Auto-antibodies to anpep 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) detecting the presence or absence of an antibody to ANPEP (Aminopeptidase N, anti-ANPEP antibody) in a blood, serum or plasma sample from the subject, and assessing colorectal cancer or the precancerous condition thereof based on the presence or absence of an antibody to ANPEP determined in step a). The present invention further relates to computer-implemented methods, databases, devices, kits and uses related thereto.

Description

[0001] Roche Diagnostics International AG RD39651PC Roche Diagnostics GmbH

[0002] Auto-antibodies to ANPEP for assessing colorectal cancer (CRC) or a precancerous condition thereof

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a method for assessing colorectal cancer or a precancerous condition thereof, said method comprising a) detecting the presence or absence of an antibody to ANPEP (Aminopeptidase N, anti- ANPEP antibody) in a blood, serum or plasma sample from the subject, and assessing colorectal cancer or the precancerous condition thereof based on the presence or absence of an antibody to ANPEP determined in step a). The present invention further relates to computer-implemented methods, databases, devices, kits and uses related thereto.

[0005] BACKGROUND OF THE INVENTION

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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).

[0013] 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).

[0014] 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).

[0015] An asssay for detecting autoantibodies against human proteins is described by Venkataraman et al. (A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors. Nat Methods 15, 330-338 (2018). https: / / doi.org / 10.1038 / nmeth.4632). Auto-antibodies have an usually highly specific clinical performance with moderate sensitivity. For example, for an auto-antibody with a sensitivity of 10-12% at a specificity of 97%, there are 3 to 4 times more cancer patients with auto-antibody response compared to the control group. In addition, out of all cancer patients 10-12% exhibit an auto-antibody response. Specificity is a measure of a diagnostic test's ability to correctly identify those who do not have the disease (true negatives). When a test has 97% specificity, it means that 97% of the individuals who do not have the disease will correctly be identified as disease-free by the test. In other words, the test will produce false positive results in 3% of the cases where individuals do not actually have the disease.

[0016] ANPEP (Aminopeptidase N, UniProt ID: P15144) belongs to the group of Zn2+dependent exopeptidases. It is a multifunctional glycoprotein that acts as a peptidase, receptor, and signaling molecule in a tissue-dependent manner. ANPEP hydrolyses the N-terminal amino acids of peptides and has a unique preference for neutral amino acids. It is expressed in a variety of cells / tissues and is therefore associated with diverse physiological functions, including proliferation, differentiation, migration, angiogenesis, invasion, metastasis, vasoconstriction, and the regulation of normal and impaired immune function.

[0017] ANPEP is a 150 kDa type II membrane dependent metalloprotease. The membrane-bound form exists as a homodimer. A slightly shorter, but still functional, soluble form is generated via MMP 14-mediated shedding in, e.g., serum (Barnieh et al. Is tumour-expressed aminopeptidase N (APN / CD13) structurally and functionally unique? BBA - Reviews on Cancer. 2021; 1876. https: / / doi.Org / 10.1016 / j.bbcan.2021.188641, Lendeckel et al. The Role of the Ectopeptidase APN / CD13 in Cancer. Biomedicines 2023. 11, 724. https: / / doi.org / 10.3390 / biomedicinesl l030724).

[0018] ANPEP is expressed on CMV-susceptible cells (i.e. certain PBMC, epithelial cells, fibroblasts, smooth muscle cells and endothelial cells) and plays a role during CMV infection. It is described to be emanated from CMV infected cells (Soderberg et al., Transplantation, 61 (4) (1996), pp. 594-60 10.1097 / 00007890-199602270-00014, Soderberg-Naucler et al. A Novel Mechanism for Virus-Induced Autoimmunity in Humans. Immunological Reviews 1996. 10.1 H l / j. l600-065x. l996.tb00916.x).

[0019] Serum ANPEP levels were substantially higher in pancreatic cancer patients than in controls (Pang et al. Serum APN / CD13 as a novel diagnostic and prognostic biomarker of pancreatic cancer. Oncotarget, Vol. 7, No. 47. 2016. 10.18632 / oncotarget. l2835).

[0020] In colorectal cancer tissue, mRNA and protein levels of ANPEP were lower in colorectal adenomas and adenocarcinomas than in the surrounding uninvolved mucosa (Sanz et al. Aminopeptidase N Activity Predicts 5-Year Survival in Colorectal Cancer Patients. J Investig Med 2015;63: 740-746. 10.1097 / JIM.0000000000000199, Ha et al. Reduced expression of alanyl aminopeptidase is a robust biomarker of non-familial adenomatous polyposis and non-hereditary nonpolyposis colorectal cancer syndrome early-onset colorectal cancer. Cancer Med. 2023 Apr;12(8): 10091-10104. doi: 10.1002 / cam4.5675). ANPEP expression is significantly lower at mRNA and protein level in early onset CRC (below 50 years of age) compared to late onset (see Ha et al., cited above).

[0021] In colorectal cancer, higher ANPEP activity in tumor tissues is correlated with better overall patient survival. Data for higher ANPEP activity in plasma of colorectal cancer patients are inconsistent and correlated with worse overall survival (Barnieh et al. Is tumour-expressed aminopeptidase N (APN / CD13) structurally and functionally unique? BBA - Reviews on Cancer. 2021; 1876. https: / / doi.Org / 10.1016 / j.bbcan.2021.188641) or better overall survival. ANPEP activity in plasma samples of CRC patients was similar to healthy individuals (see Sanz et al., cited above).

[0022] Anti-ANPEP autoantibodies were identified in bone marrow transplant patients, who had acute CMV infection or CMV reactivation in the post-transplantation period. CMV infection in these immunocompromised patients induces the formation of ANPEP-specific autoantibodies and is clinically clearly associated with the development of extensive chronic graft versus host disease (Soderberg-Naucler et al., cited above). A second study identified anti-ANPEP autoantibodies in IBD- and Crohns disease patients, who have CMV infections. CMV can be reactivated from latency in macrophages by an involvement of inflammatory cytokines, suggesting that viral reactivation occurs in inflammatory tissues (Rahbar et al. Detection of cytotoxic CD13-specific autoantibodies in sera from patients with ulcerative colitis and Crohn’s disease. Journal of Autoimmunity 26 (2006) 155el64. 10.1016 / j.jaut.2006.02.003). To the best of our knowledge, however, a link between anti-ANPEP autoantibodies and CRC has not been known or suggested.

[0023] There is a need for biomarkers, in particular of 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.

[0024] DESCRIPTION OF THE INVENTION

[0025] The present invention, therefore, provides means and methods complying with these needs.

[0026] In the studies underlying the present invention, auto-antibodies against ANPEP (anti- ANPEP antibodies) were, due to their high specificity, found as the best auto-antibodies to increase the performance of the state of the art reference biomarker carcinoembryonic antigen (CEA) in distinguishing controls from CRC and pTis cases (see Example 5). Additionally, ANPEP autoantibodies were shown to increase the performance of other biomarkers, such as SOX9, anti-p53, CLEC3B, DNER, CFB, Ferritin and AREG, or biomarker combinations (see Examples 7 and 8). Further, for distinguishing controls from CRC, pTis and HG, the presence of anti-ANPEP antibodies was again found to increase the performance of CEA and also of other biomarkers.

[0027] Using the auto-antibody, e.g., in combination with other biomarkers enhances the non- invasive detection and differentiation 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.

[0028] THE FIGURES SHOW

[0029] Figure 1: Distribution of Age in Years for the different subgroups. Overall similar age distribution between the different groups with a slight increase in age for Cases (pTis and CRC). Figure 2: Distribution of male and female subjects for the different subgroups. Overall similar distribution. Slightly more cases (CRC) and LG findings in male subjects, while more HG findings are observed in females.

[0030] Figure 3: CEA in plasma samples of subjects with CRC Cases and pTis (pathological tumor in situ) compared to controls, with other findings, and low grade adenoma.

[0031] Figure 4: 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.

[0032] Figure 5: Comparison of number of true positives detected with CEA (alone), CEA in combination with ap53 (CEA+ap53), in combination with anti-ANPEP autoantibodies (CEA+ANPEP) or in combination with both, anti-p53 autoantibodies and anti-ANPEP autoantibodies (CEA+ap53+ANPEP). (a) shows the number of true positives for distinguishing CRC + pTis cases from controls, (b) shows the number of true positives for distinguishing CRC + pTis + HG from controls, and (c) shows the number of true positives for distinguishing HG cases from controls. anti-ANPEP autoantibodies improve the clinical predictive performance over the currently established benchmark biomarkers for all endpoints.

[0033] Figure 6: Comparison of number of true positives of biomarkers used individually (BM X) or in combination with anti-ANPEP autoantibodies (BM X + ANPEP).

[0034] (a) shows the number of true positives for distinguishing CRC + pTis cases from controls, (b) shows the number of true positives for distinguishing CRC + pTis + HG from controls, and (c) shows the number of true positives for distinguishing HG cases from controls. anti-ANPEP autoantibodies improves each combination for at least one endpoint. For the first endpoint (a), for 8 out of 9 combinations, anti-ANPEP autoantibodies improve the clinical predictive performance. When adding HGs to the case group (b), anti-ANPEP autoantibodies improve the clinical predictive performance for all combinations. For just distinguishing HG Cases from controls (c), anti-ANPEP autoantibodies improve the clinical predictive performance for 7 out of 9 combinations. BRIEF SUMMARY OF THE PRESENT INVENTION

[0035] 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) detecting the presence or absence of an anti-ANPEP antibody (anti- Aminopeptidase N antibody) in a sample, such as a blood, serum or plasma sample, from the subject, and b) assessing colorectal cancer or the precancerous condition thereof.

[0036] The assessment made in step b) of the present invention is, typically, on the presence or absence of the anti-ANPEP antibody. In a preferred embodiment, the presence of the anti- ANPEP antibody in the sample is detected. The presence of the anti-ANPEP antibody is indicative for a subject who suffers from colorectal cancer or a precancerous condition thereof and thus for a subject is eligible to further diagnostic measures for assessing colorectal cancer or a precancerous condition thereof.

[0037] 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.

[0038] In a preferred embodiment of the method of the present invention, the test subject is a human subject.

[0039] In a preferred embodiment of the present invention, the anti-ANPEP antibody to be detected is an IgG antibody that specifically binds ANPEP.

[0040] 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.

[0041] 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.

[0042] 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) detecting the presence or absence of an anti-ANPEP antibody (anti- Aminopeptidase N antibody) in a sample from the subject, and b) diagnosing colorectal cancer or the precancerous condition thereof based on the presence or absence of the anti-ANPEP antibody in the sample. In an embodiment the above diagnostic method, the diagnosis is made in order to rule in colorectal cancer or a precancerous condition thereof in a subject.

[0043] 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) detecting the presence or absence of an anti-ANPEP antibody (anti- Aminopeptidase N antibody) in a sample from the subject, and b) ruling in colorectal cancer or the precancerous condition thereof based on the presence of the anti-ANPEP antibody in the sample.

