Nordlinger B, Beretta GD, Mosconi Set al on behalf of the ESMO Guidelines Working Group* Early colon cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of Oncology 24 (Supplement 6): vi64-vi72, 2013.

[0144] In a particularly preferred embodiment, the diagnostic measure is colonoscopy.

[0145] In case, the patient is identified by the above method, the method may comprise the further step of recommending said one or more further diagnostic measures. Alternatively, the method may comprise the further step of subjecting the identified patient to said one or more diagnostic measures to diagnose whether the subject suffers from colorectal cancer or a precancerous condition thereof, or not. For example, the subject may be subjected to Colonoscopy, i.e. examined by Colonoscopy. Moreover, a suitable therapeutic measure may be initiated once the disease is diagnosed.

[0146] The subject to be tested

[0147] The subject or subject to be tested in accordance with the methods of the present invention can include, but is not limited to, mammals such as bovine, avian, canine, equine, feline, ovine, porcine, or primate animals (including humans and non-human primates). In a preferred embodiment, the subject is a human subject. The terms “subject” and “patient” are used interchangeably herein.

[0148] In case the assessment is the diagnosis, the differentiation or the identification as described above, the subject to be tested is, preferably, a subject who is suspected to suffer from colorectal cancer or a precancerous condition thereof. In an embodiment, such a subject is a subject, who is at average or increased risk of having colorectal cancer or a precancerous condition thereof. In a preferred embodiment, the subject is a subject aged 50 and above (such as aged 55 and above), a subject with a family history of colorectal cancer and / or a subject who shows symptoms of colorectal cancer or a precancerous condition thereof. Symptoms of colorectal cancer or a precancerous condition thereof, in particular of CRC, may include blood in the stool, a change in bowel movements, weight loss, abdominal pain and fatigue. Moreover, the patient may be an asymptomatic patient, i.e. a patient who does not show symptoms of colorectal cancer or a precancerous condition thereof.

[0149] Preferably, a subject who is suspected to suffer from colorectal cancer or a precancerous condition thereof is a subject who is indicated for Colorectal Cancer Screening. For example, the American Cancer Society Guideline recommends that subjects at average risk and increased risk of having colorectal cancer are subjected to Colorectal Cancer Screening.

[0150] A subject who is at average risk of having colorectal cancer is a subject who is 45 years or older, but who does preferably not have a personal history of colorectal cancer, a family history of colorectal cancer, a personal history of inflammatory bowel disease (ulcerative colitis or Crohn’s disease), a confirmed or suspected hereditary colorectal cancer syndrome, such as familial adenomatous polyposis (FAP) or Lynch syndrome (hereditary nonpolyposis colon cancer or HNPC and a history of getting radiation to the abdomen (belly) or pelvic area to treat a prior cancer. Accordingly, the subject may be a subject with an age of 45 years or older.

[0151] Subjects at increased risk of having colorectal cancer might need to start colorectal cancer screening before age 45. A subject who is at increased risk is a subject who has a family history of colorectal cancer, a history of colorectal cancer, a history of inflammatory bowel disease (e.g. ulcerative colitis or Crohn’s disease), a known family history of a hereditary colorectal cancer syndrome, and / or a personal history of radiation to the abdomen or pelvic area to treat a prior cancer. Thus, the subject to be tested may be indicated for the testing, because he is considered as a subject with an average risk or increased risk or CRC.

[0152] In an embodiment of the present invention, the subject to be tested does not suffer from stage I, II, III or IV colorectal cancer. The sample

[0153] The sample to be tested in accordance with the present invention is a sample of a body fluid. Samples of body fluids can be obtained by well-known techniques and include, samples of blood, plasma, serum, urine, lymphatic fluid, sputum, ascites, or any other bodily secretion or derivative thereof. Instead of a body fluid sample, the sample might be a stool sample obtained from the subject.

[0154] In a preferred embodiment of the present invention, the sample is a blood (i.e. whole blood), serum or plasma sample. In particular, the sample is a serum or plasma sample.

[0155] Serum is the liquid fraction of whole blood that is obtained after the blood is allowed to clot. For obtaining the serum, the clot is removed by centrifugation and the supernatant is collected. Plasma is the liquid component of blood in which blood cells are absent. However, plasma contains proteins and other constituents of whole blood in suspension. For obtaining a plasma sample, whole blood can be collected in anticoagulant-treated tubes (e.g. citrate- treated or EDTA-treated tubes). Cells are removed from the sample by centrifugation. Afterwards the supernatant (i.e. the plasma sample) is obtained. Plasma can be also obtained from whole blood by filtration or agglutination.

[0156] In a preferred embodiment, the sample is plasma sample. In an embodiment, the plasma sample is a plasma sample that has not been subjected to further protein purification steps or protein enrichment steps prior to determining the level of the biomarker(s) as referred to herein. In a preferred embodiment, the plasma sample to be tested has been obtained by providing a whole blood sample from the test subject in a container comprising an anticoagulant, separating the blood plasma from the cellular fractions of blood by centrifugation and obtaining the supernatant (i.e. the blood plasma). The level of the biomarker(s) is (are) determined in a portion of the obtained supernatant, i.e. in a portion of the obtained plasma, without subjecting the supernatant to further protein enrichment or purification steps prior to determining the level of the biomarker(s).

[0157] The anticoagulant can be any anticoagulant deemed appropriate. In an embodiment, the anticoagulant is EDTA, citrate or heparin, and, in particular, EDTA. The container comprising an anticoagulant is typically an anticoagulant treated tube, such as an EDTA- treated tube, a citrate-treated tube or a heparinized tube.

[0158] In another preferred embodiment, the sample is serum sample. In an embodiment, the serum sample is a serum sample that has not been subjected to further protein purification steps or protein enrichment steps prior to determining the level of the biomarker(s) as referred to herein. In a preferred embodiment, the serum sample to be tested has been obtained by providing a whole blood sample from the test subject in a container lacking anticoagulants, thereby allowing clot formation and separating the serum from the formed clot and obtaining the supernatant (i.e. the blood plasma). The level of the biomarker(s) is (are) determined in a portion of the obtained supernatant, i.e. in a portion of the obtained serum, without subjecting the supernatant to further protein enrichment or purification steps prior to determining the level of the biomarker(s).

[0159] Since it is envisaged that the serum or plasma sample has not been subjected to further protein enrichment steps or protein purification steps, it is to be understood that the sample is not an isolated exosome preparation. Further, it is envisaged that the sample is not essentially a cellular sample, or the like. For example, the blood sample is not a sample which has been enriched for blood cells, such as a sample of peripheral blood mononuclear cells.

[0160] The biomarkers

[0161] The assessment made in accordance with the present invention, typically, is based on the level of biomarker CLEC3B, and optionally, on the level of at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, CLEC3B, DNER, CFB, Ferritin and AREG. The term “biomarker”, as used herein, refers to a protein which serves as an indicator for a disease or physiological state as referred to herein. The biomarker may be a derivative of the polypeptide or protein, may be a fragment of the polypeptide or protein, or may be a complex of the polypeptide, e.g. an immunocomplex of the polypeptide or a protein comprising more than one polypeptide. Preferably, however, the biomarker is the full-length polypeptide (in particular, as expressed in humans). Also, in case the biomarker has an activity, e.g. a catalytic activity and / or an activating activity, e.g. on target cells, the biomarker may also be determined via said activity, e.g. in an enzymatic assay. It is to be understood that in the aforesaid cases, the analyte may represent the actual biomarker and has the same potential as an indicator for the respective medical condition as the biomarker would have. Preferred modes of determination and analytes for the biomarkers of the present description are described in the context of the respective biomarkers herein below. A biomarker according to the present invention need not necessarily correspond to one molecular species. As known by the skilled person, a polypeptide may comprise variant molecular species, e.g. translated from splice variants, glycosylation variants, and the like. In an embodiment, the variants share at least one determinable feature, e.g. an epitope or an activity.

[0162] The biomarkers to be determined in accordance with the present invention are as such known in the art. Moreover, methods for the determination of the biomarkers of the biomarkers are known to the skilled person as well. C-type lectin domain family 3 member B (CLEC3B) also referred to as Tetranectin is plasminogen kringle-4 binding protein in plasma and extracellular matrix that enhances proteolytic processes enabling tumor cells to invade and metastasize. The amino acid sequence of the human CLEC3B protein is known in the art and can be e.g. assessed via UniProt ID: P05452 (in particular Entry version 214). The protein level is, typically, decreased in samples from patients with colorectal cancer or a precancerous condition thereof (as compared to the reference).

[0163] SOX9 (transcription factor SOX9) is a transcription factor that recognizes the sequence CCTTGAG along with other members of the HMG-box class DNA-binding proteins. It acts during chondrocyte differentiation and, with steroidogenic factor 1, regulates transcription of the anti-Muellerian hormone (AMH) gene. The amino acid sequence of the human SOX9 is known in the art and can be e.g. assessed via UniProt ID: P48436 (in particular Entry version 223). The protein level is, typically, increased in patients with colorectal cancer or a precancerous condition thereof (as compared to the reference).

[0164] Delta and Notch-like epidermal growth factor-related receptor (DNER) is a neuron-specific transmembrane protein carrying extracellular EGF-like repeats. The amino acid sequence of the human DNER protein is known in the art and can be e.g. assessed via UniProt ID: Q8NFT8 (in particular Entry version 172). The protein is, typically, decreased in samples from patients with colorectal cancer or a precancerous condition thereof (as compared to the reference).

[0165] Complement factor B (CFB) is a protein involved in the alternative pathway of complement activation. Factor B circulates in the blood as a single chain polypeptide. Upon activation of the alternative pathway, it is cleaved by complement factor D yielding the non-catalytic chain Ba and the catalytic subunit Bb. The amino acid sequence of the human CFB protein is known in the art and can be e.g. assessed via UniProt ID: P00751 (in particular Entry version 265). The protein is, typically, increased in samples from patients with colorectal cancer or a precancerous condition thereof (as compared to the reference).

[0166] Amphiregulin, also known as AREG, is a member of the epidermal growth factor family. It is an autocrine growth factor as well as a mitogen for astrocytes, Schwann cells and fibroblasts. The protein interacts with the EGF / TGF-alpha receptor to promote the growth of normal epithelial cells, and it inhibits the growth of certain aggressive carcinoma cell lines. The amino acid sequence of the human Amphiregulin protein is known in the art and can be e.g. assessed via UniProt ID: P15514 (in particular Entry version 206). The protein is, typically, increased in samples from patients with colorectal cancer or a precancerous condition thereof (as compared to the reference). Ferritin is a universal intracellular protein that stores iron and releases it in a controlled fashion. It is a hollow globular protein comprising 24 subunits. Typically it has internal and external diameters of about 8 and 12 nm. In humans, the protein comprises subunits of two types, light (L) and heavy (H). The amino acid sequence of the light and heavy subunits can be accessed via Uniprot (heavy subunit: UniProt ID: P02794 (in particular Entry version 255), light subunit: UniProt ID: P02792(in particular Entry version 235)). In an embodiment, the level of the light subunit is determined. The protein is, typically, decreased in samples from patients with colorectal cancer or a precancerous condition thereof (as compared to the reference).

[0167] Carcinoembryonic antigen (CEA) is elevated in various malignancies such as colorectal cancer, medullary thyroid cancer, breast cancer, mucinous ovarian cancer, etc. CEA belongs to the immunoglobulin family called CEA-related cell adhesion molecules (CEACaMs). CEA is closely associated with various functions of endothelial cells, including adhesion, proliferation, and migration of cells both in vivo and in vitro. CEA is, typically, increased in samples from patients with colorectal cancer or a precancerous condition thereof (as compared to the reference). The level of the marker can be e.g. determined with Roche’s Elecsys® CEA assay. anti-p53 is an autoantibody to p53 (anti-p53 antibody, see e.g. WO2017153336A1). Anti- p53 autoantibodies (anti-p53 (ap53)) are found in blood samples during tumorigenesis and are associated with several types of cancer, including breast, lung, colorectal, prostate, nonmelanoma, and melanoma skin and gastric cancer. A meta-analysis anti-p53 autoantibody in blood as a diagnostic biomarker for colorectal cancer has been provided by Liu at al. Scand J Immunol. 2020; 91 :el2829. The biomarker is, typically, increased in samples from patients with colorectal cancer or a precancerous condition thereof (as compared to the reference). The biomarker can be e.g. determined by using Elecsys® anti-p53 assay. Moreover, WO2017153336A1 describes assays to determine the level of the marker as well as suitable capture and detection agents. In accordance with the present invention, it is envisaged to use the detection agents and the detection method as described in WO2017153336A1 for determining the level of ap53.