[0044] In a second aspect of the method of the present invention, the assessment of colorectal cancer or the precancerous condition thereof is

[0045] A. the differentiation whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, or

[0046] B. the differentiation whether a subject i) suffers from colorectal cancer or ii) does not suffer from colorectal cancer.

[0047] 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) detecting the presence or absence of an anti-ANPEP antibody (anti- Aminopeptidase N antibody) in a sample from the subject, and b) differentiating whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, based on the presence or absence of the anti-ANPEP antibody in the sample.

[0048] In a preferred embodiment, the presence of the anti-ANPEP antibody in the sample is detected. The presence of the anti-ANPEP antibody is indicative for a subject who suffers from colorectal cancer or a precancerous condition thereof,

[0049] 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) detecting the presence or absence of an anti-ANPEP antibody in a sample from the subject, and b) differentiating whether a subject i) suffers from CRC or ii) does not suffer from CRC based on the presence or absence of the anti-ANPEP antibody in the sample. In a preferred embodiment, the presence of the anti-ANPEP antibody in the sample is detected. The presence of the anti-ANPEP antibody is indicative for a subject who suffers from colorectal cancer.

[0050] 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.

[0051] 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) detecting the presence or absence of an anti-ANPEP antibody in a sample from the subject, and b) identifying a subject who is eligible to said one or more diagnostic measures based on the presence or absence of the anti-ANPEP antibody in the sample.

[0052] In a preferred embodiment, the presence of the anti-ANPEP antibody in the sample is detected. The presence of the anti-ANPEP antibody is indicative for a subject who is eligible to further diagnostic measures for assessing colorectal cancer or a precancerous condition thereof.

[0053] 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.

[0054] 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 measures in order to diagnose colorectal cancer or a precancerous condition thereof.

[0055] In accordance with the present invention, the assessment of colorectal cancer or the precancerous condition thereof (such as the diagnosis, the differentiation, the identification as set forth herein) may be further based on the additional assessment of at least one further biomarker for colorectal cancer or a precancerous condition thereof. The additional assessment of at least one further biomarker for colorectal cancer or a precancerous condition thereof, typically, is the detection of the presence or absence of said at least one further biomarker or the determination of the level of said at least one further biomarker. Preferably, the presence or absence is detected for biomarkers which are autoantibodies, e.g. if the biomarker is anti-p53. For other biomarkers, such as, for example, carcinoembryonic antigen (CEA), of CLEC3B (C-type lectin domain family 3 member B), SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin), typically, the level is determined.

[0056] Accordingly, step a) may comprise i) detecting the presence or absence of the at least one further biomarker or ii) determining the level of the at least one further biomarker. For example, the at least further biomarker can be a polypeptide or an auto-antibody. The assessment is then based on the presence or absence of the anti-ANPEP antibody and the at least one further biomarker, i.e. its level or presence or absence.

[0057] Accordingly, the method of the present invention may comprise the following steps, al) detecting the presence or absence of an anti-ANPEP antibody (anti-Aminopeptidase N antibody) in a sample from the subject, such as a blood serum or plasma sample, and a2) detecting the presence or absence or determining the level(s) of at least one further biomarker for colorectal cancer or a precancerous condition thereof, and b) assessing colorectal cancer or the precancerous condition thereof.

[0058] The assessment in step b) is typically based on the results of step a), i.e. of steps al) and a2). Step al) and a2) could be carried out in any order.

[0059] In a preferred embodiment, the at least one further biomarker for colorectal cancer or a precancerous condition thereof is selected from the group consisting of carcinoembryonic antigen (CEA), of CLEC3B (C-type lectin domain family 3 member B), SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), anti-p53 (ap53), Ferritin and AREG (Amphiregulin) in the sample, such as in the blood, serum or plasma sample.

[0060] In some embodiments, the at least one further biomarker is selected form carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9, DNER, CFB, Ferritin and AREG (e.g. for the diagnosis of CRC). In some embodiments, the at least one further biomarker is selected form carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9, CFB, Ferritin and CLEC3B (e.g. for the diagnosis of advanced adenoma).

[0061] Accordingly, the method of the present invention may comprise the following steps, al) detecting the presence or absence of an anti-ANPEP antibody (anti-Aminopeptidase N antibody) in a sample from the subject, such as a blood serum or plasma sample, and a2) detecting the presence or absence or determining the level(s) of at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), of CLEC3B (C-type lectin domain family 3 member B), SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), anti-p53, Ferritin and AREG (Amphiregulin) in the sample from the subject, and b) assessing colorectal cancer or the precancerous condition thereof.

[0062] The assessment in step b) is typically based on the results of step a), i.e. of steps al) and a2). Step al) and a2) could be carried out in any order.

[0063] In an embodiment, the level of SOX9 is determined in step a2).

[0064] In another embodiment, the level of Ferritin is determined in step a2).

[0065] In yet another embodiment, the level of CLEC3B is determined in step a2).

[0066] In yet another embodiment, the level of CFB is determined in step a2).

[0067] In yet another embodiment, the level of AREG is determined in step a2).

[0068] In yet another embodiment, the level of AREG is determined in step a2).

[0069] In yet another embodiment, the level of DNER is determined in step a2).

[0070] In yet another embodiment, the level of CEA or is determined in step a2).

[0071] In yet another embodiment, the levels of CEA and anti-p53 are determined in step a2).

[0072] The marker anti-p53 is an autoantibody. As for the anti-ANPEP antibody, the presence or absence of the anti-p53 antibody can be detected. The presence of the anti-p53 antibody is indicative for a subject who is suffering from colorectal cancer or a precancerous condition thereof. Thus, the presence of the anti-p53 antibody is indicative for a subject who is eligible to a further measure for diagnosing colorectal cancer or a precancerous condition thereof.

[0073] Thus, in an embodiment the presence or absence of the anti-p53 antibody is detected in step a2). In yet another embodiment, the level of CEA is determined and the presence or absence of the anti-p53 antibody is detected in step a2). 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, information on the presence or absence of an anti-ANPEP antibody in a sample from a subject, and, optionally information on at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), SOX9 (SRY-box transcription factor 9), of CLEC3B (C-type lectin domain family 3 member B), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), anti-p53, Ferritin and AREG (Amphiregulin) in said sample, wherein the information on the at least one further biomarker is information on the presence or absence or a value for the level of said at least one further biomarker, b) assessing, preferably by the processing unit, colorectal cancer or the precancerous condition thereof based on the information on the presence or absence of an anti-ANPEP antibody and, optionally, on the information on the at least one further biomarker received in step a).

[0074] Moreover, the present invention relates to the use of i. an anti-ANPEP antibody, or one or more detection agents for said anti- ANPEP antibody, and ii. optionally at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, CLEC3B, 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.

[0075] Moreover, the present invention relates to the use of an anti-ANPEP antibody, or one or more detection agents for said anti-ANPEP antibody, 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, SOX9, DNER, CFB, CLEC3B, Ferritin or AREG, or a panel (i.e. a combination) thereof. In an embodiment, the performance increase is an increased sensitivity. In another particularly preferred 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 an anti-ANPEP antibody and optionally at least one detection agent for at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, CLEC3B, Ferritin and AREG.

[0077] Moreover, the present invention relates to a database comprising one or more stored reference for anti-ANPEP antibody and, optionally one or more stored references for at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9, DNER, CFB, CLEC3B, Ferritin and AREG. Preferably, the one or more stored references allow for assessing colorectal cancer or a precancerous condition thereof.

[0078] 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.

[0079] Moreover, the present invention relates to a device for assessing colorectal cancer or a precancerous condition thereof, said device comprising: a) at least one detection unit for detecting an anti-ANPEP antibody and, optionally, for detecting at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CFB, CLEC3B, Ferritin and AREG 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 anti-ANPEP antibody 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 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 assessing colorectal cancer or a precancerous condition thereof based on the presence or absence of the anti-ANPEP antibody and, optionally, based on the level or the presence or absence of the of at least one further biomarker.

[0080] The present invention further relates to a method of treating a subject, said method comprising al) detecting the presence or absence of an anti-ANPEP antibody (anti- Aminopeptidase N antibody) and, a2) optionally detecting the presence or absence or determining the level(s) of at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG in a sample from the subject, and b) diagnosing colorectal cancer or the precancerous condition thereof based on the presence or absence of the anti-ANPEP antibody and, optionally, the presence or absence or 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.

[0081] In an embodiment of the above method of treatment, the diagnosis of colorectal cancer or a precancerous condition thereof may be based on the presence or absence of the anti-ANPEP antibody and, optionally the presence or absence or level(s) of the further CRC markers only. In an alternative embodiment, the diagnosis may be based on the presence or absence of the anti-ANPEP antibody and, optionally presence or absence or the level(s) of the further CRC markers 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.

[0082] In preferred embodiment of the above method, the sample is from a subject suffering from colorectal cancer or a precancerous condition.

[0083] The present invention further relates to a method for biomarker detection in a sample, such as 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 sample with one or more detection agents which specifically bind to an anti-ANPEP antibody to allow the formation of a complex comprising the anti-ANPEP antibody present in the sample and the one or more detection agents, and ii) detecting said complex (e.g. via a detectable label of the detection agent)

[0084] In preferred embodiment, said method comprises contacting the portion of the sample with an anti-ANPEP capture antigen and an anti-ANPEP detection antigen as detection agents, whereby a complex comprising the anti-ANPEP capture antigen, the anti-ANPEP antibody and the anti-ANPEP detection antigen is formed, i) separating the complex formed in i) from unbound detection antigen, ii) detecting the complex obtained in step ii) via the detection antigen comprised therein, thereby detecting the anti-ANPEP antibody comprised in the sample.

[0085] In another preferred embodiment, said method comprises contacting a portion of the sample with an anti-ANPEP detection antigen as detection agent, whereby a complex comprising the anti-ANPEP antibody and the anti-ANPEP detection antigen is formed, i) separating the complex formed in i) from unbound detection antigen, ii) detecting the complex obtained in step ii) via the anti-ANPEP detection antigen comprised therein, thereby detecting the anti-ANPEP antibody comprised in the sample.

[0086] Preferably, the method further comprises detecting the presence or absence or determining the level of at least one further biomarker for CRC or a precancerous conditions thereof, e.g. selected from the group consisting of carcinoembryonic antigen (CEA), SOX9 (SRY-box transcription factor 9), CLEC3B, anti-p53, DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin) in the same or in a different portion of said blood, serum or plasma sample from said subject.

[0087] In a preferred embodiment of the above method, the sample is from a subject who suffers from colorectal cancer or a precancerous condition thereof.

[0088] DETAILED SUMMARY OF THE PRESENT INVENTION / DEFINITIONS

[0089] 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.

[0090] 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’.

[0091] 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.

[0092] 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 %.