[0168] Step a) of the method of the present invention comprises the determination of the level of at least one further biomarker selected from the group consisting of CEA, anti-p53, CLEC3B, DNER, CFB, Ferritin and AREG. It is to be understood that the level of the protein biomarker(s) is determined, rather than of the mRNA. Preferred combination of biomarkers in accordance with the methods, uses, devices, kits and databases of the present invention are those combinations tested in the Examples section (see Examples 6 to 9).

[0169] In preferred embodiment, the levels of the biomarkers CLEC3B and CEA are determined (see e.g. Table 9a, 9b, 10a and 10b in the Examples section).

[0170] In another preferred embodiment, the levels of the biomarkers CLEC3B and ap53 are determined (see e.g. Table 9a, 9b, 10a and 10b).

[0171] In yet another preferred embodiment, the levels of the biomarkers CLEC3B and SOX9 are determined (see e.g. Table 9a, 9b).

[0172] In yet another preferred embodiment, the levels of the biomarkers CLEC3B and DNER are determined (see e.g. Table 9a, 9b and 10a).

[0173] In yet another preferred embodiment, the levels of the biomarkers CLEC3B and Ferritin are determined (see e.g. Table 9a and 9b).

[0174] In yet another preferred embodiment, the levels of the biomarkers CLEC3B and CFB are determined (Table 9a, 9b, 10a and 10b).

[0175] In yet another preferred embodiment, the levels of the biomarkers CLEC3B and AREG are determined (Table 9a, 10a and 10b).

[0176] In yet another preferred embodiment, the levels of the biomarkers CLEC3B, ap53 and CEA are determined (see e.g. Example 9, Table I la and 1 lb).

[0177] The determination of biomarker levels

[0178] Step a) of the method of the present invention comprises the determination of the level of the biomarker CLEC3B, and optionally, the level of at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, Ferritin and AREG. It is to be understood that the level of the protein biomarker(s) is determined, rather than the level of the mRNA.

[0179] Typically, the level of a biomarker can be determined by determining a complex of the analyte with a detection compound, in particular an antibody or fragment thereof, i.e. in an immunoassay. Said determining of a complex of the analyte may be performed in any format deemed appropriate by the skilled person, in particular a sandwich, competition, or other assay format. Said assays will develop a signal which is indicative for the level of a biomarker. Thus, determining may include micro-plate ELISA-based methods, fully- automated or robotic immunoassays (available, e.g., from Roche). Suitable measurement methods may also include precipitation (particularly immunoprecipitation), electrochemiluminescence (electro-generated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), fluorescent immunoassay (FIA), electrochemiluminescence sandwich immunoassays (ECLIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA), scintillation proximity assay (SPA), turbidimetry, nephelometry, latex-enhanced turbidimetry or nephelometry, or solid phase immune tests. Further methods known in the art such as gel electrophoresis, 2D gel electrophoresis, SDS polyacrylamid gel electrophoresis (SDS-PAGE) or Western Blotting. More typically, techniques particularly envisaged for determining the biomarkers referred to herein are described herein below.

[0180] The biomarker anti-p53 is an autoantibody against p53. Typically, a detection agent that specifically binds an anti-p53 antibody is a polypeptide, or fragment thereof, which is capable of being bound by the anti-p53 antibodies present in the sample. In a preferred embodiment, the polypeptide comprises the sequence of the human p53, or a fragment thereof which is capable of being bound by the anti-p53 antibodies present in the sample. Preferably, said fragment comprises an epitope that is bound by a significant percentage of those antibodies in a sample that also bind to the full-length p53 polypeptide. Said p53 polypeptide, or fragment thereof, may further comprise a detectable label. The sequence of the human p53 is disclosed in WO2017153336A1. Typically, the presence of the marker in the sample is indicative for a subject who suffers from colorectal cancer or a precancerous condition thereof.

[0181] The determination of a biomarker as set forth herein may comprise mass spectrometry (MS) which is carried out after the separation step (e.g. by LC or HPLC). Mass spectrometry as used herein encompasses all techniques which allow for the determination of the molecular weight (i.e. the mass) or a mass variable corresponding to a compound, i.e. a biomarker, to be determined in accordance with the present invention. Preferably, mass spectrometry as used herein relates to GC-MS, LC-MS, direct infusion mass spectrometry, FT-ICR-MS, GEMS, HPLC-MS, quadrupole mass spectrometry, any sequentially coupled mass spectrometry such as MS-MS or MS-MS-MS, ICP-MS, Py-MS, TOF or any combined approaches using the aforementioned techniques. How to apply these techniques is well known to the person skilled in the art. Moreover, suitable devices are commercially available. More preferably, mass spectrometry as used herein relates to LC-MS and / or HPLC-MS, i.e. to mass spectrometry being operatively linked to a prior liquid chromatography separation step. Preferably, the mass spectrometry is tandem mass spectrometry (also known as MS / MS). Tandem mass spectrometry, also known as MS / MS involves two or more mass spectrometry step, with a fragmentation occurring in between the stages. In tandem mass spectrometry two mass spectrometers in a series connected by a collision cell. The mass spectrometers are coupled to the chromatographic device. The sample that has been separated by a chromatography is sorted and weighed in the first mass spectrometer, then fragmented by an inert gas in the collision cell, and a piece or pieces sorted and weighed in the second mass spectrometer. The fragments are sorted and weighed in the second mass spectrometer. Identification by MS / MS is more accurate. In an embodiment, the level of a biomarker as set forth herein is determined by Proximity Extension Assay. The biomarker is detected by a matched pair of antibodies that are conjugated to partially complementary oligonucleotides. When both antibodies bind to the biomarker, a partially double-stranded nucleic acid is formed by the hybridization of the partially complementary oligonucleotides. The level of said partially double-stranded nucleic acid can be measured by quantitative real-time PCR or next generation sequencing (see e.g. (Assarsson et al. PLoS One 2014 Apr 22;9(4), doi: 10.1371 / joumal. pone.0095192). The determined level indicates the level of the biomarker.

[0182] In another embodiment, the level of a biomarker as set forth herein is determined by Proximity Ligation Assay which is similar to the Proximity Extension Assay. In this assay, the oligonucleotides are ligated by a ligase to a closed, circle DNA template that can be used for rolling-circle amplification (RCA).

[0183] In step b) of the method of the present invention, colorectal cancer or a precancerous condition thereof is assessed. Typically, the assessment (such as the diagnosis, differentiation, or identification) is based on the level of CLEC3B and, optionally, the level of the at least one further biomarker as set forth herein above. In an embodiment, colorectal cancer or a precancerous condition thereof is assessed by comparing the determined level of CLEC3B to a reference level for this marker, and optionally, by comparing the determined level of the at least one further biomarker to a reference level for the at least one further biomarker (e.g. the level of CEA to a reference level of CEA, or the level of anti-p53 to a reference level of antib-p53 etc.) Alternatively, the colorectal cancer or a precancerous condition thereof is assessed by calculating a score for assessing colorectal cancer or the precancerous condition thereof based on the determined level of CLEC3B and optionally based on the level of the at least one further biomarker.

[0184] As set forth above, it is also envisaged to calculate a score based on the level(s) of the biomarker(s), in particular a single score. The assessment of colorectal cancer or a precancerous condition thereof is then made based on the score. The assessment may include a comparison of the score to a reference score which allows for the assessment of colorectal cancer or a precancerous condition thereof as referred to herein. The calculated score, in an embodiment combines, information on the level(s) of the biomarker(s). For example, if two biomarkers are determined, the score is calculated based on the levels of the two markers. In the score, the biomarkers may be weighted in accordance with their contribution to the establishment of the differentiation, wherein the weighting factor of the individual biomarkers may be different. The calculated score combines information on the level(s) of the biomarkers determined in step a) of method of the present invention, (e.g. of two or three biomarkers). Moreover, in the score, the biomarkers are, preferably, weighted in accordance with their contribution to the establishment of the assessment. Thus, the values for the individual markers are weighted and the weighted values are used for calculating the score. Suitable coefficients (weights) can be determined by the skilled person without further ado, e.g. by training the coefficients using the corresponding biomarker information from a representative reference population comprising controls and subjects suffering from the respective medical condition (e.g. CRC or a precancerous condition thereof). When using absence or presence information of a biomarker (e.g. anti-p53 antibody), the value instead of a biomarker level value could be a predetermined value representative for absence or presence, wherein the values for absence or presence are different (e.g. the value for presence is higher than for absence). For example, for the absence of the biomarker a value (instead of a biomarker level value) of 0 could be assigned and for the presence a value of 1 (instead of a biomarker level value) could be assigned. A score can also be calculated from a decision tree or a set (ensemble) of decision trees that has been trained the two biomarkers. Alternatively, a score can be also calculated by any other supervised and / or unsupervised machine-learning methods used for classification. This includes all kinds of suited artificial neural networks, support vector machines, k-nearest neighbor, boosting and bagging regression and tree-based models. Further, such a score could be created by statistical or closed-form methods like linear and quadratic discriminant analysis or likelihood ratio functions. Based on the combination of biomarkers applied in the method of the invention, the weight of an individual biomarker as well as the structure of machine learning model or mathematical formula may be different. In an embodiment, to calculate a score, the levels of the biomarkers determined may be linearly combined (e.g. Score = c0+ c * [Biomarker + c2* [Biomarker2] + •••), preferably with corresponding coefficients (c1;c2, ...) and intercept (c0). In preferred embodiments, the levels of the biomarkers ([Biomarker , [Biomarker2], ...) are log transformed (e.g. natural logarithm, log 2 or log 10, preferably log 10) for the linear combination to a score. The output of such a linear combination may be directly used as score. Alternatively or additionally, the output of this linear combination may be used as input to a mathematical transformation (e.g. a sigmoid transformation such as ( / (%) = 1 / (1 + exp(— %)))) providing a risk score, which maps the output range of the score to 0-1.

[0185] As set forth above, the score can be regarded as a classifier parameter for assessing colorectal cancer or a precancerous condition thereof as set forth herein. In particular, it enables the person who provides the assessment based on a single score. The reference score is preferably a value, in particular a cut-off value which allows for assessing a subject with suspected infection as set forth herein. Preferably, the reference is a single value. Thus, the person does not have to interpret the entire information on the levels of the individual biomarkers. Using a scoring system as described herein, advantageously, values of different dimensions or units for the biomarkers may be used since the values will be mathematically transformed into the score. Accordingly, e.g. values for absolute concentrations may be combined in a score with peak area ratios. The reference score to be applied may be elected based on the desired sensitivity or the desired specificity. How to elect a suitable reference score is well known in the art.

[0186] The score as referred to herein is not limited to the biomarkers specifically mentioned herein. For example, the score can also take into account further biomarkers and / or parameters such as the age and / or sex of the subject to be tested.

[0187] The term “reference” or “reference level”, as used herein, relates to a value, e.g. a level or any value derived therefrom, e.g. a score, which can be correlated to a medical condition and, in an embodiment, which allows for the assessment of the invention to be made, in a further embodiment enables allocation of a subject into either a group of subjects suffering from a disease or condition, or a group of subjects which do not suffer from said disease or condition. Such a reference can be a threshold value, e.g. a threshold level, which separates these groups from each other. For example, the reference may be a value which allows for allocation of a subject into a group of subjects suffering from colorectal cancer or a precancerous condition thereof, or not suffering from colorectal cancer or a precancerous condition thereof. The reference may, however, also be a reference range, e.g., in an embodiment, a range of values for which colorectal cancer or a precancerous condition thereof can be excluded. Furthermore, the reference may be a value calculated from the aforesaid values, e.g. from the levels of two or more biomarkers, in an embodiment to provide a score. A suitable reference separating the two groups can be provided without further ado e.g. by the statistical tests referred to herein elsewhere based on values of biomarkers from suitable reference groups as specified herein below. As the skilled person understands, it may not always be possible, although particularly envisaged, to provide a reference unambiguously allocating each and every possible value of a biomarker to one of the aforesaid groups; thus, there may be a range of values for which a clear assessment cannot be provided. In an embodiment, however, as indicated above, a reference enables the assessment to be made for each and every value of a biomarker or set of biomarkers which may be measured. As the skilled person understands, the specific value of a reference may depend on the assessment intended and on parameters thereof; thus, the reference value for assessing colorectal cancer or a precancerous condition thereof may typically be different from the reference value for ruling in or ruling out colorectal cancer or a precancerous condition thereof.