[0093] The at least one further biomarker for colorectal cancer or a precancerous condition thereof may be any biomarker that allows for assessing colorectal cancer or a precancerous condition thereof, in particular any biomarker that can aid in the diagnosis of colorectal cancer or a precancerous condition thereof (other than the anti ANPEP antibody). Such biomarker are known in the art and described by Brenner, H., Tao, S., Haug, U. (2014). Blood-based biomarkers for detection of colorectal cancer: A systematic review. Clinical Epidemiology, 6, 319-331, by 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, by Bhardwaj M et al. 2019. Molecular Oncology 14 (2020) 8-21, or by Bhardwaj M et al., 2020.European Journal of Cancer 127 (2020) 30e40 or in EP 2 829 881 Bl.

[0094] The term “antibody” typically refers to a polypeptide ligand comprising at least a light chain and heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. Typically, the antibody of the present invention has heavy (H) chains and light (L) chains interconnected by disulfide bonds. The term “light chain” as used herein includes a full-length light chain and fragments thereof having a sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains. The term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Thus, the term “antibody” refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM. As set forth elsewhere herein, an autoantibody is an antibody as produced by the immune system of a subject to be tested.

[0095] The “anti-ANPEP antibodies” and “anti-p53 antibodies” are autoantibodies. Accordingly, they are, if present, antibodies produced by the immune system of the subject to be tested. In other words, they are not a recombinant antibody that has been added to the sample. The anti-ANPEP antibody to be detected is capable of binding, preferably specifically binding human ANPEP. The anti-ap53 antibody to be detected is capable of binding, preferably specifically binding human p53. The autoantibody can be an autoantibody of any isoform, i.e. can be IgA, IgD, IgE, IgG or IgM. Preferably, the autoantibody is an IgG or IgM antibody. Most preferably the autoantibody is an IgG antibody.

[0096] The term “determining” as used herein refers to semiquantitative or quantitative determination of a biomarker referred to herein (e.g. of CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG). 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.

[0097] 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.

[0098] The term “detecting the presence or absence of an anti-ANPEP antibody” or “detecting an anti-ANPEP antibody” as used herein, typically refers to the assessment of whether anti- ANPEP antibodies are present in the sample or not. In an embodiment, the detection of the presence of the anti-ANPEP antibody means that the level of the antibody is above a predetermined reference level for assessing colorectal cancer or a precancerous condition thereof. In an embodiment, the detection of the absence of the anti-ANPEP antibody means that the level of the antibody is below a predetermined reference level for assessing colorectal cancer or a precancerous condition thereof. In some embodiments, the reference level is the limit of detection of the assay.

[0099] In an embodiment, the expression “detecting the presence or absence of an anti-ANPEP antibody” “detecting an anti-ANPEP antibody” as used herein refers to the determining of the level of the antibody. In this case, the level is compared to a suitable reference level or taken into account for the calculation of a score as described elsewhere herein.

[0100] 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 score. 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.

[0101] 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.

[0102] 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.

[0103] The term “detection agent”, for which also "detection compound" may be used, as used herein, refers to any agent which allows the detection or the 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.

[0104] “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.

[0105] Typically, a detection agent that specifically binds an anti-ANPEP antibody is a polypeptide, or fragment thereof, which is capable of being bound by the anti-ANPEP antibodies present in the sample. In a preferred embodiment, the polypeptide comprises the sequence of the human Aminopeptidase N polypeptide, or a fragment thereof which is capable of being bound by the anti-ANPEP 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 ANPEP polypeptide. Said ANPEP polypeptide, or fragment thereof, may further comprise a detectable label. The sequence of the human Aminopeptidase N polypeptide under Uniprot accession Number Pl 5144 (AMPN HUMAN, Last updated:2007-04-03 v4, Checksum 37B6BC1BF0D6B1F2, and ii) a detectable label.

[0106] Moreover, the amino acid sequence of the human aminopeptidase N polypeptide is shown in SEQ ID NO: 1

[0107] MAKGFYISKSLGILGILLGVAAVCTI IALSWYSQEKNKNANSSPVASTTPSASATTNPA SATTLDQSKAWNRYRLPNTLKPDSYRVTLRPYLTPNDRGLYVFKGSSTVRFTCKEATDVI I IHSKKLNYTLSQGHRWLRGVGGSQPPDIDKTELVEPTEYLWHLKGSLVKDSQYEMDS E EE GE LADDLAG FYRS E YME GNVRKWAT T QMQAADARKS FPC FDE PAMKAE FN I T L I HP KDLTALSNMLPKGPSTPLPEDPNWNVTEFHTTPKMSTYLLAFIVSEFDYVEKQASNGVLI RIWARPSAIAAGHGDYALNVTGPILNFFAGHYDTPYPLPKSDQIGLPDFNAGAMENWGLV TYRENSLLFDPLSSSSSNKERWTVIAHELAHQWFGNLVTIEWWNDLWLNEGFASYVEYL GADYAEPTWNLKDLMVLNDVYRVMAVDALASSHPLSTPASEINTPAQISELFDAISYSKG ASVLRMLSSFLSEDVFKQGLASYLHTFAYQNTIYLNLWDHLQEAVNNRS IQLPTTVRDIM NRWTLQMGFPVITVDTSTGTLSQEHFLLDPDSNVTRPSEFNYVWIVPITS IRDGRQQQDY WLIDVRAQNDLFSTSGNEWVLLNLNVTGYYRVNYDEENWRKIQTQLQRDHSAIPVINRAQ 11 NDAFNLAS AHKVP VT LALNNT L FL I E E RQ YMP WE AAL S S L S Y FKLM FDRS E VYG PMKN YLKKQVTPLFIHFRNNTNNWREIPENLMDQYSEVNAISTACSNGVPECEEMVSGLFKQWM ENPNNNPIHPNLRSTVYCNAIAQGGEEEWDFAWEQFRNATLVNEADKLRAALACSKELWI LNRYLS YTLNPDL IRKQDATS T 11 S I TNNVI GQGLVWDFVQSNWKKLFNDYGGGS FS FSN LIQAVTRRFSTEYELQQLEQFKKDNEETGFGSGTRALEQALEKTKANIKWVKENKEWLQ WFTENSK ( SEQ ID NO : 1 )

[0108] The detection agent may be fused or linked permanently or reversibly to a detectable label. Suitable labels are well known to the skilled person. 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. 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 protein as well as exemplary detection methods for anti-p53 are disclosed in WO2017153336A1

[0109] As regards to the biomarkers CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG, the detection agent is preferably an antibody or antigen-binding fragment thereof that specifically binds the marker.

[0110] “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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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. Further, they typically apply to the method for biomarker detection.

[0115] 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 testinal 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.

[0116] 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.

[0117] 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.

[0118] 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).

[0119] The assessment made in accordance with the method of the present invention is preferably based on the presence or absence of the anti-ANPEP antibody. Accordingly, the method of the present invention comprises step a) of detecting the presence or absence of an anti- ANPEP antibody (anti- Aminopeptidase N antibody) 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 for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), anti-p53 (ap53), SOX9, DNER , CFB, Ferritin and AREG.

[0120] Thus, the present invention relates to method for assessing colorectal cancer or a precancerous condition thereof (in particular advanced adenoma) in a subject, comprising the steps of al) detecting the presence or absence of an anti-ANPEP antibody (anti- Aminopeptidase N antibody), a2) optionally assessing the presence or absence or determining the level(s) of at least one further biomarker for CRC or the precancerous condition thereof (e.g. a biomarker selected from the group consisting of CEA, anti- p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG) in a sample from the subject, and b) assessing colorectal cancer or the precancerous condition thereof based on the presence or absence of the anti-ANPEP antibody and optionally based on the presence or absence or the level of the at least one further biomarker as determined in step a2).

[0121] In an embodiment, the assessment in step b) of the above method is based on the presence or absence of the anti-ANPEP antibody and, optionally, on the comparison of 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. Thus, step b) may comprise a comparison step. 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 presence or absence of the anti-ANPEP antibody and, optionally, the level(s) of the at least one further biomarker for colorectal cancer or the precancerous condition thereof. Thereby, colorectal cancer or the precancerous condition thereof is assessed (i.e. based on the calculated score).

[0122] 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.

[0123] 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

[0124] • 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),

[0125] • the diagnosis of colorectal cancer (such as the rule-in or rule-out of colorectal cancer),

[0126] • the differentiation whether a subject i) suffers from colorectal cancer or ii) does not suffer from colorectal cancer,

[0127] • the differentiation whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma, or

[0128] • the identification of a subject who is eligible to one or more further diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof.

[0129] 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.

[0130] 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 based on the presence or absence of the anti-ANPEP antibody in the sample and optionally on the presence or absence or the determined level of at least one further biomarker for colorectal cancer or a precancerous condition thereof. However, in a further embodiment, the assessment is based on the presence or absence of the anti-ANPEP antibody in the sample and optionally on the presence or absence or the determined level of the at least one further biomarker for colorectal cancer or a precancerous condition thereof 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.

[0131] 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.

[0132] Alternatively, the anti-ANPEP antibody 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 (di ckkopf homolog 3), M2PK (pyruvate kinase muscle

[0133] 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.

[0134] In an embodiment, the anti-ANPEP antibody is combined with DKK-3 (dickkopf homolog

[0135] 3), M2PK (pyruvate kinase muscle 2), and IGFBP2 (insulin-like growth factor binding protein-2).

[0136] In another embodiment, the anti-ANPEP antibody is combined with M2PK, IGFBP2, and EpCAM (epithelial cell adhesion molecule). In yet another embodiment, the anti-ANPEP antibody is combined with M2PK, IGFBP2 and IL- 13 (interleukin- 13).

[0137] In yet another embodiment, the anti-ANPEP antibody is combined with M2PK, IGFBP2 and IL-8 (interleukin-8).

[0138] Diagnosing colorectal cancer or a precancerous condition thereof

[0139] 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.

[0140] Accordingly, the present invention envisages a method for diagnosing colorectal cancer or a precancerous condition thereof in a subject, comprising the steps of a) detecting the presence or absence of an anti-ANPEP antibody (anti- Aminopeptidase N antibody) and, optionally, detecting the presence or absence or determining the level of at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG in a sample from the subject, and b) diagnosing colorectal cancer or the precancerous condition thereof based on the presence or absence of the anti-ANPEP antibody and, optionally, the presence or absence or the level of the at least one further biomarker determined in step a).

[0141] In a preferred embodiment, step b) of the above diagnostic method comprises

[0142] (b) diagnosing colorectal cancer or the precancerous condition thereof based on the presence or absence of the anti-ANPEP antibody and, optionally based the presence or absence of the at least one further biomarker and / or on the comparison of the level of the at least one further biomarker to a reference level for said at least one further biomarker.

[0143] Thus, step b) may comprise a comparison step. Alternatively, a score for the diagnosis of colorectal cancer or a precancerous condition thereof may be calculated. Thus, the diagnosis is based on the score. Accordingly, in an another preferred embodiment, step b) of the above diagnostic method comprises

[0144] (b) calculating a score for diagnosing colorectal cancer or the precancerous condition thereof, wherein the score is calculated based on the presence or absence of the anti- ANPEP antibody and, optionally, the presence or absence or the level(s) of the at least one further biomarker, whereby colorectal cancer or a precancerous condition thereof is to be diagnosed.

[0145] 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).

[0146] 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).

[0147] 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.

[0148] 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)

[0149] 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.

[0150] 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).

[0151] The direction of the individual biomarkers for the diagnosis of colorectal cancer or a precancerous condition thereof, i.e. whether an increased or decreased level of the biomarkers (as compared to the reference) is indicative for the diagnosis is disclosed elsewhere herein or known in the art.

[0152] Method for differentiation

[0153] 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 intraepithelial neoplasia) when undergoing colonoscopy.

[0154] 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 intraepithelial neoplasia) 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.

[0155] 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) detecting the presence or absence of an anti-ANPEP antibody (anti- Aminopeptidase N antibody) and, optionally, detecting the presence or absence or determining the level of at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG in a sample from the subject, and b) differentiating whether a subject i) suffers from CRC or advanced adenoma or ii) does not suffer from CRC or advanced adenoma based on the presence or absence of the anti-ANPEP antibody and optionally the presence or absence or the level of the at least one further biomarker,

[0156] In preferred embodiment the level of the at least one further biomarker is compared a reference level for said at least one further biomarker.

[0157] Alternatively, step b) may comprise calculating a score for differentiating between i) and ii), wherein the score is calculated based on the presence or absence of the anti-ANPEP antibody and, optionally, the presence or absence or the level(s) of the at least one further biomarker.

[0158] 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) detecting the presence or absence of an anti-ANPEP antibody (anti- Aminopeptidase N antibody) and, optionally, detecting the presence or absence or determining the level of at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG in a sample from the subject, and a) differentiating whether a subject i) suffers from CRC or ii) does not suffer from CRC based on the presence or absence of the anti-ANPEP antibody and optionally the level of the at least one further biomarker presence or absence of an anti-ANPEP antibody and, optionally, based on the presence or absence or the level of the at least one further biomarker to a reference level for said at least one further biomarker.

[0159] In preferred embodiment the level of the at least one further biomarker is compared a reference level for said at least one further biomarker.

[0160] Alternatively, step b) may comprise calculating a score for differentiating between i) and ii), wherein the score is calculated based on the presence or absence of the anti-ANPEP antibody and, optionally, based on the presence or absence or on the level(s) of the at least one further biomarker.

[0161] 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.

[0162] Method for identifying a subject who is eligible to further diagnostic measures

[0163] 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, anti-ANPEP antibodies and optionally further biomarker(s) for CRC or precancerous conditions thereof 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 presence or absence of an anti-ANPEP antibody 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 or absence of colorectal cancer or a precancerous condition thereof, in said subject.

[0164] 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) detecting the presence or absence of an anti-ANPEP antibody (anti- Aminopeptidase N antibody) and, optionally, detecting the presence or absence or determining the level of at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example 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) identifying a subject who is eligible to said one or more diagnostic measures thereof based on the presence or absence of the anti-ANPEP antibody and optionally based on the presence or absence or the level of the at least one further biomarker.

[0165] In a preferred embodiment, the presence of the anti-ANPEP antibody in the sample is detected. The presence of the anti-ANPEP antibody is indicative for a subject who is eligible to further diagnostic measures for assessing colorectal cancer or a precancerous condition thereof.

[0166] In preferred embodiment, the level of the at least one further biomarker is compared a reference level for said at least one further biomarker.

[0167] Alternatively, step b) may comprise calculating a score for identifying a subject who is eligible to one or more diagnostic measures for diagnosing colorectal cancer or a precancerous condition thereof, wherein the score is calculated based on the presence or absence of the anti-ANPEP antibody and, optionally, based on the presence or absence of the or on the level(s) of the at least one further biomarker for CRC or a precancerous condition thereof, whereby it is assessed whether a subject is eligible to one or more diagnostic measures for diagnosing colorectal cancer or a precancerous condition thereof.

[0168] 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.

[0169] Specifically, the measure is ruled in, if the patient is likely to suffer from colorectal cancer or a precancerous condition thereof. Thus, the patient is eligible to said one or more diagnostic measures.

[0170] 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.

[0171] In a particularly preferred embodiment, the diagnostic measure is colonoscopy. 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.

[0172] The subject to be tested

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] The sample

[0179] The sample to be tested in accordance with the present invention typically 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.

[0180] 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.

[0181] 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.

[0182] 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).

[0183] 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.

[0184] 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).

[0185] 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.

[0186] In an embodiment, the sample is colorectal tissue sample from a subject as referred to herein. For example, the sample is colorectal cancer tissue sample or an colorectal tissue sample comprising adenoma tissue.

[0187] The biomarkers

[0188] The assessment made in accordance with the present invention, typically, is based on the presence or absence of anti-ANPEP antibodies, and optionally, based on the presence or absence or based on the level of at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example 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 an antibody or protein which serves as an indicator for a disease or physiological state as referred to herein. If the biomarker is a protein (e.g. selected from the group consisting of CEA, SOX9, CLEC3B, DNER, CFB, Ferritin and AREG) it may be the protein as such or 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 as expressed in human. 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. Further, as known by the skilled person, autoantibodies may be heterogeneous.

[0189] The biomarker molecules to be determined in accordance with the present invention are as such known in the art, yet not for the uses as described herein. Moreover, methods that can be applied for the detection or determination of the biomarkers, e.g. immunoassays, are known to the skilled person as well.

[0190] In accordance with the present invention, the presence or absence of an anti-ANPEP antibody shall be detected, i.e. the presence or absence of anti-ANPEP antibodies in the sample. The presence of anti-ANPEP antibodies in the sample is indicative for a subject who is likely to suffer from colorectal cancer or a precancerous condition thereof and thus for a subject who eligible to further diagnostic measures for diagnosing colorectal cancer or a precancerous condition thereof

[0191] The terms “anti-ANPEP antibody”. “anti-ANPEP auto-antibody", "anti-ANEP antibody" or "ANEP antibody" are used interchangeably herein. The antibody to be detected is an autoantibody, i.e. an antibody produced by the immune system of the subject to be tested. In other words, it is not a recombinant antibody that has been added to the sample. The anti- ANPEP antibody to be detected is capable of binding, preferably specifically binding human ANPEP. For example, it binds the membrane bound form of human ANPEP or the soluble form. ANPEP (Aminopeptidase N, UniProt ID: Pl 5144) belongs to the group of Zn2+ dependent exopeptidases. It is a multifunctional glycoprotein that acts as a peptidase, receptor, and signaling molecule in a tissue-dependent manner. ANPEP hydrolyses the N- terminal amino acids of peptides and has a unique preference for neutral amino acids. Preferably, the anti-ANPEP antibody to the detected herein is an IgG antibody. 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).

[0192] 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).

[0193] 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).

[0194] 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).

[0195] 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 are 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).

[0196] 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 which is herewith is incorporated by reference in its entirety describes assays to determine the level of the marker as well as suitable capture and detection agents.

[0197] 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.

[0198] The determination of biomarker levels

[0199] The anti-ANPEP antibody can be detected by methods known in the art. Preferably, the anti- ANPEP antibody is detected by an immunoprecipitation assay, an immunoblot assay, or, in particular, an immunoassay. The same applies to the anti-p53 antibody. In the following methods for detecting the anti-ANPEP antibody are summarized. The methods can be used for detecting anti-p53 antibodies mutatis mutandis. It is to be understood that instead of anti-ANPEP detection antigen and / or an anti-ANPEP capture antigen, an anti-p53 detection antigen and / or an anti p53 capture antigen are used, respectively.

[0200] More preferably, the detection of the anti-ANPEP antibody in the sample comprises the step of contacting the sample with an anti-ANPEP detection antigen to allow the formation of a complex comprising the anti-ANPEP detection antigen and the anti-ANPEP antibody present in the sample and the further step of detecting said complex. Detection of this complex indicates presence of anti-ANPEP antibodies.

[0201] Preferably, the anti-ANPEP detection antigen comprises i) the Aminopeptidase N polypeptide, or a fragment thereof which is capable of being bound by the anti-ANPEP antibody, and ii) a detectable label as described below, such as an enzymatic label, a luminescent label, a fluorescent label, a chemiluminescent label or an electrochemiluminescent label.

[0202] More preferably, the detection of the anti-ANPEP antibody in the sample comprises i) contacting the sample with an anti-ANPEP capture antigen and an anti-ANPEP detection antigen, whereby a complex comprising the anti-ANPEP capture antigen, the anti-ANPEP antibody and the anti-ANPEP detection antigen is formed, ii) separating the complex formed in i) from unbound detection antigen, iii) detecting the complex obtained in step ii) via the anti-ANPEP detection antigen (i.e. via the detectable label of the anti-ANPEP detection antigen) comprised therein, thereby detecting the anti-ANPEP antibody comprised in the sample.

[0203] Preferably, the anti-ANEP capture antigen is the Aminopeptidase N polypeptide, or a fragment thereof which is capable of being bound by the anti-ANPEP antibody. Preferably, the anti-ANPEP capture antigen is bound to a solid phase as described herein below.

[0204] Also preferably, the detection of the anti-ANPEP antibody in the sample comprises i) contacting the sample with an anti-ANPEP capture antigen and anti-human antibody antibody comprising a detectable label, whereby a complex comprising the anti- ANPEP capture antigen, the anti-ANPEP antibody and the anti-human antibody antibody comprising a detectable label is formed, ii) separating the complex formed in i) from unbound anti-human antibody antibody, iii) detecting the complex obtained in step ii) via the detectable label of the antibody comprised therein, thereby detecting the anti-ANPEP antibody comprised in the sample.

[0205] The anti-human antibody antibody shall be capable of binding the anti-ANPEP antibody present in the sample. Preferably, the anti-human antibody antibody is an antibody that that binds the constant region of human antibody, such as the Fc region, i.e. its binding does not not interfere with the binding of the autoantibody in the sample to ANPEP. The specificity of the anti human antibody antibody is selected dependent on the isotype of the autoantibodies detected. For example, for detecting an IgG autoantibody, an anti-human IgG antibody is used..

[0206] As set forth above, the detection of anti-ANPEP antibodies may be based on the use of both, an anti ANPEP capture antigen and of an anti-ANPEP detection antigen, respectively. An antibody against ANPEP present in a sample to be investigated binds to both these antigens, thereby forming a complex comprising the anti-ANPEP capture antigen, the anti-ANPEP antibody and the anti-ANPEP detection antigen.