[0188] As indicated herein above, a reference may in particular be derived from at least one reference group, the term "reference group" relating to a group of subjects with known status with regard to the assessment. Thus the reference group may e.g. be a group of subjects for which it is known whether they suffer from colorectal cancer or a precancerous condition thereof or not. The population of subjects in a reference group in an embodiment comprises a plurality of subjects, e.g. at least 5, 10, 50, 100, 1,000, or 10,000 subjects. Typically, the subject to be diagnosed and the subjects of the said reference group are of the same species. The reference applicable for an individual subject may vary depending on various physiological parameters such as age, gender, or subpopulation. As is understood by the skilled person, the prevalence of colorectal cancer or a precancerous condition thereof in the population is low, thus, a reference may be derived also from the average population. Assuming that contribution of actually afflicted subjects is low, such an average population reference group may be treated as a reference group known not to suffer from colorectal cancer or a precancerous condition thereof; in an embodiment, in such case, the size of the reference group is sufficiently high, e.g. at least 100, in a further embodiment at least 1000, in a further embodiment at least 10000 subjects. In view of the description herein, the skilled person understands that a reference group may, in principle, also be a mixed population of subjects with regard to colorectal cancer or a precancerous condition thereof, provided that the status of each member of said mixed population with regards to colorectal cancer or a precancerous condition thereof is or becomes known before deriving a reference from such group. In an embodiment, the reference cohort is a cohort that is similar to the cohort tested in the Examples section (see Example 1).

[0189] Reference levels can, in principle, be calculated for a cohort of subjects based on the average or mean values for a given parameter such as biomarker level by applying standard statistically methods. In particular, accuracy of a test such as a method aiming to diagnose an event, or not, is best described by its receiver-operating characteristics (ROC) (see especially Zweig 1993, Clin. Chem. 39:561-577). The ROC graph is a plot of all of the sensitivity / specificity pairs resulting from continuously varying the decision threshold over the entire range of data observed. The clinical performance of a diagnostic method depends on its accuracy, i.e. its ability to correctly allocate subjects to a certain prognosis or diagnosis. The ROC plot indicates the overlap between the two distributions by plotting the sensitivity versus 1 -specificity for the complete range of thresholds suitable for making a distinction. On the y-axis is sensitivity, or the true-positive fraction, which is defined as the ratio of number of true-positive test results to the product of number of true-positive and number of false-negative test results. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup. On the x-axis is the false-positive fraction, or 1 -specificity, which is defined as the ratio of number of false-positive results to the product of number of true-negative and number of falsepositive results. It is an index of specificity and is calculated entirely from the unaffected subgroup. Because the true- and false-positive fractions are calculated entirely separately, by using the test results from two different subgroups, the ROC plot is independent of the prevalence of the event in the cohort. Each point on the ROC plot represents a sensitivity / - specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap in the two distributions of results) has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity). The theoretical plot for a test with no discrimination (identical distributions of results for the two groups) is a 45° diagonal line from the lower left comer to the upper right corner. Most plots fall in between these two extremes. If the ROC plot falls completely below the 45° diagonal, this is easily remedied by reversing the criterion for "positivity" from "greater than" to "less than" or vice versa. Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test. Dependent on a desired confidence interval, a threshold can be derived from the ROC curve allowing for the diagnosis or prediction for a given event with a proper balance of sensitivity and specificity, respectively. Accordingly, the reference to be used for the aforementioned method of the present invention, i.e. a threshold which allows to discriminate between subjects suffering from a disease or not suffering from disease can be generated, usually, by establishing a ROC for said cohort as described above and deriving a threshold level therefrom. Dependent on a desired sensitivity and specificity for a diagnostic method, the ROC plot allows deriving suitable thresholds. It will be understood that an optimal sensitivity may be desired for excluding a subject from suffering from colorectal cancer or a precancerous condition thereof (i.e. a rule-out), whereas an optimal specificity may be envisaged for a subject to be assessed as suffering from colorectal cancer or a precancerous condition thereof (i.e. a rule-in).

[0190] As will be understood by the skilled person, the reference level shall be suitable for the assessment are referred to herein, such as for the diagnosis of colorectal cancer or a precancerous condition thereof, or the differentiation. Thus, the reference level is, in an embodiment, a predetermined value for the level of biomarker which allows for assessing colorectal cancer or a precancerous condition thereof.

[0191] In accordance with the present invention, colorectal cancer or a precancerous condition thereof is typically assessed based on the comparison and / or the calculation made in step b). The term "assessing" has been specified herein above. Typically, the assessment may in particular be based on the status of the reference group(s) and the reference derived therefrom. As the skilled person understands, a biomarker may be decreased or increased in an afflicted subject compared to a control subject. Thus, in case the reference is derived from a group of subjects known to be afflicted with the disease or condition, a biomarker value essentially identical to the reference in an embodiment leads to an assessment of the subject under investigation being afflicted with the disease or condition, and a value different, in an embodiment significantly different, from said reference in a further embodiment leads to an assessment of the subject under investigation not being afflicted with the disease or condition. Conversely, in case the reference is derived from a group of subjects known not to be afflicted with the disease or condition, a biomarker value essentially identical to the reference in an embodiment leads to an assessment of the subject under investigation not being afflicted with the disease or condition, and a value different, in an embodiment significantly different, from said reference in a further embodiment leads to an assessment of the subject under investigation being afflicted with the disease or condition.

[0192] In a preferred embodiment, the reference may be a threshold value; such a threshold value may be derived from a reference group known to suffer from the disease or condition. In another preferred embodiment, such a threshold value may be derived from a reference group known not to suffer from the disease of condition. In a preferred embodiment, a threshold value is derived from a reference group known to suffer from the disease or condition and from a reference group known not to suffer from the disease of condition, e.g. from a ROC plot as specified herein above and as detailed herein in the Examples. Typically, in case a biomarker value of a subject under investigation exceeds the aforesaid threshold reference toward the values of the reference group known to suffer from the disease or condition, it will be assumed that the subject is likely to suffer from the disease of condition; and in case a biomarker value of a subject under investigation exceeds the aforesaid threshold toward the values of the reference group known to not suffer from the disease or condition, it will be assumed that the subject is likely not to suffer from the disease or condition. Thus, depending on the specific biomarker and its correlation with a disease or condition, values found in a sample which are higher than or equal to a threshold may be indicative for the presence of a medical condition while those being lower may be indicative for the absence of the medical condition; or, it may also be that values found in a sample to be investigated which are lower or identical than the threshold are indicative for the presence of a medical condition while those being higher are indicative for the absence of the medical condition.

[0193] As the skilled person understands in view of the description herein, the above applies mutatis mutandis to a score, which may incorporate levels of more than one biomarker, in an embodiment all biomarkers. A reference score may be derived from a reference group known to suffer from the disease or condition, or from a reference group known not to suffer from the disease or condition. Alternatively, reference score may be derived from a mixed population comprising subjects known to suffer from the disease or condition and subjects known not to suffer from the disease or condition.

[0194] In an embodiment, a calculated score which is larger than the reference score indicates that the subject is likely to suffer from the disease or condition. Also, a calculated score which is lower than the reference score indicates that the subj ect is not likely to suffer from the disease or condition. As specified herein above, a reference score may in particular be calculated by the same mathematical operations applied to calculate a score, but using values from one or more reference group(s). The reference score also may be e.g. a threshold score or a reference score range. As the skilled person will understand as well, the assessment following from the comparison of a score to a reference score will depend on the specificities of score calculation; thus, whether a score above or below a threshold score is indicative of colorectal cancer or a precancerous condition thereof will depend on the specific way of calculating the score. E.g. in the exemplary score calculated according to the Examples, a score higher than the cutoff score is indicative of colorectal cancer or a precancerous condition thereof.

[0195] In an embodiment of the present invention, the assessment is made with a pre-selected sensitivity or specificity. Accordingly, the reference is a level which allows the assessment, such as the diagnosis, the differentiation, the identification of a subject with the preselected sensitivity or specificity.

[0196] Preferred diagnostic algorithms

[0197] As regards to the diagnosis as referred to herein, an increased level of the biomarker CLEC3B as compared to the reference level is, typically, indicative for a subject who is not likely to suffer from colorectal cancer or a precancerous condition thereof, whereas a decreased level is indicative for a subject who is likely to suffer from colorectal cancer or a precancerous condition thereof, wherein said reference level in a preferred embodiment is a threshold value as specified herein above. This applies, e.g., if the reference level is a predetermined value for the level of biomarker which allows for diagnosing colorectal cancer or a precancerous condition thereof (in particular advanced adenoma).

[0198] In another embodiment, the reference level is a predetermined value for the level of biomarker which allows for ruling out colorectal cancer or a precancerous condition thereof. Typically, an increased level of CLEC3B as compared to a reference level is indicative for a subject who does not suffer from colorectal cancer or a precancerous condition thereof. Thus, the diagnosis can be ruled out. In another embodiment, the reference level is a predetermined value for the level of biomarker which allows for ruling in colorectal cancer or a precancerous condition thereof. Typically, a decreased level of CLEC3B as compared to a reference level is indicative for a subject who does suffer from colorectal cancer or a precancerous condition thereof, wherein said reference level in a preferred embodiment is a threshold value as specified herein above. Thus, the diagnosis can be ruled in.

[0199] In another embodiment, the reference level is a predetermined value for the level of biomarker which allows for

[0200] A. the differentiation whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, or

[0201] B. the differentiation whether a subject i) suffers from colorectal cancer or ii) does not suffer from colorectal cancer, wherein said reference level in a preferred embodiment is a threshold value as specified herein above.

[0202] With respect to the differentiation under A., a decreased level of the biomarker CLEC3B as compared to the reference level is indicative for a subject who suffers from colorectal cancer or advanced adenoma, whereas an increased level is indicative for a subject who does not suffer from colorectal cancer or advanced adenoma. With respect to the differentiation under B., a decreased level of the biomarker CLEC3B as compared to the reference level is indicative for a subject who suffers from colorectal cancer, whereas an increased level is indicative for a subject who does not suffer from colorectal cancer.

[0203] In another embodiment, the reference level is a predetermined value for the level of biomarker which allows for identifying a subject who is eligible to one or more diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof. Typically, a decreased level of the biomarker CLEC3B as compared to the reference level is indicative for a subject who is eligible to one or more diagnostic measures, whereas an increased level is indicative for a subject who is not eligible to said one or more diagnostic measures, wherein said reference level in a preferred embodiment is a threshold value as specified herein above.

[0204] The biomarkers Ferritin and DNER are (as CLEC3B) decreased in colorectal cancer or a precancerous condition thereof. Thus, a level of CLEC3B below the reference level for CLEC3B in combination with a level of the at least one further biomarker selected from Ferritin and DNER below the reference level for said marker indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof, wherein said reference levels in a preferred embodiment are threshold values as specified herein above. For example, a decreased level of both markers indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof, wherein an increased level of both markers (as compared to the reference levels) indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof.

[0205] In contrast, the biomarkers CEA, anti-p53, CFB and AREG are SOX9 are increased in colorectal cancer or a precancerous condition thereof. Thus, a particular assessment, such as the diagnosis can be made if the level of CLEC3B is decreased as compared to the reference level and the level of the at least one further biomarker (i.e. CEA, anti-p53, CFB and AREG are SOX9) is increased as compared to the respective reference level, wherein said reference levels in a preferred embodiment are threshold values as specified herein above.

[0206] This is exemplified for the diagnosis of colorectal cancer or a precancerous condition thereof Preferably, the following applies, if the biomarkers CLEC3B and DNER are determined in the method of the present invention: a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of DNER above the reference level for DNER indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, whereas a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of DNER below the reference level for DNER indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof, wherein said reference levels in a preferred embodiment are threshold values as specified herein above.

[0207] Preferably, the following applies, if the biomarkers CLEC3B and Ferritin are determined in the method of the present invention: a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of Ferritin above the reference level for Ferritin indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, whereas a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of Ferritin below the reference level for Ferritin indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof, wherein said reference levels in a preferred embodiment are threshold values as specified herein above.

[0208] Preferably, the following applies, if the levels of the biomarkers CLEC3B and CEA are determined: a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of CEA below the reference level for CEA indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, whereas a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of CEA above the reference level for CEA indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof, wherein said reference levels in a preferred embodiment are threshold values as specified herein above.