[0207] The term "capture antigen" is familiar to a person skilled in the art. In order to perform a double antigen sandwich immunoassay it is required that one provides an epitope at least twice, at least once on the capture antigen to mediate binding to a solid phase, and at least once on the detection antigen to allow for detection of the sandwich complex (antigenantibody-antigen) formed.

[0208] In one embodiment the anti-ANPEP capture antigen is capable of binding to a solid phase. Materials for manufacturing a solid phase (solid support) are well-known in the art. and include, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and / or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, wells and walls of reaction vessels, plastic tubes etc. In one embodiment the solid phase is represented by the wells and walls of a microtitre plate. In one embodiment the solid phase is in the form of particles. The particle may be a microparticle which is or which comprises magnetic or ferromagnetic metals, alloys or compositions. In further embodiments, the solid phase material may have specific properties and e.g. be hydrophobic, or hydrophilic. In one embodiment of the present invention, the microparticles are paramagnetic microparticles and the separation of such particles in the measurement method according to the present disclosure is facilitated by magnetic forces. Magnetic forces are applied to pull the paramagnetic or magnetic particles out of the solution / suspension and to retain them as desired while liquid of the solution / suspension can be removed and the particles can e.g. be washed. In order for the capture antigen to be capable of binding to a solid phase it is preferred to employ a specific binding pair. One partner of such binding pair is bound to the capture antigen and the other partner of such binding pair is bound to the solid phase. A "binding pair" as used herein consists of two partners binding to each other with high affinity, i.e. with one nanomolar affinity or better. Embodiments for binding pairs are for example the binding pairs consisting of receptor and ligand, antigen and antibody, hapten and anti-hapten antibody, and binding pairs based on naturally occurring high affinity binding pairs. One example of a receptor-ligand binding pair is a pair consisting of a steroid hormone receptor and the corresponding steroid hormone. One further type of a binding pair which is suitable for the method according to the present invention is a hapten and anti-hapten antibody binding pair. A hapten is an organic molecule with a molecular weight of 100 to 2000 Dalton, preferably of 150 to 1000 Dalton. Such small molecule which if used as such is non- immunogenic but can be rendered immunogenic by coupling it to a carrier molecule and anti- hapten antibodies can be generated according to standard procedures. The hapten may be selected from the group comprising sterols, bile acids, sexual hormones, corticoids, cardenolides, cardenolide-glycosides, bufadienolides, steroid- sapogenines and steroid alkaloids, cardenolides and cardenolide-glycosides. Representatives of these substance classes are digoxigenin, digitoxigenin, gitoxigenin, strophanthidin, digoxin, digitoxin, ditoxin, strophanthin. Another suitable hapten is for example fluorescein. Examples of binding pairs based on naturally occurring high affinity binding pairs are biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin and avidin or streptavidin as well as the FimG and DsF binding pair. The biotin- (strept)avidin binding pair is well-known in the art. The basic principles of the FimG-DsF binding pair are e.g. described in WO2012 / 028697.

[0209] In one embodiment, the ANPEP capture antigen is covalently bound to a first partner of a binding pair and the second partner of said binding pair is bound to the solid phase.

[0210] In one embodiment the first partner of the binding pair is selected from hapten, biotin or a biotin analogue such as aminobiotin, iminobiotin or desthiobiotin, DsF and the ligand for a receptor and the second partner of said binding pair is selected from anti-hapten antibody, avidin or streptavidin, FimG and a ligand-receptor.

[0211] In one embodiment first partner of the binding pair is selected from hapten, biotin or a biotin analogue such as aminobiotin, iminobiotin or desthiobiotin and DsF, and the second partner of said binding pair is selected from anti-hapten antibody, avidin or streptavidin and FimG. In one embodiment first partner of the binding pair is selected from biotin or a biotin analogue, such as aminobiotin, iminobiotin or desthiobiotin, and DsF, and the second partner of said binding pair is selected from, avidin or streptavidin and FimG.

[0212] In one embodiment first partner of the binding pair is biotin or a biotin analogue such as aminobiotin, iminobiotin or desthiobiotin and the second partner of said binding pair is selected from avidin or streptavidin. In one embodiment first partner of the binding pair is biotin and the second partner of said binding pair is streptavidin.

[0213] In one embodiment the method according to the present invention is practiced with an ANPEP capture antigen that is biotinylated and a solid phase that is coated with avidin or streptavidin.

[0214] It is, however, also possible to directly bind the capture antigen to a solid phase. In one embodiment the method according to the present invention is practiced with an ANPEP capture antigen that is bound to the solid phase. Antibodies are at least bivalent, i.e. having the potential to bind with one 'arm' to the capture antigen and with the other arm (or any of the other arms for multivalent antibodies) to the detection antigen.

[0215] An antibody to ANPEP present in a sample to be investigated binds to both these antigens, thereby forming a complex comprising the capture antigen, the anti-ANPEP antibody and the detection antigen.

[0216] The skilled person is familiar with the term “detection antigen”. The term detection antigen implies that an antigen to which it is specific can be detected. Such detection can be accomplished by various types of labels which are either directly or indirectly detectable. In one embodiment, the anti-ANPEP detection antigen comprises a directly detectable label. The term detectably labeled encompasses labels that can be directly or indirectly detected. Directly detectable labels either provide a detectable signal or they interact with a second label to modify the detectable signal provided by the first or second label, e.g. to give FRET (fluorescence resonance energy transfer). Labels such as fluorescent dyes and luminescent (including chemiluminescent and electrochemiluminescent) dyes (Briggs et al "Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J. Chem. Soc,Perkin-Trans. 1 (1997) 1051-1058) provide a detectable signal and are generally applicable for labeling. In one embodiment detectably labeled refers to a label providing or inducible to provide a detectable signal, i.e. to a luminescent label, to a fluorescent label, to a chemiluminescent label or to an electrochemiluminescent label, respectively.

[0217] In one embodiment, the microparticle-based analyte-specific binding assay makes use of a chemiluminescent or an electrochemiluminescent label and a corresponding light detection system. The light produced by the label is measured and directly or indirectly indicates the presence or quantity of the analyte.

[0218] Electrochemiluminescent (ECL) assays provide a sensitive and precise measurement of the presence and concentration of an analyte of interest. Such techniques use labels or other reactants that can be induced to luminesce when electrochemically oxidized or reduced in an appropriate chemical environment. Such electrochemiluminescense is triggered by a voltage imposed on a working electrode at a particular time and in a particular manner. In one embodiment the directly detectable label (used to label the detection antigen in a method of the present disclosure) is selected from the group consisting of a luminescent label, a fluorescent label, a chemiluminescent label or an electrochemiluminescent label. In one embodiment, the directly detectable label is a chemiluminescent or an electrochemiluminescent label.

[0219] In one embodiment the double-antigen sandwich assay according to the present disclosure uses the full length ANPEP polypeptide both as the capture as well as the detection antigen Further, a fragment of the human ANPEP, representing at least one important epitope of the ANPEP polypeptide is used. 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 ANPEP polypeptide.

[0220] In some embodiments of the method of the present invention, step a) comprises the determination the level of at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CLEC3B, 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.

[0221] 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.

[0222] The determination of a biomarker as set forth herein, e.g. of the anti-ANPEP antibody, CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG 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, CE-MS, 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.

[0223] In an embodiment, the level of a biomarker as set forth herein, such as of CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG, 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 / journal. pone.0095192). The determined level indicates the level of the biomarker. In another embodiment, the level of a biomarker as set forth herein, such as of CEA, anti- p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG, 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).

[0224] The assessment step

[0225] 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 presence or absence of the anti-ANPEP antibody and, optionally, based presence or absence or the level of the at least one further biomarker as set forth herein above. In an embodiment, colorectal cancer or a precancerous condition thereof based on the presence or absence of the anti-ANPEP antibody, and optionally, based presence or absence or the comparison 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 anti-p53, the level of SOX9 to a reference level of SOX9, the level of Ferritin to a reference level of Ferritin, the level of AREG to a reference level of AREG, the level of DNER to a reference level of DNER, the level of CLEC3B o a reference level of CLEC3B). Further (and as described) elsewhere herein, anti-p53 can also be assessed on presence or absence. 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 presence or absence of the anti-ANPEP antibody and optionally on the level or the presence or absence of the at least one further biomarker. Further (and as described elsewhere herein), anti-p53 can also be assessed on presence or absence.

[0226] As set forth above, it is also envisaged to calculate a score based on based on the presence or absence of the anti-ANPEP antibody, and optionally, based presence or absence or level of the at least on further biomarker for CRC or a precancerous condition thereof, 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 presence or absence of the anti-ANPEP antibody and information on the presence or absence and / or on the level(s) of the at the least one further biomarker. For example, if two biomarkers are detected or determined, the score is calculated based on the information of the two biomarkers (such as the presence or absence or the level). 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.

[0227] The calculated score combines information as assessed in step b). (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 information the individual markers is weighted and the weighted information is 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-ANPEP antibody or 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(-x)))) providing a risk score, which maps the output range of the score to 0-1. If the biomarker is a autoantibody and only presence or absence is assessed, the biomarker concentration value 0 may be used for absence and the biomarker concentration value 1 may be used for presence. How to weight the individual parameters may be determined using a representative reference group. The term “reference group” is defined herein below. In an embodiment, the reference group comprises control and disease subjects (e.g. as described in Examples).

[0228] 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 presence or absence or 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.

[0229] The score as referred to herein is not limited to the biomarker specifically mentioned herein. For examples, the score can also take into account further biomarkers or parameters such as, for example age and / or sex.

[0230] 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 level, 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.

[0231] 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).

[0232] 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).

[0233] 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. In accordance with the present invention, colorectal cancer or a precancerous condition thereof is typically assessed based on the presence or absence of an anti-ANPEP antibody and optionally the comparison (i.e. of the level of the at least one further biomarker to a suitable reference level) 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. Typically, the presence of anti-ANPEP antibodies in a sample of a subject is indicative that the subject is suffering from colorectal cancer or a precancerous condition thereof. Further, a biomarker, such as CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG, 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.

[0234] 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 of 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.

[0235] As the skilled person understands in view of the description herein, the above applies mutatis mutandis to a score, which may incorporate information on the presence or absence or on the level 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.

[0236] 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.

[0237] 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.

[0238] As regards to the diagnosis as referred to herein, the presence of anti-ANPEP antibodies (in the sample) is, typically, indicative for a subject who is suffering from colorectal cancer or a precancerous condition thereof.

[0239] As regards to the identification as referred to herein, the presence of anti-ANPEP antibodies is, typically, indicative for a subject who is eligible to one or more diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof.. Also typically, the absence of anti-ANPEP antibodies is indicative for a subject who is eligible to one or more diagnostic measures for diagnosing colorectal cancer or the precancerous condition thereof. As regards to the differentiation as referred to herein, the presence of anti-ANPEP antibodies is, typically, indicative for a subject that suffers from CRC or advanced adenoma. As regards to the differentiation as referred to herein, the presence of anti-ANPEP antibodies is, typically, indicative for a subject suffers from CRC. Also typically, the absence of anti- ANPEP antibodies is indicative for a subject who does not suffer from CRC.