[0209] Preferably, the following applies, if the levels of the biomarkers CLEC3B and anti-p53 are determined: a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of anti-p53 below the reference level for anti-p53 indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, whereas a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of anti-p53 above the reference level for anti-p53 indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof, wherein said reference levels in a preferred embodiment are threshold values as specified herein above. Preferably, the following applies, if the levels of the biomarkers CLEC3B and CFB are determined: a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of CFB below the reference level for CFB indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, whereas a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of CFB above the reference level for CFB indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof, wherein said reference levels in a preferred embodiment are threshold values as specified herein above.

[0210] Preferably, the following applies, if the levels of the biomarkers CLEC3B and AREG are determined: a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of AREG below the reference level for AREG indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, whereas a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of AREG above the reference level for AREG indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof, wherein said reference levels in a preferred embodiment are threshold values as specified herein above.

[0211] Preferably, the following applies, if the levels of the biomarkers CLEC3B and SOX9 are determined: a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of SOX9 below the reference level for SOX9 indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, whereas a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of SOX9 above the reference level for SOX9 indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof, wherein said reference levels in a preferred embodiment are threshold values as specified herein above.

[0212] Preferably, the following applies, if the levels of the biomarkers CLEC3B, ap53 and CEA are determined: a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of CEA above the reference level for CEA and a level of ap53 above the reference level for ap53 indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of CEA below the reference level for CEA and a level of ap53 below the reference level for ap53 indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, wherein said reference levels in a preferred embodiment are threshold values as specified herein above. If the assessment is the identification of a subject who is eligible to one or more diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof, the diagnostic algorithm in principle correspond to the above algorithm, with the proviso that a subject who is likely (or who is not likely) to suffer from colorectal cancer or a precancerous condition thereof is a subject who is eligible (or who is not eligible) to said one or more diagnostic measures.

[0213] For example, the following applies if the biomarkers CLEC3B and DNER are determined: a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of DNER below the reference level for DNER indicates that the subject is eligible to said one or more diagnostic measures, whereas a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of DNER above the reference level for DNER indicates that the subject is not eligible to said one or more diagnostic measures, wherein said reference levels in a preferred embodiment are threshold values as specified herein above.

[0214] In connection with the rule out, a level of CLEC3B which is above the reference level for ruling out colorectal cancer or the precancerous condition, in combination with a level of CEA, anti-p53, SOX9, CFB or AREG which is below the reference level for the respective marker for ruling out colorectal cancer or the precancerous condition, is indicative for the rule out of colorectal cancer or the precancerous condition, wherein said reference levels in a preferred embodiment are threshold values as specified herein above.

[0215] In connection with the rule out, a level of CLEC3B which is above the reference level for ruling out colorectal cancer or the precancerous condition, in combination with a level of Ferritin and DNER which is above the reference level for the respective marker for ruling out colorectal cancer or the precancerous condition, is indicative for the rule out of colorectal cancer or the precancerous condition, wherein said reference levels in a preferred embodiment are threshold values as specified herein above.

[0216] Alternatively, a subject suffers from CRC or advanced adenoma or is eligible to one or more further diagnostic measures if a) the level of CLEC3B is decreased as compared to the reference, b) the presence of anti-p53 antibodies is detected in the sample, or c) the level of CLEC3B is increased as compared to the reference and anti-p53 antibodies are detected in the sample.

[0217] The result of the assessment in the last step of the method of the present invention, in an embodiment, is a statement concerning the status of the subject with regards to colorectal cancer or a precancerous condition thereof. Said result may be implicit, e.g. by juxtaposing the value determined in the sample to one or more reference(s), which may be further evaluated, e.g. by a medical practitioner. The result may, however, also be explicit, e.g. by annunciating that the comparison suggests a specific status with regards to colorectal cancer or a precancerous condition thereof. Thus, the method may further comprise a step of annunciating the result of the assessment. Alternatively or in addition, the result of the assessment may also be used in further assessments.

[0218] Additional steps of the method of the present invention (therapeutic and diagnostic methods).

[0219] The result of the assessment is step b) of the method of the present invention may form the basis for a further diagnostic measure for diagnosing colorectal cancer or a precancerous condition thereof in the test subject or a recommendation of said further diagnostic measure. Thus, in case a subject is diagnosed to be likely suffer from colorectal cancer or a precancerous condition thereof, a suitable diagnostic assessment is recommended or initiated in order to confirm the assessment made by the method of the present invention (and thus e.g. to diagnose that the subject suffers from colorectal cancer or a precancerous condition thereof, or not). Preferably, the diagnostic measures is selected from Colonoscopy, Sigmoidoscopy, CT CoIonography (Virtual Colonoscopy), Double-Contrast Barium Enema, Capsule Endoscopy, a fecal immunochemical test and a stool DNA test. In particular, the further diagnostic measure is Colonoscopy. Thus, the diagnosis as referred to herein can be based on the biomarker(s) and images obtained by colonoscopy from the test subject. In the absence of colorectal cancer or a precancerous condition on said images, the test subject does not suffer from colorectal cancer or a precancerous condition. In the presence of colorectal cancer or a precancerous condition on said images, the test subject suffers from colorectal cancer or a precancerous condition.

[0220] The result of the assessment of the method of the present invention may form the basis for a treatment of the subject or a decision thereon. In particular, in case a subject is diagnosed to suffer from colorectal cancer or a precancerous condition thereof, a suitable therapeutic measure is recommended or initiated.

[0221] Typically, the therapeutic measure shall aim to treat colorectal cancer or a precancerous condition thereof. At an early stage, colorectal cancer may be removed during a colonoscopy using one of several techniques, including endoscopic mucosal resection or endoscopic submucosal dissection. The same applies to advanced adenomas. For subjections with localized such as stage 0 or I, the preferred treatment is complete surgical removal with adequate margins, with the attempt of achieving a cure. The procedure of choice is a partial colectomy. In patients suffering from stage II CRC, stage III CRC or stage IV CRC, the therapeutic measure is, preferably, the administration of a medicament selected from capecitabine, fluorouracil, irinotecan and oxaliplatin. Moreover, an antiangiogenic drugs such as bevacizumab and epidermal growth factor receptor inhibitors such as aflibercept, cetuximab and panitumumab can be administered. Also, the patient might be subjected to radiation therapy.

[0222] Computer-implemented method

[0223] Moreover, the present invention relates to a computer-implemented method for assessing colorectal cancer or a precancerous condition thereof, comprising a) receiving, at a processing unit, a value for the level of the biomarker CLEC3B is a sample (such as blood, serum or plasma) from a subject, and, optionally at least one further value for the level of at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, Ferritin and AREG in a sample, b) comparing, by said processing unit, the value or values received in step a) to a reference or to references and / or calculating, by said processing unit, a score for assessing colorectal cancer or the precancerous condition thereof, wherein the score is based on the value or values received in step a), and c) assessing, preferably, by said processing unit, colorectal cancer or the precancerous condition thereof based on the result of step b).

[0224] Terms, such as “computer-implemented”, "receiving" and “sample” have been defined above. The definitions apply accordingly. Moreover, preferred references, scores and diagnostic algorithms have been disclosed in connection with the method for assessing colorectal cancer or a precancerous condition thereof.

[0225] Moreover, the present invention relates to a database comprising one or more stored references for the biomarker CLEC3B and, optionally one or more stored references for at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, Ferritin and AREG. Preferably, the one or more stored references allow for assessing colorectal cancer or a precancerous condition thereof.

[0226] The term “database” has been defined above. The definition applies accordingly.

[0227] Moreover, the present invention relates to a device for assessing colorectal cancer or a precancerous condition thereof, said device comprising: a) at least one measuring unit for determining a level of the biomarker CLEC3B and, optionally, a level of at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, Ferritin and AREG in a sample (such as blood, serum or plasma) from a subject, said at least one measuring unit comprising at least one detection agent for the biomarker CLEC3B and, optionally, at least one detection agent for the biomarker CEA, at least one detection agent for the biomarker anti-p53, at least one detection agent for the biomarker CLEC3B, at least one detection agent for the biomarker Ferritin, at least one detection agent for the biomarker DNER, at least one detection agent for the biomarker CFB and / or at least one detection agent for the biomarker AREG, and b) an evaluation unit operably linked to the measuring unit, said evaluation unit comprising a data processor comprising instructions for i) carrying out a comparison of the level of the biomarker CLEC3B and, optionally, of the level of said at least one further biomarker to a reference or references and / or for ii) carrying out a calculation of a score for assessing colorectal cancer or a precancerous condition thereof, wherein the score is calculated based on the level of CLEC3B and, optionally, on the level(s) of the at least one further biomarker.

[0228] The term “device” has been defined herein above. The definition applies accordingly.

[0229] The measuring unit, herein also referred to as “analyzing unit”, typically, comprises at least one reaction zone having a first detection agent for the first biomarker and a second detection agent for the second biomarker. The device may comprise one or more further detection agent(s) for one or more further, optional biomarker(s). The detection agent may be comprised in immobilized form on a solid support or carrier which is to be contacted to the sample. The detection agent may, however, also be comprised e.g. in liquid form in the device, e.g. as a stock solution, in particular in case the biomarker is determined in an activity assay. Moreover, in the reaction zone, it is in an embodiment possible to apply conditions which allow for the specific binding of the detection agent(s) to the biomarkers comprised in the sample and / or for a catalytic reaction to occur. The reaction zone may either allow directly for sample application or it may be connected to a loading zone where the sample is applied. In the latter case, the sample can be actively or passively transported via a connection between the loading zone and the reaction zone to the reaction zone. Moreover, the reaction zone shall be also connected to a detector. The connection shall be such that the detector can detect the result of a detection reaction, e.g. the binding of the biomarkers to their detection agents or an enzymatic reaction. Suitable detectors depend on the techniques used for measuring the presence or level of the biomarkers. For example, for optical detection, transmission of light may be required between the detector and the reaction zone while for electrochemical determination a fluidal connection may be required, e.g., between the reaction zone and an electrode. The detector shall be adapted to allow determination of the level of the biomarker(s). The determined level can be subsequently transmitted to the evaluation unit. The evaluation unit comprises at least one data processor, which may also be referred to as a data processing unit, such as a computer, with an implemented algorithm for determining the level present in the sample. Appropriate data processing units are known in the art and include in particular a Central Processing Unit (CPU), a Graphics Processing Units (GPU), an Application Specific Integrated Circuit (ASIC), a Tensor Processing Unit (TPU), a field- programmable gate array (FPGA), and other data processing units know in the art. A data processor may e.g. be a general purpose computer or a portable computing device. It should also be understood that multiple computing devices may be used together, such as over a network or other methods of transferring data, for performing one or more steps of the methods disclosed herein. Exemplary computing devices include desktop computers, laptop computers, personal data assistants (“PDA”), cellular devices, smart or mobile devices, tablet computers, servers, and the like. In general, a data processing unit comprises a processor capable of executing a plurality of instructions (such as a program of software).

[0230] The evaluation unit, typically comprises or has access to a memory. A memory is a computer readable information storage medium and may comprise a single storage device or multiple storage devices, located either locally with the computing device or accessible to the computing device across a network, for example. Computer-readable media may be any available media that can be accessed by the computing device and includes both volatile and non-volatile media. Further, computer readable-media may be one or both of removable and non-removable media. By way of example, and not limitation, computer-readable media may comprise computer storage media. Exemplary computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or any other memory technology, CD- ROM, Digital Versatile Disk (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used for storing a plurality of instructions capable of being accessed by the computing device and executed by the processor of the computing device. The evaluation unit, in an embodiment, further comprises a database as specified herein above.

[0231] According to embodiments of the instant disclosure, software may include instructions which, when executed by a processor of the computing device, may perform one or more steps of the methods disclosed herein. Some of the instructions may be adapted to produce signals that control operation of other units of the device, e.g. a measuring unit and / or other devices and thus may operate through those control signals to transform materials far removed from the device itself. These descriptions and representations are the means used by those skilled in the art of data processing, for example, to most effectively convey the substance of their work to others skilled in the art.

[0232] The plurality of instructions may also comprise an algorithm which is generally conceived to be a self-consistent sequence of steps leading to a desired result. These steps may include those requiring physical manipulations of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic pulses or signals capable of being stored, transferred, transformed, combined, compared, and otherwise manipulated. It proves convenient at times, principally for reasons of common usage, to refer to these signals as values, characters, display data, numbers, or the like as a reference to the physical items or manifestations in which such signals are embodied or expressed. It should be borne in mind, however, that all of these and similar terms are to be associated with the appropriate physical quantities and are merely used here as convenient labels applied to these quantities.

[0233] The evaluation unit may also comprise or have access to an output device. Exemplary output devices include displays, printers, files, and telecommunication devices, such as fax devices, data servers, and the like. According to some embodiments, a computing device may perform one or more steps of a method disclosed herein, and thereafter provide an output, via an output device, relating to an assessment result, indication, ratio or other factor of the method.

[0234] The term “detection agent”, for which also "detection compound" may be used, as used herein, refers to any agent which allows determination, in an embodiment specific determination, of an amount of at least one biomarker. Thus, the detection agent may in particular be a reaction substrate, e.g. in case the biomarker has catalytic activity, e.g. is an enzyme; or the detection agent may be an agent binding, in an embodiment specifically, to a biomarker or analyte thereof.

[0235] The skilled person selects suitable detection substrates in dependence on the biomarker, i.e. catalytic activity, to be detected, based on information available in the art. For specific biomarkers, example substrates are provided herein above.

[0236] Also, binding agents for specific antigens, as well as methods for providing them, are known in the art. As indicated herein above, the detection agent being a binding agent in an embodiment specifically binds to a biomarker, i.e. does not cross-react with other components present in the sample. Typically, a detection agent specifically binding a biomarker as referred to herein may be an antibody, an antibody fragment or derivative, an aptamer, a ligand for the biomarker, a receptor for the biomarker, an enzyme known to bind and / or convert the biomarker, or a small molecule known to specifically bind to the biomarker. For example, antibodies as referred to herein as detection agents include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F(ab)2 fragments that are capable of binding antigen or hapten. Aptamer detection agents, e.g., may be nucleic acid or peptide aptamers. Methods to prepare such aptamers are well- known in the art. The detection agent may be fused or linked permanently or reversibly to a detectable label. Suitable labels are well known to the skilled artisan. Suitable detectable labels are any labels detectable by an appropriate detection method. Typical labels include gold particles, latex beads, acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels ("e.g. magnetic beads", including paramagnetic and superparamagnetic labels), and fluorescent labels.

[0237] “Specific binding” of a detection agent means that it should not bind substantially to, i.e. cross-react with, another peptide, polypeptide or substance present in the sample to be analyzed. Preferably, the specifically bound biomarker should be bound with at least 3 times higher, more preferably at least 10 times higher and even more preferably at least 50 times higher affinity than any other components of the sample. Non-specific binding may be tolerable, if it can still be distinguished and measured unequivocally, e.g. according to its size on a Western Blot, or by its relatively higher abundance in the sample.

[0238] Moreover, the present invention relates to a kit for assessing colorectal cancer or a precancerous condition thereof, said kit comprising at least one detection agent for the biomarker CLEC3B and optionally at least one detection agent for at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, Ferritin and AREG.

[0239] The term “detection agent” has been defined above. In a preferred embodiment, the detection agent is an antibody, or antigen-binding fragments thereof, which specifically binds to the biomarker as referred to herein.

[0240] Moreover, the present invention relates to the use of i. the biomarker CLEC3B, or at least one detection agent thereof, and optionally ii. at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, Ferritin and AREG, or at least one detection agent for said at least one further biomarker, in a sample (such as a blood, serum or plasma sample) from a subject for assessing colorectal cancer or a precancerous condition thereof.

[0241] Moreover, the present invention relates to the use of i. at least one detection agent for CLEC3B, and ii. optionally at least one detection agent for at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, SOX9, CFB, Ferritin and AREG, in for determining the level of CLEC3B and optionally the level of said at least one further biomarker in a sample (such as a blood, serum or plasma) from a subject as referred herein. Preferably, said use is an in vitro use. The term “detection agent” has been defined above. In a preferred embodiment, the detection agent is an antibody, or antigen-binding fragments thereof, which specifically binds to the biomarker as referred to herein (in particular for CEA, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG).

[0242] Finally, the present invention relates to a method for determining the level of a first biomarker, the level of a second biomarker and, optionally, the level of a third biomarker in a blood, serum or plasma sample from a subject suspected to suffer from colorectal cancer or a precancerous condition thereof comprising i) contacting a portion of said blood, serum or plasma sample with one or more detection agents that specifically bind to the biomarker CLEC3B present in the sample, thereby allowing the formation of first complex comprising said biomarker and said one or more detection agents, and ii) contacting the same or a different portion of said blood, serum or plasma sample with one or more detection agents that specifically bind a second biomarker selected from the group consisting of SOX9 (SRY-box transcription factor 9), carcinoembryonic antigen (CEA), anti-p53 (ap53), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), and thereby allowing the formation of a second complex comprising said second biomarker and said one or more detection agents, and optionally iii) contacting the same portion as in i) or ii), or a different portion of said blood, serum or plasma sample with one or more detection agents that specifically bind a third biomarker selected from the group consisting of SOX9 (SRY-box transcription factor 9), carcinoembryonic antigen (CEA), anti-p53 (ap53), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), and thereby allowing the formation of a third complex comprising said third biomarker and said one or more detection agents, and iv) determining the level of the first biomarker, the second biomarker and, optionally, the third biomarker by determining the level of the first complex, the level of the second complex, and optionally the level of third complex. Preferably, the sample is a blood, serum or plasma sample from a subject suspected to suffer from colorectal cancer or a precancerous condition thereof. More preferably, the sample is a blood, serum or plasma sample from a subject who suffers from colorectal cancer or a precancerous condition thereof.

[0243] The invention further relates to a computer program including computer-executable instructions for performing the method according to the present invention in one or more of the embodiments enclosed herein when the program is executed on a computer or computer network or a device as specified herein. Specifically, the computer program may be stored on a computer-readable data carrier. Thus, specifically, one, more than one or even all of method steps of the method of the present invention may be performed by using a computer or a computer network, in an embodiment by using a computer program. Thus, the present invention in particular proposes a computer program comprising instructions which, when the program is executed by a computer, cause the computer to carry out a method as specified herein above; and to a computer-readable medium comprising instructions which, when executed by a computer, cause the computer to carry out a method as specified herein above; to a computer-readable data carrier having stored thereon the computer program as specified herein above; and to a data carrier signal carrying the computer program as specified herein above. The present invention also relates to a data processing apparatus, device, or system comprising means for carrying out performing the method according to the present invention; to a data processing apparatus, device, or system comprising a processor configured to perform the method according to the present invention.

[0244] The invention further discloses and proposes a computer program product having program code means, in order to perform the method according to the present invention in one or more of the embodiments enclosed herein when the program is executed on a computer or computer network. Specifically, the program code means may be stored on a computer- readable data carrier.

[0245] In the following, an overview on the embodiments of the present invention is provided. The definitions and explanations provided herein above preferably apply mutatis mutandis to the following:

[0246] 1. A method for assessing colorectal cancer or a precancerous condition thereof in a subject, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B), and optionally the level of at least one further biomarker selected from the group consisting of SOX9 (SRY-box transcription factor 9), carcinoembryonic antigen (CEA), anti-p53 (ap53), DNER (Delta and Notch- like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin) in a sample from the subject, and b) assessing colorectal cancer or the precancerous condition thereof based on the level of CLEC3B, and, optionally, the level of at least one further biomarker determined in step a).

[0247] 2. The method of embodiment 1, wherein the subject is suspected to suffer from colorectal cancer or the precancerous condition thereof.

[0248] 3. The method of any one of embodiment 1 or 2, wherein step b) comprises i. comparing the level of CLEC3B to a reference level and, optionally the level of the at least one further biomarker to a reference level for said at least one biomarker, and / or ii. calculating a score for assessing colorectal cancer or the precancerous condition thereof, wherein the score is calculated based on the level of CLEC3B and optionally the level of the at least one further biomarker.

[0249] 4. The method of embodiment 3, wherein the reference level for CLEC3B is a predetermined value for the level of CLEC3B which allows for assessing colorectal cancer or a precancerous condition thereof, and optionally wherein the reference level for said at least one further biomarker is a predetermined value for the level of said at least one further biomarker which allows for assessing colorectal cancer or a precancerous condition thereof.

[0250] 5. The method of any one of embodiments 1 to 4, wherein the precancerous condition of colorectal cancer is advanced adenoma.

[0251] 6. The method of any one of embodiments 1 to 5, wherein the colorectal cancer encompasses stage 0 (pTis), stage I, stage II, stage III and stage IV colorectal cancer.

[0252] 7. The method of any one of embodiments 1 to 6, wherein the levels of i. CLEC3B and CFB, ii. CLEC3B and SOX9, iii. CLEC3B and Ferritin, iv. CLEC3B and AREG, v. CLEC3B and anti-p53, vi. CLEC3B and DNER, vii. CLEC3B and CEA, or viii. CLEC3B, CEA and anti-p53 are determined.

[0253] 8. The method of any one of embodiments 1 to 7, wherein the assessment of colorectal cancer or the precancerous condition thereof is the diagnosis of colorectal cancer or the precancerous condition thereof.

[0254] 9. The method of embodiment 8, wherein the level of the biomarker CLEC3B is determined, and wherein preferably a level of the biomarker CLEC3B below the reference level indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof and / or wherein a level of the biomarker CLEC3B above the reference level indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, wherein said reference level in a preferred embodiment is a threshold value.

[0255] 10. The method of embodiment 8, wherein i. the levels of the biomarkers CLEC3B and CEA are determined, wherein preferably a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of CEA above the reference level for CEA indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof and / or wherein preferably a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of CEA below the reference level for CEA indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, ii. the levels of the biomarkers CLEC3B and ap53 are determined, wherein preferably a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of ap53 above the reference level for ap53 indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof and / or wherein preferably a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of ap53 below the reference level for ap53 indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, iii. the levels of the biomarkers CLEC3B and SOX9 are determined, wherein preferably a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of SOX9 above the reference level for ap53 indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof and / or wherein preferably a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of SOX9 below the reference level for SOX9 indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, iv. the levels of the biomarkers CLEC3B and DNER are determined, wherein preferably a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of DNER below the reference level for DNER indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof and / or wherein preferably a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of DNER above the reference level for DNER indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, v. the levels of the biomarkers CLEC3B and Ferritin are determined, wherein preferably a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of Ferritin below the reference level for Ferritin indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof and / or wherein preferably a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of Ferritin above the reference level for Ferritin indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, vi. the levels of the biomarkers CLEC3B and AREG are determined, wherein preferably a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of AREG above the reference level for AREG indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof and / or wherein preferably a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of AREG below the reference level for AREG indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, or vii. the levels of the biomarkers CLEC3B, ap53 and CEA are determined, wherein preferably a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of CEA above the reference level for CEA and a level of ap53 above the reference level for ap53 indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof and / or wherein preferably a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of CEA below the reference level for CEA and a level of ap53 below the reference level for ap53 indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof wherein in each of i. to vii. said reference levels in a preferred embodiment are threshold values.

[0256] 11. The method of embodiment 8, wherein the diagnosis is the rule in of colorectal cancer or the precancerous condition thereof.

[0257] 12. The method of any one of embodiments 8 to 11, further comprising initiating or recommending one or more further diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof for a subject who has been diagnosed as likely suffering from colorectal cancer or the precancerous condition thereof.

[0258] 13. The method of any one of embodiments 8 to 12, further comprising initiating or recommending a suitable therapeutic measure for a subject diagnosed to suffer from colorectal cancer or the precancerous condition thereof.

[0259] 14. The method of any one of embodiments 8 to 10, wherein the diagnosis is the rule out of colorectal cancer or the precancerous condition thereof.

[0260] 15. The method of any one of embodiments 1 to 7, wherein the assessment of colorectal cancer or the precancerous condition thereof is a) the differentiation whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, or b) the differentiation whether a subject (i) suffers from colorectal cancer or ii) does not suffer from colorectal cancer.

[0261] 16. The method of any one of embodiments 1 to 15, wherein sample is a body fluid sample.