[0240] As set forth above, the method of the present invention may also encompass the detection / determination of biomarker combinations.

[0241] In case the biomarker combination is a combination of anti-ANPEP antibodies and SOX9 is determined in step a2) typically the following applies. Typically, a subject suffers from CRC or advanced adenoma or is eligible to one or more further diagnostic measures if a) the presence of anti-ANPEP antibodies is detected in the sample, b) the level of SOX9 is increased as compared to the reference, or c) the presence of anti-ANPEP antibodies is detected in the sample and the level of SOX9 is increased as compared to the reference, wherein said reference in a preferred embodiment is a threshold value as specified herein above.

[0242] As regards item a) the level of SOX9 may be decreased as compared to the reference. As regards item b), anti-ANPEP antibodies may be absent in the sample.

[0243] In case the biomarker combination is a combination of anti-ANPEP antibodies and CEA, typically the following applies. Typically, a subject suffers from CRC or advanced adenoma or is eligible to one or more further diagnostic measures if a) the presence of anti-ANPEP antibodies is detected in the sample, b) the level of CEA is increased as compared to the reference (i.e. the reference level), or c) the presence of anti-ANPEP antibodies is detected in the sample and the level of CEA is increased as compared to the reference, wherein said reference in a preferred embodiment is a threshold value as specified herein above.

[0244] As regards item a) the level of CEA may be decreased as compared to the reference. As regards item b), anti-ANPEP antibodies may be absent in the sample.

[0245] In case the biomarker combination is a combination of anti-ANPEP antibodies and CFB, typically the following applies. Typically, a subject suffers from CRC or advanced adenoma or is eligible to one or more further diagnostic measures if a) the presence of anti-ANPEP antibodies is detected in the sample, b) the level of CFB is increased as compared to the reference (i.e. the reference level), or c) the presence of anti-ANPEP antibodies is detected in the sample and the level of CFB is increased as compared to the reference, wherein said reference in a preferred embodiment is a threshold value as specified herein above.

[0246] As regards item a) the level of CFB may be decreased as compared to the reference. As regards item b), anti-ANPEP antibodies may be absent in the sample.

[0247] In case the biomarker combination is a combination of anti-ANPEP antibodies and AREG, typically the following applies. Typically, a subject suffers from CRC or advanced adenoma or is eligible to one or more further diagnostic measures if a) the presence of anti-ANPEP antibodies is detected in the sample, b) the level of AREG is increased as compared to the reference, or c) the presence of anti-ANPEP antibodies is detected in the sample and the level of AREG is increased as compared to the reference, wherein said reference in a preferred embodiment is a threshold value as specified herein above.

[0248] As regards item a), the level of AREG may be decreased as compared to the reference. As regards item b), anti-ANPEP antibodies may be absent in the sample.

[0249] In case the biomarker combination is a combination of anti-ANPEP antibodies and anti-p53, typically the following applies. Typically, a subject suffers from CRC or advanced adenoma or is eligible to one or more further diagnostic measures if a) the presence of anti-ANPEP antibodies is detected in the sample, b) the level of anti-p53 is increased as compared to the reference, or c) the presence of anti-ANPEP antibodies is detected in the sample and the level of anti- p53 is increased as compared to the reference, wherein said reference in a preferred embodiment is a threshold value as specified herein above.

[0250] As regards item a), the level of anti-p53 may be decreased as compared to the reference. As regards item b), anti-ANPEP antibodies may be absent in the sample.

[0251] Alternatively, a subject suffers from CRC or advanced adenoma or is eligible to one or more further diagnostic measures if a) the presence of anti-ANPEP antibodies is detected in the sample, b) the presence of anti-p53 antibodies is detected in the sample, or c) the presence of anti-ANPEP antibodies and anti-p53 antibodies is detected in the sample.

[0252] In case the biomarker combination is a combination of anti-ANPEP antibodies, CEA and anti-p53, typically the following applies. Typically, a subject suffers from CRC or advanced adenoma or is eligible to one or more further diagnostic measures if a) the presence of anti-ANPEP antibodies is detected in the sample, b) the level of anti-p53 and / or of CEA is increased as compared to the reference, or c) the presence of anti-ANPEP antibodies is detected in the sample and the level of anti- p53 and / or CEA is increased as compared to the reference, wherein said reference in a preferred embodiment is a threshold value as specified herein above.

[0253] As regards item a), the level of level of anti-p53 and / or of CEA may be decreased as compared to the reference. As regards item b), anti-ANPEP antibodies may be absent in the sample.

[0254] Alternatively, a subject suffers from CRC or advanced adenoma or is eligible to one or more further diagnostic measures if a) the presence of anti-ANPEP antibodies is detected in the sample b) the presence of anti-p53 antibodies is detected in the sample, and / or b) the level CEA is increased as compared to the reference, wherein said reference in a preferred embodiment is a threshold value as specified herein above.

[0255] In case the biomarker combination is a combination of anti-ANPEP antibodies and Ferritin, typically the following applies. Typically, a subject suffers from CRC or advanced adenoma or is eligible to one or more further diagnostic measures if a) the presence of anti-ANPEP antibodies is detected in the sample, b) the level of Ferritin is decreased as compared to the reference, or c) the presence of anti-ANPEP antibodies is detected in the sample and the level of Ferritin is decreased as compared to the reference, wherein said reference in a preferred embodiment is a threshold value as specified herein above.

[0256] As regards item a), the level of Ferritin may be increased as compared to the reference. As regards item b), anti-ANPEP antibodies may be absent in the sample.

[0257] In case the biomarker combination is a combination of anti-ANPEP antibodies and CLEC3B, typically the following applies. Typically, a subject suffers from CRC or advanced adenoma or is eligible to one or more further diagnostic measures if a) the presence of anti-ANPEP antibodies is detected in the sample, b) the level of CLEC3B is decreased as compared to the reference, or c) the presence of anti-ANPEP antibodies is detected in the sample and the level of CLEC3B is decreased as compared to the reference, wherein said reference in a preferred embodiment is a threshold value as specified herein above.

[0258] As regards item a), the level of CLEC3B may be increased as compared to the reference. As regards item b), anti-ANPEP antibodies may be absent in the sample.

[0259] In case the biomarker combination is a combination of anti-ANPEP antibodies and DNER, typically the following applies. Typically, a subject suffers from CRC or advanced adenoma or is eligible to one or more further diagnostic measures if a) the presence of anti-ANPEP antibodies is detected in the sample, b) the level of DNER is decreased as compared to the reference, or c) the presence of anti-ANPEP antibodies is detected in the sample and the level of DNER is decreased as compared to the reference, wherein said reference in a preferred embodiment is a threshold value as specified herein above.

[0260] As regards item a), the level of DNER may be increased as compared to the reference. As regards item b), anti-ANPEP antibodies may be absent in the sample.

[0261] 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.

[0262] Additional steps of the method of the present invention (therapeutic and diagnostic methods).

[0263] 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 measure 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) as referred to herein, i.e. anti-ANPEP antibody and, optionally one or more further biomarkers for CRC, such as CEA, anti-p53, SOX9, DNER, CFB, CLEC3B, Ferritin and AREG, 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.

[0264] 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.

[0265] 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.

[0266] Moreover, the patient may be subjected to combinations of the above treatments.

[0267] Computer-implemented method

[0268] 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, information on the presence or absence of an anti-ANPEP antibody in a sample from a subject, and, optionally information for at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), SOX9 (SRY-box transcription factor 9), of CLEC3B (C-type lectin domain family 3 member B), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), anti-p53, Ferritin and AREG (Amphiregulin) in said sample, wherein the information for said at least one further biomarker is a value for the level or information on the present or absence of said at least one further biomarker, b) assessing, preferably by the processing unit, colorectal cancer or the precancerous condition thereof based on the information on the presence or absence of an anti-ANPEP antibody and, optionally, based on the information for said at least one further biomarker received in step a).

[0269] 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.

[0270] Moreover, the present invention relates to a database comprising one or more stored references for anti-ANPEP antibodies and, optionally one or more stored references for at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG. Preferably, the one or more stored references allow for assessing colorectal cancer or a precancerous condition thereof.

[0271] The term “database” has been defined above. The definition applies accordingly.

[0272] 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.

[0273] 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.

[0274] Devices, Kits, uses and detection methods

[0275] Moreover, the present invention relates to a device for assessing colorectal cancer or a precancerous condition thereof, said device comprising: a) at least one detection unit for detecting an anti-ANPEP antibody and, optionally, for detecting the level of at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), CLEC3B (C-type lectin domain family 3 member B), Anti-p53, 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 detection unit comprising at least one detection agent for the anti-ANPEP antibody and, optionally, at least one detection agent for the biomarker CEA, at least one detection agent for the biomarker CLEC3B, at least one detection agent for the 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 detection unit, said evaluation unit comprising a data processor comprising instructions for assessing colorectal cancer or a precancerous condition thereof based on the presence or absence of the anti-ANPEP antibody and, optionally, based on presence or absence or the level of at least one further biomarker. The term “device” has been defined herein above. The definition applies accordingly.

[0276] The detection unit (herein also referred to as analyzing unit or measuring unit), typically, comprises at least one reaction zone having a detection agent for anti-ANPEP antibody and 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.

[0277] 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).

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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. 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.

[0282] 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.

[0283] 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 person. 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.

[0284] “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.

[0285] 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 anti- ANPEP antibody and at least one detection agent for at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG.

[0286] The term “detection agent” has been defined elsewhere herein.

[0287] In a preferred embodiment, an anti-ANPEP detection antigen as defined elsewhere herein is used as detection agent for the anti-ANPEP antibody. In another preferred embodiment, an anti-ANPEP capture antigen as defined elsewhere herein is used as detection agent for the anti-ANPEP antibody. In yet another preferred embodiment, an anti-ANPEP capture antigen and an anti-ANPEP detection antigen are used as detection agents for the anti-ANPEP antibody. In yet another preferred embodiment, an anti-ANPEP capture antigen and an a anti-human antibody with a detectable lable are used as detection agent for the anti-ANPEP antibody.

[0288] In a preferred embodiment, an anti-p53 detection antigen is used as detection agent for the anti-p53 antibody. In another preferred embodiment, an anti-p53 capture antigen as herein is used as detection agent for the anti-p53 antibody. In yet another preferred embodiment, an anti-p53 capture antigen and an anti-p53 detection antigen are used as detection agents for the anti-p53 antibody. In yet another preferred embodiment, an anti-p53 capture antigen and an a anti-human antibody with a detectable lable are used as detection agent for the anti- p53 antibody.

[0289] In a preferred embodiment, the detection agent for CEA, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG, is an antibody, or antigen-binding fragments thereof, which specifically binds to the biomarker as referred to herein.