[0262] 17. The method of embodiment 15, wherein the sample is a blood, serum or plasma sample. The method of any one of embodiments 1 to 7, wherein the assessment of colorectal cancer or the precancerous condition thereof is the identification of a subject who is eligible to one or more diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof. The method of embodiment 18, wherein said one or more diagnostic methods are ruled in or ruled out. The method of any one of embodiments 12, 18 or 19, wherein the one or more further diagnostic measures are selected from Colonoscopy, Sigmoidoscopy, CT CoIonography (Virtual Colonoscopy), Double-Contrast Barium Enema, Capsule Endoscopy, a fecal immunochemical test and a stool DNA test. The method of embodiment 20, wherein the further diagnostic measure is colonoscopy. A computer-implemented method for assessing colorectal cancer or a precancerous condition thereof, comprising a) receiving, at a processing unit, a value for the level of CLEC3B in a sample from a subject, and, optionally at least one further value for the level of at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin) in said sample, b) comparing, by said processing unit, the value or values received in step a) to a reference or to references and / or calculating, by said processing unit, a score for assessing colorectal cancer or the precancerous condition thereof, wherein the score is based on the value or values received in step a), and c) assessing, preferably by the processing unit, colorectal cancer or the precancerous condition thereof based on the result of step b). The computer-implemented method of embodiment 22, wherein the subject is suspected to suffer from colorectal cancer or the precancerous condition thereof. 24. The computer-implemented method of embodiment 22 or 23, wherein the reference level is a predetermined value for the level the respective biomarker which allows for assessing colorectal cancer or a precancerous condition thereof.

[0263] 25. The computer-implemented method of any one of embodiments 22 to 24, wherein the precancerous condition of colorectal cancer is advanced adenoma.

[0264] 26. The computer-implemented method of any one of embodiments 22 to 25, wherein the colorectal cancer encompasses stage 0 (pTis), stage I, stage II, stage III and stage IV colorectal cancer.

[0265] 27. The method of any one of embodiments 22 to 26, wherein the assessment of colorectal cancer or the precancerous condition thereof is the diagnosis of colorectal cancer or the precancerous condition thereof.

[0266] 28. The computer-implemented method of embodiment 27, wherein the value for level of the biomarker CLEC3B is received in step a), and wherein preferably a level of the biomarker CLEC3B below the reference level indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof and / or wherein a level of the biomarker CLEC3B above the reference level indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof.

[0267] 29. The computer-implemented method of embodiment 27, wherein values for the biomarker combinations as set forth in embodiment 7 are obtained, and wherein the diagnostic algorithm is the diagnostic algorithm as defined in embodiment 10.

[0268] 30. The computer-implemented method of any one of embodiments 27 to 29, wherein the diagnosis is the rule in of colorectal cancer or the precancerous condition thereof.

[0269] 31. The computer-implemented method of any one of embodiments 22 to 30, further comprising recommending, by said processing unit, one or more further diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof for a subject who is likely to suffer from colorectal cancer or the precancerous condition thereof, for example a diagnostic measure as set forth in embodiment 20 or 21.

[0270] 32. The computer-implemented method of any one of embodiments 27 to 29, wherein the diagnosis is the rule out of colorectal cancer or the precancerous condition thereof. 33. The computer-implemented method of any one of embodiments 22 to 26, wherein the assessment of colorectal cancer or the precancerous condition thereof is a) the differentiation whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, or b) the differentiation whether a subject (i) suffers from colorectal cancer or ii) does not suffer from colorectal cancer.

[0271] 34. The computer-implemented method of any one of embodiments 22 to 33, wherein the sample is a blood, plasma or serum sample.

[0272] 35. The computer-implemented method of any one of embodiments 22 to 34, wherein the subject is a human subject.

[0273] 36. The computer-implemented method of any one of embodiments 22 to 26, wherein the assessment of colorectal cancer or the precancerous condition thereof is the identification of a subject who is eligible to one or more diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof, for example a diagnostic measure as set forth in embodiment 20 or 21.

[0274] 37. The computer-implemented method of embodiment 36, wherein said one or more diagnostic methods are ruled in or ruled out.

[0275] 38. Use of i. the biomarker CLEC3B, or at least one detection agent thereof, and optionally ii. at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), or at least one detection agent for said at least one further biomarker, in a sample from a subject for assessing colorectal cancer or a precancerous condition thereof. 39. The use of embodiment 38, wherein the assessment is the diagnosis of colorectal cancer or a precancerous condition thereof, in particular wherein the assessment is the diagnosis colorectal cancer.

[0276] 40. The use of embodiment 38, wherein the assessment is the differentiation a) whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, or b) whether a subject i) suffers from colorectal cancer or ii) does not suffer from colorectal cancer.

[0277] 41. The use of embodiment 38, wherein the assessment of colorectal cancer or the precancerous condition thereof is the identification of a subject who is eligible to one or more diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof, for example a diagnostic measure as set forth in embodiment 20 or 21.

[0278] 42. A device for assessing colorectal cancer or a precancerous condition thereof, said device comprising: a) at least one measuring unit for determining a level of the biomarker CLEC3B and, optionally, a level of at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin) in a sample from a subject, said at least one measuring unit comprising at least one detection agent for the biomarker CLEC3B and, optionally, at least one detection agent for the biomarker CEA, at least one detection agent for the biomarker anti-p53, at least one detection agent for the biomarker SOX9, at least one detection agent for the biomarker DNER, at least one detection agent for the biomarker Ferritin, at least one detection agent for the biomarker CFB and / or at least one detection agent for the biomarker AREG, and b) an evaluation unit operably linked to the measuring unit, said evaluation unit comprising a data processor comprising instructions for i) carrying out a comparison of the level of the biomarker CLEC3B and, optionally, of the level of said at least one further biomarker to a reference or references and / or for ii) carrying out a calculation of a score for assessing colorectal cancer or a precancerous condition thereof, wherein the score is calculated based on the level of CLEC3B and, optionally, on the level(s) of the at least one further biomarker.

[0279] 43. The device of embodiment 42, wherein the assessment is the diagnosis of colorectal cancer or a precancerous condition thereof, in particular wherein the assessment is the diagnosis of colorectal cancer.

[0280] 44. The device of embodiment 42, wherein the assessment is the differentiation a) whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, or b) whether a subject i) suffers from colorectal cancer or ii) does not suffer from colorectal cancer.

[0281] 45. The device of embodiment 42, wherein the assessment of colorectal cancer or the precancerous condition thereof is the identification of a subject who is eligible to one or more diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof, for example a diagnostic measure as set forth in embodiment 20 or 21.

[0282] 46. The method, the computer-implemented method, the use or the device of any one of the preceding embodiments, wherein the subject is a human subject.

[0283] 47. The method, the computer-implemented method, the use or the device of any one of the preceding embodiments, wherein the sample is a body fluid sample.

[0284] 48. The method, the computer-implemented method, the use or the device of any one of the preceding embodiments, wherein the sample is blood, serum or plasma sample.

[0285] 49. The method, the computer-implemented method, the use or the device of embodiment 48, wherein the sample is a plasma sample.

[0286] 50. The method, the computer-implemented method, the use or the device of embodiment 48, wherein the sample is a serum sample.

[0287] 51. A kit for assessing colorectal cancer or a precancerous condition thereof, said kit comprising at least one detection agent for the biomarker CLEC3B and at least one detection agent for at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9 (SRY-box transcription factor 9), DNER , CFB, Ferritin and AREG. The kit of embodiment 51, wherein the assessment is an assessment as set forth in embodiment 43, 44 or 45. The use, the kit or the device of any one of the preceding embodiments, wherein the detection agent is an antibody that specifically detects the biomarker, or an antigenbinding fragment thereof. A database comprising one or more stored references for the biomarker CLEC3B and, optionally one or more stored references for at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9 (SRY-box transcription factor 9), DNER , CFB, Ferritin and AREG. The database of embodiment 54, wherein the one or more stored references allow for assessing colorectal cancer or a precancerous condition thereof, in particular wherein the assessment is an assessment as set forth in embodiment 43, 44 or 45. A computer program including computer-executable instructions for performing the computer-implemented method according to any one of embodiments 22 to 37, when the program is executed on a computer or computer network. The method, the computer-implemented method, or the use of any one of the preceding embodiments, wherein the subject does not suffer from stage I, II, III or IV colorectal cancer. A method for determining the level of a first biomarker, the level of a second biomarker and, optionally, the level of a third biomarker in a blood, serum or plasma sample from a subject suspected to suffer from colorectal cancer or a precancerous condition thereof comprising i) contacting a portion of said blood, serum or plasma sample with one or more detection agents that specifically bind to the biomarker CLEC3B present in the sample, thereby allowing the formation of first complex comprising said biomarker and said one or more detection agents, and ii) contacting the same or a different portion of said blood, serum or plasma sample with one or more detection agents that specifically bind a second biomarker selected from the group consisting of SOX9 (SRY-box transcription factor 9), carcinoembryonic antigen (CEA), anti-p53 (ap53), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), and thereby allowing the formation of a second complex comprising said second biomarker and said one or more detection agents, and optionally iii) contacting the same portion as in i) or ii), or a different portion of said blood, serum or plasma sample with one or more detection agents that specifically bind a third biomarker selected from the group consisting of SOX9 (SRY-box transcription factor 9), carcinoembryonic antigen (CEA), anti-p53 (ap53), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), and thereby allowing the formation of a third complex comprising said third biomarker and said one or more detection agents, and iv) determining the level of the first biomarker, the second biomarker and, optionally, the third biomarker by determining the level of the first complex, the level of the second complex, and optionally the level of third complex.

[0288] 59. The method of embodiment 58, wherein the subject suffers from colorectal cancer or a precancerous condition thereof.

[0289] All patents, patent applications, and publications or public disclosures referred to or cited herein are incorporated by reference in their entirety.

[0290] The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.

[0291] Example 1: Cohort Descriptions

[0292] Biomarker levels using immunoassays were measured in two distinct cohorts: Potsdam CRC and Blitz. The two cohorts are briefly described in the following sections.

[0293] Example la. Potsdam CRC Cohort Biomarker levels using immunoassays were measured in samples from two cohorts, the Potsdam S4 screening cohort and the MSKK (Molecular Signature of Colorectal Cancer) cohort. The Potsdam S4 cohort is a prospective colonoscopy screening cohort from Potsdam, Germany. Inclusion criteria for enrollment were age > 55 years old, and informed consent. Exclusion criteria were colonoscopy, rectoscopy, or sigmoidoscopy in the last 5 years, diagnosis of colorectal cancer before the study, or active malignancy. Asymptomatic and symptomatic persons meeting the inclusion criteria were enrolled into the study, followed by a blood draw, and were screened with colonoscopy. The findings were pathologically confirmed with a report from the colonoscopy. In total, 1636 patients were enrolled in the Potsdam S4 study.

[0294] The MSKK cohort is a cohort of symptomatic patients with a high suspicion of colorectal cancer or large advanced adenoma. Enrollment for this cohort was carried out in a surgical setting. The MSKK cohort comprises a total of 2452 subjects. Parts of the cohorts were used to enrich the number of colorectal cancer cases and achieve the pre-specified sample size.

[0295] From a total of 4088 subjects that underwent colonoscopy a subset of 559 (554 S4, 5 MSKK) patients have been selected. 325 of the selected subjects had either no findings (nonfindings), other findings (e.g. inflammatory bowel disease) or only benign findings (low grade adenoma) (‘Controls’) while 116 had malignant findings (CRC + pTis (pathological tumor in situ)) and 118 were diagnosed with a high risk of potential future malignant findings (i.e. high grade intraepithelial neoplasia / advanced adenoma (HG)). For a detailed overview of the distribution in the categories, see Table 1. In the following analysis we distinguish between two endpoints, one focusing only on CRC and pTis as cases and the other including high grade intraepithelial neoplasia cases as well.

[0296] Potsdam CRC cohort is further referred to as ‘Cohort 1’ and figures (a) correspond to the Potsdam cohort. Table 1: Overview of Patient Characteristics. Distribution of number of subjects for the different categories. Controls are split into subjects that had no other findings, other findings like inflammatory bowel disease, or benign findings, meaning low grade intraepithelial neoplasia. Cases are split into subjects having malignant findings either colorectal cancer cases (CRC) or having a pathological tumor in situ (pTis). Subjects with high grade intraepithelial neoplasia / advanced adenoma findings are classified as high risk patients.