[0290] Moreover, the present invention relates to the use of i. an anti-ANPEP antibody, or one or more detection agents for said anti- ANPEP antibody, and optionally ii. at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG, or at least one detection agent for said at least one further biomarker, in a sample (such as ablood, serum or plasma) from a subject for assessing colorectal cancer or a precancerous condition thereof.

[0291] Moreover, the present invention relates to the use of i. one or more detection agents for said anti-ANPEP antibody, and optionally ii. or at least one detection agent for at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of CEA, anti-p53, SOX9, DNER, CLEC3B, CFB, Ferritin and AREG, for the biomarker detection a sample (such as blood, serum or plasma) from a subject as referred herein.

[0292] Preferably, said use is an in vitro use. The term “detection agent” has been defined above.

[0293] 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.

[0294] Moreover, the present invention relates to method for biomarker detection in a sample, such as blood, serum or plasma sample from a subject suspected to suffer from colorectal cancer or a precancerous condition thereof. Preferably, the anti-ANPEP antibody is detected as described elsewhere herein. More preferably, comprising i) contacting a portion of said blood, serum or plasma sample with one or more detection agents which specifically bind to an anti-ANPEP antibody to allow the formation of a complex comprising the anti-ANPEP antibody present in the sample and the one or more detection agents, and ii) detecting said complex, preferably in order detect the presence or absence of an anti-ANPEP antibody

[0295] Preferably, an anti-ANPEP detection antigen is used as detection agent (e.g. in the kit, device or detection method). More preferably, an anti-ANPEP detection antigen and an anti-ANPEP capture antigen are used as detection agents. More preferably, anti-ANPEP capture antigen and an anti-human antibody with a detectable label (e.g anti-human IgG antibody with a detectable label) are used as detection agents.

[0296] In preferred embodiment of the above method, the method comprises i) contacting the portion of the sample with an anti-ANPEP capture antigen and an anti-ANPEP detection antigen as detection agents, whereby a complex comprising the anti-ANPEP capture antigen, the anti-ANPEP antibody and the anti-ANPEP detection antigen is formed, ii) separating the complex formed in i) from unbound detection antigen, and iii) detecting the complex obtained in step ii) via the anti-ANPEP detection antigen comprised therein, thereby detecting the anti-ANPEP antibody comprised in the sample.

[0297] In preferred embodiment of the above method, the method comprises i) contacting the portion of the sample with an anti-ANPEP capture antigen and a detection agent, whereby a complex comprising the anti-ANPEP capture antigen, the anti-ANPEP antibody and the detection agent is formed, ii) separating the complex formed in i) from unbound detection agent, and iii) detecting the complex obtained in step ii) via the detection agent comprised therein, thereby detecting the anti-ANPEP antibody comprised in the sample.

[0298] The detection agent is preferably and agent that binds to a complex formed by the ANPEP capture antigen and the anti-ANPEP antibody. Preferably, said detection agent is an agent comprising a detectable label. Preferably, said detection agent is an anti-human antibody antibody comprising a detectable label or an anti-ANPEP detection antigen as defined elsewhere herein.

[0299] In another preferred embodiment of the above method, the method comprises i) contacting the portion of the sample with an anti-ANPEP detection antigen as detection agent, whereby a complex comprising the anti-ANPEP antibody and the anti-ANPEP detection antigen is formed, ii) separating the complex formed in i) from unbound anti-ANPEP detection antigen, and iii) detecting the complex obtained in step ii) via the anti-ANPEP detection antigen comprised therein, thereby detecting the anti-ANPEP antibody comprised in the sample.

[0300] In yet another preferred embodiment of the above method, the method comprises i) contacting the portion of the sample with an anti-ANPEP capture antigen and an anti-human antibody with a detectable label as detection agents, whereby a complex comprising the anti-ANPEP capture antigen, the anti-ANPEP antibody and the anti-human antibody antibody with a detectable label is formed, ii) separating the complex formed in i) from unbound anti-human antibody antibody, and iii) detecting the complex obtained in step ii) via the anti-human antibody antibody with a detectable label comprised therein, thereby detecting the anti-ANPEP antibody comprised in the sample.

[0301] In a preferred embodiment, the method further comprising determining the level of at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), SOX9 (SRY-box transcription factor 9), CLEC3B, Anti-p53, DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin) in the same or in a different portion of said blood, serum or plasma sample from said subject. Again, one or more detection agents that bind to the markers can be used.

[0302] 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. In an embodiment, it is known that the subjects suffers from colorectal cancer or a precancerous condition thereof.

[0303] 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:

[0304] 1. A method for assessing colorectal cancer or a precancerous condition thereof in a subject, comprising the steps of a) detecting the presence or absence of an anti-ANPEP antibody (anti- Aminopeptidase N antibody) 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 presence or absence of the anti-ANPEP antibody.

[0305] 2. The method of embodiment 1, wherein the subject is suspected to suffer from colorectal cancer or the precancerous condition thereof.

[0306] 3. The method of embodiment 1 or 2, wherein step a) further comprises detecting the presence or absence or the determining the level of one or more further biomarkers for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), of CLEC3B (C-type lectin domain family 3 member B), S0X9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), anti-p53, Ferritin and AREG (Amphiregulin) in the blood, serum or plasma sample. The method of embodiment 3, wherein the assessment in step b) comprises calculating a score for assessing colorectal cancer or the precancerous condition thereof, wherein the score is calculated based on the presence or absence of the anti- ANPEP antibody and the level or the presence or absence of the at least one further biomarker, and assessing colorectal cancer or the precancerous condition thereof based on the calculated score. The method of embodiment 3, wherein colorectal cancer or the precancerous condition thereof is assessed based on i) the presence or absence of the anti-ANPEP antibody and ii) the presence or absence of the at least one further biomarker and / or the comparison of the level of the at least one further biomarker to a reference level for said at least one further biomarker. The method of embodiment 5, 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. The method of any one of embodiments 1 to 6, wherein the precancerous condition of colorectal cancer is advanced adenoma. The method of any one of embodiments 1 to 7, wherein the colorectal cancer encompasses stage 0 (pTis), stage I, stage II, stage III and stage IV colorectal cancer. The method of any one of embodiments 1 to 8, wherein the assessment of colorectal cancer or the precancerous condition thereof is the diagnosis of colorectal cancer or the precancerous condition thereof. The method of embodiment 9, wherein the presence of the anti-ANPEP antibody indicates that the subject suffers from colorectal cancer or the precancerous condition thereof. 11. The method of embodiment 9, wherein the diagnosis is the rule in of colorectal cancer or the precancerous condition thereof.

[0307] 12. The method of any one of embodiments 9 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 suffering from colorectal cancer or the precancerous condition thereof.

[0308] 13. The method of any one of embodiments 9 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.

[0309] 14. The method of any one of embodiments 1 to 8, 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.

[0310] 15. The method of any one of embodiments 1 to 8, 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.

[0311] 16. The method of embodiment 15, 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, in particular wherein the further diagnostic measure is colonoscopy.

[0312] 17. The method of any one of embodiments 1 to 16, wherein the anti-ANPEP antibody is detected by an immunoprecipitation assay, an immunoblot assay, or an immunoassay.

[0313] 18. The method of embodiment 1 to 17, wherein the detection of the anti-ANPEP antibody in the sample comprises the step of contacting the sample with an anti-ANPEP detection antigen to allow the formation of a complex comprising the anti-ANPEP detection antigen and the anti-ANPEP antibody present in the sample and the further step of detecting said complex. The method of any one of embodiments 1 to 18, wherein the detection of the anti- ANPEP antibody in the sample comprises: i) contacting the sample with an anti-ANPEP capture antigen and an anti-ANPEP detection antigen, whereby a complex comprising the anti-ANPEP capture antigen the anti-ANPEP antibody and the anti-ANPEP detection antigen is formed, ii) separating the complex formed in i) from unbound detection antigen, iii) detecting the complex obtained in step ii) via the detection antigen comprised therein, thereby detecting the anti-ANPEP antibody comprised in the sample. The method embodiment 18 or 19, wherein the anti-ANPEP detection antigen comprises i) the Aminopeptidase N polypeptide, or a fragment thereof which is capable of being bound by the anti-ANPEP antibody, and ii) a detectable label. The method of embodiment 20, wherein the detectable label is selected from the group consisting of an enzymatic label, a luminescent label, a fluorescent label, a chemiluminescent label or an electrochemiluminescent label. The method of any one of embodiments 19 to 21, wherein the anti-ANPEP capture antigen is the Aminopeptidase N polypeptide, or a fragment thereof which is capable of being bound by the anti-ANPEP antibody. The method of embodiment 22, wherein the anti-ANPEP capture antigen is bound to a solid phase. The method of any one of embodiment 1 to 23, wherein the detected antibody is an IgG antibody. A computer-implemented method for assessing colorectal cancer or a precancerous condition thereof, comprising a) receiving, at a processing unit, information on the presence or absence of an anti-ANPEP antibody in a sample from a subject, and, optionally information (such as information on the presence or absence, or the level) for at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), SOX9 (SRY-box transcription factor 9), of CLEC3B (C-type lectin domain family 3 member B), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), anti-p53, Ferritin and AREG (Amphiregulin) in said sample, b) assessing, preferably by the processing unit, colorectal cancer or the precancerous condition thereof based on the information on the presence or absence of an anti-ANPEP antibody and, optionally, on the information for the at least one further biomarker.

[0314] 26. Use of i. an anti-ANPEP antibody, or one or more detection agents for said anti- ANPEP antibody, and optionally ii. at least one biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), CLEC3B (C-type lectin domain family 3 member B), anti-p53, 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.

[0315] 27. The use of embodiment 26, wherein the assessment is the is the diagnosis of colorectal cancer or the precancerous condition thereof.

[0316] 28. The use of embodiment 26, wherein 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.

[0317] 29. The use of embodiment 26, 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. 30. The use of any one of embodiments 26 to 29, wherein a) an anti-ANPEP capture antigen or b) an anti-ANPEP capture antigen and an anti-ANPEP detection antigen is (are) used as detection agent(s) for said anti-ANPEP antibody.

[0318] 31. A device for assessing colorectal cancer or a precancerous condition thereof, said device comprising: a) at least one detection unit for detecting an anti-ANPEP antibody and, optionally, for detecting at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), CLEC3B (C-type lectin domain family 3 member B), Anti-p53, 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 detection unit comprising at least one detection agent for the anti-ANPEP antibody and, optionally, at least one detection agent for the biomarker CEA, at least one detection agent for the biomarker CLEC3B, at least one detection agent for the 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 detection unit, said evaluation unit comprising a data processor comprising instructions for assessing colorectal cancer or a precancerous condition thereof based on the presence or absence of the anti-ANPEP antibody and, optionally, based on the presence or absence or the level of at least one further biomarker.