[0297] Example lb: BLITZ Cohort

[0298] Samples were drawn from the BliTz study collective. BliTz (Begleitende Evaluierung innovativer Testverfahren zur Darmkrebs-Fruherkennung) is a prospective screening study conducted in cooperation with more than 20 gastrointestinal practices in southwestern Germany. Detailed information on the Blitz study can be found elsewhere (Hundt S, Haug U, Brenner H. Comparative evaluation of immunochemical fecal occult blood tests for colorectal adenoma detection. Ann Intern Med 2009;150: 162-9; Haug U, Hundt S, Brenner H. Quantitative immunochemical fecal occult blood testing for colorectal adenoma detection: evaluation in the target population of screening and comparison with qualitative tests. Am J Gastroenterol 2010;105:682-90; Brenner H, Tao S, Haug U. Low-dose aspirin use and performance of immunochemical fecal occult blood tests. JAMA 2010;304:2513- 20.). Briefly, since the end of 2005 pre-colonoscopy stool and blood samples from more than 7,000 participants of screening colonoscopy were collected. After colonoscopy, basic demographic and clinical data were extracted from colonoscopy and histology reports in a standardized manner by trained research assistants who were blinded to blood and stool test results. Cancer stages were classified according to the UICC (Union for International Cancer Control) classification and advanced adenomas were defined as adenomas with at least one of the following features: 1 cm in size, tubulovillous or villous components, and high-grade dysplasia. Further patient data were collected with a short patient questionnaire. A subset of 532 patients have been selected. 297 of the selected subjects had either no findings (nonfindings), other findings (e.g. inflammatory bowel disease) or only benign findings (nonadvanced adenoma) (‘Controls’) while 84 had malignant findings (CRC + pTis (pathological tumor in situ)) and 151 were diagnosed with a high risk of potential future malignant findings (i.e. advanced adenoma (AA)). For a detailed overview of the distribution in the categories, see Table 2. In the following analysis we distinguish between two endpoints, one focusing only on CRC and pTis as cases and the other including advanced adenoma cases as well.

[0299] The Blitz CRC cohort is further referred to as ‘Cohort 2’ and figures (b) correspond to the Blitz cohort.

[0300] Table 2: Overview of Patient Characteristics. Distribution of number of subjects for the different categories. Controls are split into subjects that had no other findings, other findings like inflammatory bowel disease, or benign findings, meaning low grade intraepithelial neoplasia. Cases are split into subjects having malignant findings either colorectal cancer cases (CRC) or having a pathological tumor in situ (pTis). Subjects with high grade intraepithelial neoplasia / advanced adenoma findings are classified as high risk patients.

[0301] Example 2. Immunological detection of CLEC3B

[0302] CLEC3B levels were assessed using the Olink platform based on the PEA (Proximity Extension Assay) technology.

[0303] PEA technology is the unique technology behind Olink® Explore platforms enabling high- throughput, multiplex immunoassays of proteins using minimal volumes of serum, plasma, or almost any other type of biological sample. The basis of PEA is a dual-recognition immunoassay, where two matched antibodies labeled with unique DNA oligonucleotides simultaneously bind to a target protein in solution. This brings the two antibodies into proximity, allowing their DNA oligonucleotides to hybridize, serving as template for a DNA polymerase-dependent extension step. This creates a double stranded DNA “barcode” which is unique for the specific antigen and quantitatively proportional to the initial concentration of target protein. The hybridization and extension are immediately followed by PCR amplification. The resulting DNA amplicon can then be quantified either by microfluidic qPCR on a Fluidigm® Biomark instrument, or on the Illumina® NovaSeq platform. The exponential amplification properties of PCR utilized in PEA enable to achieve a strong readout signal, providing assay sensitivity on par or better than traditional enzyme-linked immunosorbent assays (ELIS As) (Assarsson et al. PLoS One 2014 Apr 22;9(4), doi: 10.1371 / journal. pone.0095192). The data for biomarkers such as SOX9, DNER, CLEC3B, CFB and AREG were generated on the same samples using Olink® Explore 3072. Olink® Explore 3072 is a combination of 8 separate Olink® Explore 384 panels: Olink® Explore 384 Inflammation (Product Code 91101), Olink® Explore 384 Oncology (Product Code 91102), Olink® Explore 384 Cardiometabolic (Product Code 91103), Olink® Explore 384 Neurology (Product Code 91104), Olink® Explore 384 Inflammation II (Product Code 91105), Olink® Explore 384 Oncology II (Product Code 91106), Olink® Explore 384 Cardiometabolic II (Product Code 91107), Olink® Explore 384 Neurology II (Product Code 91108).

[0304] The Olink platform does not measure actual marker concentrations but relative biomarker quantifications expressed in an arbitrary unit in log2, the Olink NPX scale (Normalized Protein expression). This NPX scale is based on the measurement signals which are normalized using the standard Olink approach using a set of internal quality controls (Assarsson et al. PLoS One 2014 Apr 22;9(4), doi: 10.1371 / journal. pone.0095192).

[0305] CEA, Ferritin and anti-TP53 levels were measured using the respective Roche Elecsys® ECLIA (electrochemiluminescence immunoassay) assays on the same samples.

[0306] Example 3. Description of diagnostic performance calculation of individual marker and marker combination

[0307] Performance of individual markers is reported as “area under the receiver operating curve” (AUC) for distinguishing cases from controls. Depending on the defined endpoint, the composition of cases differs between (1) CRC + pTis cases and (2) CRC + pTis + HG / AA. For the biomarker combinations, the markers were mathematically combined via logistic regression and the AUC was again used as a general measure for marker and marker combination performance.

[0308] Example 4. Diagnostic performance and box plot analysis of CLEC3B

[0309] CLEC3B EDTA plasma samples were measured using the Olink® technology. CLEC3B EDTA plasma levels were shown to decrease in pTis and CRC cases (stage I - IV) compared to the control groups as well as compared to HG (Fig. 5a, b and Fig. 6a, b). Additionally, there were no significant differences observed in CLEC3B levels for different tumor locations (i.e. difference between rectum and colon colorectal cancer cases) (Fig. 7a and b). As CLEC3B levels were shown to be lower for CRC cases, the marker shows a good performance (AUC of 0.659 / 0.651) for separating the control groups (Controls + other findings + low grade / non-advanced adenoma) from CRC + pTis cases (Fig. 8 & 10). When adding HG / AA cases to the case group, the performance slightly drops to 0.614 / 0.591 (Fig. 9 & 11).

[0310] Example 4a. Diagnostic Performance in Cohort 1 (Potsdam CRC cohort)

[0311] Table 3: Summary of diagnostic performance of CLEC3B biomarker to discriminate subjects with no or only benign findings and subjects with malignant or potential future malignant findings through colonoscopy. The performance is analyzed using receiver operator characteristic (ROC) analysis and summarized by the area under the curve (AUC) of the ROC analysis and the associated 95% confidence interval. N depicts the number of samples tested (cases plus controls, numbers in each group vary depending on the analyses).

[0312] The results for the diagnostic performance of Cohort 1 are shown in Figures 8 and 9.

[0313] Example 4b. Diagnostic Performance in Cohort 2 (Blitz cohort)

[0314] Table 4: Summary of diagnostic performance of CLEC3B biomarker to discriminate subjects with no or only benign findings and subjects with malignant or potential future malignant findings through colonoscopy (Blitz cohort). The performance is analyzed using receiver operator characteristic (ROC) analysis and summarized by the area under the curve (AUC) of the ROC analysis and the associated 95% confidence interval. N depicts the number of samples tested (cases plus controls, numbers in each group vary depending on the analyses).

[0315] The results for the diagnostic performance Cohort 2 are also shown in Figures 10 and 11.

[0316] Overall, CLEC3B was found to be second best performing single marker in cohort 1 in distinguishing Controls from CRC + pTis cases out of 2,926 measured proteins. Additionally, for distinguishing Controls from CRC + pTis + HG / AA, CLEC3B was found as one of the top performing single markers in both cohorts. For both endpoints, CLEC3B achieved a similar AUC than the current reference marker carcinoembryonic antigen (CEA) (see Example 5) in cohort 1 and a nearly as high performance as CEA in cohort 2. CEA is so far known as the best performing single marker for CRC (Wild N. et al., 2010).

[0317] Example 5: Diagnostic performance and boxplot analysis of CEA (reference biomarker): Carcinoembryonic antigen (CEA) is so far the best performing single marker for detecting CRC cases (Wild N et al. , ClinCancer Res. 2010 Dec 15 ; 16(24): 6111 -21. doi : 10.1158 / 1078- 0432.CCR- 10-0119). To highlight the contribution of CLEC3B, CEA levels were measured in the same sample set as CLEC3B using the Roche Elecsys® CEA ECLIA (electrochemiluminescence immunoassay) method in cohort 1 and the Olink® technology in cohort 2.

[0318] For cohort 1, CEA was shown to achieve a relatively good performance with an AUC of 0.656 for distinguishing CRC + pTis from controls and an AUC of 0.604 when adding HG / AA to the case group. For both endpoints, CLEC3B achieved a similar performance (see Example 4a). For cohort 2, CEA was shown to achieve a good performance with an AUC of 0.707 for distinguishing CRC + pTis from controls and an AUC of 0.611 when adding HG / AA to the case group. For both endpoints, CLEC3B achieved a nearly as high performance as CEA (see Example 4b).

[0319] Example 5a. Results of CEA in Cohort 1 (Potsdam CRC cohort)

[0320] Figure 12 shows the results for CEA in plasma samples of subjects with CRC Cases and pTis compared to controls, with other findings, and low grade adenoma. Figure 13 shows the results for CEA in plasma samples of subjects with CRC Cases, pTis and high grade or advanced adenoma compared to controls, with other findings, and low grade adenoma.

[0321] Table 5: Summary of diagnostic performance of CEA biomarker to discriminate subjects with no or only benign findings and subjects with malignant or potential future malignant findings through colonoscopy. The performance is analyzed using receiver operator characteristic (ROC) analysis and summarized by the area under the curve (AUC) of the ROC analysis and the associated 95% confidence interval. N depicts the number of samples tested (cases plus controls, numbers in each group vary depending on the analyses).

[0322] Example 5b. Results of CEA in Cohort 2 (Blitz cohort)

[0323] Figure 14 shows the results for CEA in plasma samples of subjects with CRC Cases and pTis compared to controls, with other findings, and low grade adenoma. Figure 15 shows the results for CEA in plasma samples of subjects with CRC Cases, pTis and high grade or advanced adenoma compared to controls, with other findings, and low grade adenoma.

[0324] Table 6: Summary of diagnostic performance of CEA biomarker to discriminate subjects with no or only benign findings and subjects with malignant or potential future malignant findings through colonoscopy. The performance is analyzed using receiver operator characteristic (ROC) analysis and summarized by the area under the curve (AUC) of the ROC analysis and the associated 95% confidence interval. N depicts the number of samples tested (cases plus controls, numbers in each group vary depending on the analyses).

[0325] Example 6: Diagnostic performance of CLEC3B and CEA combined

[0326] In addition to assessing the performance of the individual biomarker CLEC3B in comparison to CEA, the two biomarkers were further combined via logistic regression and the AUC was again used as performance measurement. Adding CLEC3B on top of CEA improves the performance of CEA by 5.9% / 2.4% for detecting CRC cases + pTis compared to controls or by 4.6% / 2.0% for detecting CRC cases + pTis + HG, showing the additional value of CLEC3B.

[0327] Table 7a: Cohort 1 (Potsdam CRC): Diagnostic performance of combination of CLEC3B with CEA. Calculation of diagnostic performance of biomarker combination to discriminate subjects with no or only benign findings and subjects with malignant or potential future malignant findings through colonoscopy is shown by the area under the curve (AUC) of the ROC analysis and improvement of the combined biomarker panel to

[0328] CEA alone. N depicts the number of samples tested (cases plus controls, numbers in each group vary depending on the analyses).

[0329] Table 7b: Cohort 2 (Blitz CRC): Diagnostic performance of combination of CLEC3B with CEA. Calculation of diagnostic performance of biomarker combination to discriminate subjects with no or only benign findings and subjects with malignant or potential future malignant findings through colonoscopy is shown by the area under the curve (AUC) of the ROC analysis and improvement of the combined biomarker panel to CEA alone. N depicts the number of samples tested (cases plus controls, numbers in each group vary depending on the analyses).

[0330] Example 7: Comparison with combined diagnostic performance of CEA with ap53

[0331] To confirm specificity of the CEA performance improvement by its combination with CLEC3B, we have combined CEA with the other established biomarker, anti-p53 (ap53). Combination with ap53 did not improve diagnostic performance of CEA as much as combining it with CLEC3B for any of the analyzed groups (Table 8a, b). Individual AUC of ap53 for diagnosis of CRC + pTis was 0.547 / 0.544, for CRC + pTis + HG was 0.521 / 0.517.

[0332] Table 8a: Cohort 1 (Potsdam CRC): Diagnostic performance of combination of CEA with ap53. Calculation of diagnostic performance of CEA biomarker combined with ap53 to discriminate subjects with no or only benign findings and subjects with malignant or potential future malignant findings through colonoscopy is shown describing the area under the curve (AUC) of the ROC analysis and improvement of the combined biomarker panel to CEA alone. N depicts the number of samples tested (cases plus controls, numbers in each group vary depending on the analyses).

[0333] Table 8b: Cohort 2 (Blitz CRC) Diagnostic performance of combination of CEA with ap53. Calculation of diagnostic performance of CEA biomarker combined with ap53 to discriminate subjects with no or only benign findings and subjects with malignant or potential future malignant findings through colonoscopy is shown describing the area under the curve (AUC) of the ROC analysis and improvement of the combined biomarker panel to CEA alone. N depicts the number of samples tested (cases plus controls, numbers in each group vary depending on the analyses).

[0334] Example 8. Diagnostic performance of CLEC3B combined with other biomarkers

[0335] In addition to combining CLEC3B with established biomarkers like CEA, we identified other markers that also improved the performance. The markers were again mathematically combined via logistic regression and the AUC was used as a general measure for marker performance.

[0336] Example 8a: Cohort 1 (Potsdam CRC)

[0337] Combinations of marker pairs (bivariate marker combinations) having improved AUCs over the single markers by at least one percentage point for discriminating subjects with no or only benign findings and subjects with malignant findings.

[0338] Table 9a: Bivariate marker combinations with their joint performance (AUC.bi), the univariate performance of the first marker (AUC. l) and the second marker (AUC.2), along with the performance improvement of the bivariate marker over the best single marker (Impr.AUC) for discriminating Controls (Controls, other findings, low grade adenoma (LG) versus CRC Cases + pTis.

[0339] Combinations of marker pairs (bivariate marker combinations) having improved AUCs over the single markers by at least one percentage point for discriminating subjects with no or only benign findings and subjects with malignant or potential future malignant findings. Controls (Controls, other findings, LG) vs. CRC Cases +pTis +HG

[0340] Table 9b: Bivariate marker combinations with their joint performance (AUC.bi), the univariate performance of the first marker (AUC. l) and the second marker (AUC.2), along with the performance improvement of the bivariate marker over the best single marker (Impr.AUC) for discriminating Controls (Controls, other findings, low grade adenoma (LG) versus CRC Cases + pTis + HG (high grade adenoma) Biomarker name abbreviations:

[0341] S0X9 (UniProtID: P48436): Transcription Factor SOX9

[0342] DNER (UniProt ID: Q8NFT8): Delta and Notch-like epidermal growth factor-related receptor CFB (UniProt ID: P00751): Complement factor B

[0343] AREG (UniProt ID: P15514): Amphiregulin

[0344] Example 8b: Cohort 2 (Blitz CRC)

[0345] Combinations of marker pairs (bivariate marker combinations) having improved AUCs over

[0346] Table 10a: Bivariate marker combinations with their joint performance (AUC.bi), the univariate performance of the first marker (AUC. l) and the second marker (AUC.2), along with the performance improvement of the bivariate marker over the best single marker (Impr.AUC) for discriminating Controls (Controls, other findings, low grade adenoma (LG) versus CRC Cases + pTis.

[0347] Combinations of marker pairs (bivariate marker combinations) having improved AUCs over the single markers by at least one percentage point for discriminating subjects with no or only benign findings and subjects with malignant or potential future malignant findings. Controls (Controls, other findings, LG) vs. CRC Cases +pTis +HG

[0348] Table 10b: Bivariate marker combinations with their joint performance (AUC.bi), the univariate performance of the first marker (AUC. l) and the second marker (AUC.2), along with the performance improvement of the bivariate marker over the best single marker (Impr.AUC) for discriminating Controls (Controls, other findings, low grade adenoma (LG) versus CRC Cases + pTis + HG (high grade adenoma)

[0349] Example 9. Diagnostic performance of CLEC3B combined with CEA and ap53:

[0350] To even further confirm and highlight the added value of CLEC3B, we combined CLEC3B with the other two established biomarkers CEA and ap53. Adding CLEC3B on top of the other two markers, further increased the performance by 4.7% / 3% for distinguishing controls from CRC cases + pTis and by 4% / 2.4% for distinguishing controls from CRC cases + pTis + HG.

[0351] Table Ila: Cohort 1 (Potsdam CRC): Diagnostic performance of CEA and anti-p53 combined with CLEC3B. Calculation of diagnostic performance to discriminate subjects with no or only benign findings and subjects with malignant or potential future malignant findings through colonoscopy is shown describing the area under the curve (AUC) of the ROC analysis and improvement of the combined biomarker panel to CEA alone. N depicts the number of samples tested (cases plus controls, numbers in each group vary depending on the analyses).

[0352] Table 11b: Cohort 2 (Blitz CRC): Diagnostic performance of CEA and anti-p53 combined with CLEC3B. Calculation of diagnostic performance of CEA and anti-p53 biomarkers combined with CLEC3B to discriminate subjects with no or only benign findings and subjects with malignant or potential future malignant findings through colonoscopy is shown describing the area under the curve (AUC) of the ROC analysis and improvement of the combined biomarker panel to CEA alone. N depicts the number of samples tested (cases plus controls, numbers in each group vary depending on the analyses).

Claims

1. Roche Diagnostics International AG 29 September 2025Roche Diagnostics GmbH Our Ref.: RD39557PCClaims1. A method for assessing colorectal cancer or a precancerous condition thereof in a subject, comprising the steps of a) determining the level of CLEC3B (C-type lectin domain family 3 member B), and the level of at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), CFB (Complement factor B), anti-p53 (ap53), SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), Ferritin and AREG (Amphiregulin) in a blood, serum or plasma sample from the subject, and b) assessing colorectal cancer or the precancerous condition thereof based on the level of CLEC3B, and the level of at least one further biomarker determined in step a).

2. The method of claim 1, wherein the subject is suspected to suffer from colorectal cancer or the precancerous condition thereof.

3. The method of any one of claim 1 or 2, wherein step b) comprises i. comparing the level of CLEC3B to a reference level for CLEC3B and the level of the at least one further biomarker to a reference level for said at least one biomarker, and / or ii. calculating a score for assessing colorectal cancer or the precancerous condition thereof, wherein the score is calculated based on the level of CLEC3B and the level of the at least one further biomarker.

4. The method of claim 3, wherein the reference level for CLEC3B is a predetermined value for the level of CLEC3B which allows for assessing colorectal cancer or a precancerous condition thereof, and wherein the reference level for said at least one further biomarker is a predetermined value for the level of said at least one further biomarker which allows for assessing colorectal cancer or a precancerous condition thereof.

5. The method of any one of claims 1 to 4, wherein the precancerous condition of colorectal cancer is advanced adenoma and / or wherein the colorectal cancer encompasses stage 0 (pTis), stage I, stage II, stage III and stage IV colorectal cancer.

6. The method of any one of claims 1 to 5, wherein the assessment of colorectal cancer or the precancerous condition thereof is the diagnosis of colorectal cancer or the precancerous condition thereof.

7. The method of claim 6, wherein the levels of the biomarkers CLEC3B and CEA are determined, and wherein preferably a level of the biomarker CLEC3B below the reference level for CLEC3B in combination with a level of CEA above the reference level for CEA indicates that the subject is likely to suffer from colorectal cancer or a precancerous condition thereof and / or wherein a level of the biomarker CLEC3B above the reference level for CLEC3B in combination with a level of CEA below the reference level for CEA indicates that the subject is not likely to suffer from colorectal cancer or a precancerous condition thereof, wherein said reference levels are threshold values.

8. The method of any one of claims 1 to 5, wherein the assessment of colorectal cancer or the precancerous condition thereof is a) the differentiation whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, or b) the differentiation whether a subject (i) suffers from colorectal cancer or ii) does not suffer from colorectal cancer.

9. The method of any one of claims 1 to 5, wherein the assessment of colorectal cancer or the precancerous condition thereof is the identification of a subject who is eligible to one or more diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof, wherein optionally the one or more further diagnostic measures are selected from Colonoscopy, Sigmoidoscopy, CT CoIonography (Virtual Colonoscopy), Double-Contrast Barium Enema, Capsule Endoscopy a fecal immunochemical test and a stool DNA test.

10. A method for determining the level of a first biomarker, said first biomarker being CLEC3B, the level of a second biomarker and, optionally, the level of a third biomarker in a blood, serum or plasma sample from a subject suspected to suffer from colorectal cancer or a precancerous condition thereof comprisingi) contacting a portion of said blood, serum or plasma sample with one or more detection agents that specifically bind to the first biomarker CLEC3B present in the sample, thereby allowing the formation of first complex comprising said biomarker and said one or more detection agents, and ii) contacting the same or a different portion of said blood, serum or plasma sample with one or more detection agents that specifically bind the second biomarker selected from the group consisting of carcinoembryonic antigen (CEA), SOX9 (SRY-box transcription factor 9), anti-p53 (ap53), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), and thereby allowing the formation of a second complex comprising said second biomarker and said one or more detection agents, and iii) optionally contacting the same portion as in i) or ii), or a different portion of said blood, serum or plasma sample with one or more detection agents that specifically bind a third biomarker selected from the group consisting of SOX9 (SRY-box transcription factor 9), carcinoembryonic antigen (CEA), anti-p53 (ap53), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), and thereby allowing the formation of a third complex comprising said third biomarker and said one or more detection agents, and iv) determining the level of the first biomarker, the second biomarker and, optionally, the third biomarker by determining the level of the first complex, the level of the second complex, and optionally the level of third complex.

11. The method of any one of claims 1 to 10, wherein i. CLEC3B and CEA, ii. CLEC3B and SOX9, iii. CLEC3B and Ferritin, iv. CLEC3B and AREG, v. CLEC3B and anti-p53, vi. CLEC3B and DNER, vii. CLEC3B and CFB, or viii. CLEC3B, CEA and anti-p53 are determined.

12. A computer-implemented method for assessing colorectal cancer or a precancerous condition thereof, comprising a) receiving, at a processing unit, a value for the level of CLEC3B in a blood, serum or plasma sample from a subject, and at least one further value for the level of at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin) in said sample, b) comparing, by said processing unit, the values received in step a) to a reference or to references and / or calculating, by said processing unit, a score for assessing colorectal cancer or the precancerous condition thereof, wherein the score is based on the values received in step a), and c) assessing, preferably by the processing unit, colorectal cancer or the precancerous condition thereof based on the result of step b).

13. Use of i. the biomarker CLEC3B, or at least one detection agent thereof, and ii. at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), or at least one detection agent for said at least one further biomarker, in a blood, serum or plasma sample from a subject for assessing colorectal cancer or a precancerous condition thereof.

14. A kit for assessing colorectal cancer or a precancerous condition thereof, said kit comprising at least one detection agent for the biomarker CLEC3B and at least one detection agent for at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), CFB, anti-p53 (ap53), SOX9 (SRY-box transcription factor 9), DNER, Ferritin and AREG, wherein optionally the detection agent is an antibody that specifically detects the biomarker, or an antigen-binding fragment thereof.

15. A device for assessing colorectal cancer or a precancerous condition thereof, said device comprising:a) at least one measuring unit for determining a level of the biomarker CLEC3B and a level of at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin) in a blood, serum or plasma sample from a subject, said at least one measuring unit comprising (I) at least one detection agent for the biomarker CLEC3B and (II) at least one detection agent for the biomarker CEA, at least one detection agent for the biomarker anti-p53, at least one detection agent for the biomarker SOX9, at least one detection agent for the biomarker DNER, at least one detection agent for the biomarker Ferritin, at least one detection agent for the biomarker CFB and / or at least one detection agent for the biomarker AREG, and b) an evaluation unit operably linked to the measuring unit, said evaluation unit comprising a data processor comprising instructions for i) carrying out a comparison of the level of the biomarker CLEC3B and of the level of said at least one further biomarker to a reference or references and / or for ii) carrying out a calculation of a score for assessing colorectal cancer or a precancerous condition thereof, wherein the score is calculated based on the level of CLEC3B and on the level(s) of the at least one further biomarker.

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