[0319] 32. A kit for assessing colorectal cancer or a precancerous condition thereof, said kit comprising at least one detection agent for anti-ANPEP antibody and at least one detection agent for at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), CLEC3B, Anti-p53 SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin). 33. A method for biomarker detection in a sample, such as 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 which specifically bind to an anti-ANPEP antibody to allow the formation of a complex comprising the anti-ANPEP antibody present in the sample and the one or more detection agents, and ii) detecting said complex.

[0320] 34. The method of embodiment 33, comprising i) contacting the portion of the sample with an anti-ANPEP capture antigen and an anti-ANPEP detection antigen as detection agents, whereby a complex comprising the anti-ANPEP capture antigen, the anti-ANPEP antibody and the anti-ANPEP detection antigen is formed, ii) separating the complex formed in i) from unbound detection antigen, iii) detecting the complex obtained in step ii) via the detection antigen comprised therein, thereby detecting the anti-ANPEP antibody comprised in the sample.

[0321] 35. The method of embodiment 33 or 34, further comprising determining the level of at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), SOX9 (SRY-box transcription factor 9), CLEC3B, Anti-p53, DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin) in the same or in a different portion of said blood, serum or plasma sample from said subject.

[0322] 36. The method of any one of embodiments 33 to 35, wherein the sample is from a subject who suffers from colorectal cancer or a precancerous condition thereof.

[0323] 37. A computer program including computer-executable instructions for performing the computer-implemented method according to embodiment 26, when the program is executed on a computer or computer network.

[0324] All patents, patent applications, and publications or public disclosures referred to or cited herein are incorporated by reference in their entirety. 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.

[0325] Example 1: Study setup

[0326] Biomarker levels using immunoassays were measured in samples from the Potsdam CRC study that comprises samples from 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.

[0327] 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. From a total of 4088 subjects that underwent colonoscopy a subset of 531 (526 S4, 5 MSKK) patients have been selected. 323 of the selected subjects had either no findings (nonfindings), other findings (e.g. inflammatory bowel disease) or only benign findings (low grade intraepithelial neoplasia) (‘Controls’) while 116 had malignant findings (CRC + pTis (pathological tumor in situ)) and 92 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 adenoma cases as well, unless specifically stated otherwise.

[0328] 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.

[0329] Example 2: Immunological detection of anti-ANPEP autoantibodies

[0330] ANPEP (Aminopeptidase N) (UniProt ID: P15144) levels were assessed using the CDI HuProt technology.

[0331] HuProt is the microarray-based technology by CDI Labs Inc. which enables it to screen autoantibody response profiles against more than 21000 unique human proteins or iso forms proteins, covering more than 81% of the human proteome. The basis of the platform are recombinantly produced full-length human proteins in yeast which are GST-purified and subsequently immobilized on microarray chips. For a HuProt assay, plasma samples of individuals are incubated with the microarray, allowing a patient’s antibodies to react and bind to the exposed immobilized proteins. Afterwards, fluorescence labeled secondary antibodies against the primary patient antibodies are incubated and their binding events are measured using a fluorescence scanner, which in turn sheds light on auto-antibody reactivity against the human proteome (Venkataraman, A., Yang, K., Irizarry, J. et al. A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors. Nat Methods 15, 330-338 (2018). https: / / doi.org / 10.1038 / nmeth.4632).

[0332] The ANPEP data for this patent were created using HuProt 4.0 microarrays against IgG response. Data was preprocessed and normalized using quantile normalization by CDI Labs and subsequently log transformed. Due to the fact that auto-antibodies typically contribute clinical predictive performance with high specificity, we binarized the signal intensities. To that end, we selected batch-specific 97%-specificity thresholds and set values exceeding this threshold to one and otherwise to zero.

[0333] CEA, Ferritin and anti-TP53 levels were measured using the respective Roche Elecsys® ECLIA (electrochemiluminescence immunoassay) assays on the same samples. The data of other biomarkers (including 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).

[0334] 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).

[0335] Example 3: Description of diagnostic performance calculation of individual marker and marker combination

[0336] Performance of individual markers is reported as “area under the receiver operating curve” (AUC) for distinguishing cases from controls. Additionally, the sensitivity at 90% specificity of the respective marker or marker panel is reported. Depending on the defined endpoint, the composition of cases differs between (1) CRC + pTis cases and (2) CRC + pTis + HG. For the biomarker combinations, the markers were mathematically combined via logistic regression and the AUC and the sensitivity were again used as general measures for marker and marker combination performance.

[0337] Overall, ANPEP was found as one of the best auto-antibodies to increase the performance of the current reference marker carcinoembryonic antigen (CEA) in distinguishing Controls from CRC + pTis cases out of —20,000 measured auto-antibodies (see Example 5). Additionally, ANPEP was shown to increase the performance of other biomarkers or biomarker combinations (see Example 7, 8). Further, for distinguishing Controls from CRC + pTis + HG, ANPEP was again found to increase the performance of CEA and also of other biomarkers (see Example 7, 8). CEA is so far known as the best performing single marker for CRC (Wild N. et al., 2010).

[0338] Example 4: 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 have a baseline comparator performance, CEA levels were measured in the same sample set as ANPEP using the Roche Elecsys® CEA ECLIA (electrochemiluminescence immunoassay) method. CEA was shown to achieve a relatively good performance with an AUC of 0.655 for distinguishing CRC + pTis from controls and a sensitivity at 90% specificity of 33.6%, amounting to 39 true positive predictions in our dataset (see Table 2 and Figure 3). When adding HG to the case group, the AUC slightly decreased to 0.601 and the sensitivity decreased to 23.1%, amounting to 48 true positive predictions in our dataset (see Table 2 and Figure 4).

[0339] Figure 3 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 4 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.

[0340] Table 2: 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 as well as the sensitivity at 90% specificity and number of true positive predictions (TP) for both endpoints. N depicts the number of samples tested (cases plus controls, numbers in each group vary depending on the analyses). Example 5: Diagnostic performance of ANPEP and CEA combined:

[0341] In order to assess the added value of ANPEP on top of CEA, a known benchmark biomarker, the two biomarkers were combined via logistic regression and the AUC as well as the sensitivity at 90% specificity were again used as performance measurements. Adding ANPEP to CEA improves the performance of CEA alone from 39 true positive predictions to 45 true positives (representing an improved sensitivity of 5.2%) for detecting CRC cases + pTis compared to controls. Likewise, for detecting CRC cases + pTis + HG, the number of true positive predictions improves from 48 to 60 (representing an improved sensitivity of 5.7%), when using CEA alone and in combination with ANPEP, respectively (see Table 3 and Figure 5).

[0342] Table 3: Calculation of diagnostic performance of CEA biomarker combined with ANPEP 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, the number of true positives (TP), as well as the sensitivity at 90% specificity and the 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). Example 6: Comparison with combined diagnostic performance of CEA with ap53:

[0343] To confirm specificity of the CEA performance improvement by its combination with ANPEP, we have combined CEA with another established auto-antibody, anti-p53 (ap53), as a reference. The combination of CEA and ap53 results in a slight performance improvement compared to using CEA alone for both endpoints. With ap53, two additional true positives are identified (improved sensitivity of 1.7%) for separating CRC tumor cases + pTis from controls and one additional true positive is identified (improved sensitivity of 0.5%) for discriminating CRC tumor cases + pTis + HG / AA from controls (see Table 4 and Figure 5). On the other hand, CEA and ANPEP lead substantially greater performance improvement for both endpoints (see Figure 5).

[0344] Table 4: 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, the number of true positives (TPs), as well as the sensitivity at 90% specificity 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)

[0345] Example 7. Diagnostic performance of ANPEP combined with other biomarkers:

[0346] In addition to combining ANPEP with an established biomarker like CEA, we analyzed combinations of ANPEP with other markers. The markers were again mathematically combined via logistic regression and the AUC as well as the sensitivity at 90% specificity was used as general measures for marker performance. We observe that combining the markers with ANPEP leads to a performance improvement for at least one endpoint or performance metric. For instance when combining SOX9 and ANPEP, the number of true positive predictions increases by 4 and 12 when considering CRC tumor + pTis or CRC tumor + pTis + HG / AA vs controls in our cohort, which otherwise would have remained false negative predictions (see Figure 5, Table 5 and Table 6). In a cancer screening setting this result would mean that these patients might not receive the necessary followup diagnostic care and early treatment.

[0347] Biomarker name abbreviations:

[0348] DNER (UniProt ID: Q8NFT8): Delta and Notch-like epidermal growth factor-related receptor

[0349] CLEC3B (UniProt ID: P05452): Tetranectin, C-type lectin domain family 3 member B CFB (UniProt ID: P00751): Complement factor B AREG (UniProt ID: P15514): Amphiregulin

[0350] SOX9 (UniProtID: P48436): Transcription Factor SOX9

[0351] Example 7a: Combinations of marker pairs (bivariate marker combinations) having improved AUCs and sensitivity over the single markers by at least one percentage point for discriminating subjects with no or only benign findings and subjects with malignant findings.

[0352] Table 5: Bivariate marker combinations of ANPEP with an additional marker for discriminating CRC Cases + pTis versus Controls, other findings, low grade adenoma (LG). The performances are shown in AUC, number of true positives (TP) and Sensitivity. For each metric, the performance improvement over the other marker (without ANPEP) is listed, showing that ANPEP improves the performance for at least one metric for every combination. Example 7b: Combinations of marker pairs (bivariate marker combinations) having improved AUCs and sensitivity 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.

[0353] Controls (Controls, other findings, LG) v.v. CRC Cases + pTis + HG

[0354] Table 6: Bivariate marker combinations of ANPEP with an additional marker for discriminating CRC Cases + pTis + HG / AA versus Controls, other findings, low grade adenoma (LG). The performances are shown in AUC, number of true positives (TP) and Sensitivity. For each metric, the performance improvement over the other marker (without ANPEP) is listed, showing that ANPEP improves the performance for at least one metric for every combination. Example 7c: Combinations of marker pairs (bivariate marker combinations) having improved AUCs and sensitivity over the single markers by at least one percentage point for discriminating subjects with no or only benign findings and subjects with potential future malignant findings.

[0355] Controls (Controls, other findings, LG) v.v. HG cases Table 7: Bivariate marker combinations of ANPEP with an additional marker for discriminating HG / AA Cases versus Controls, other findings, low grade adenoma (LG). The performances are shown in AUC, number of true positives (TP) and Sensitivity. For each metric, the performance improvement over the other marker (without ANPEP) is listed, showing that ANPEP improves the performance for at least one metric for every combination.

[0356] Example 8. Diagnostic performance of ANPEP combined with CEA and ap53:

[0357] To further confirm and highlight the added value of ANPEP, we combined ANPEP with the other two established biomarkers CEA and ap53. Adding ANPEP on top of the other two markers, further increased the sensitivity by 4.4% / 5.2% for distinguishing controls from CRC cases + pTis / CRC cases + pTis + HG. Similarly, we observe 5 and 11 additional true positive predictions, which in clinical practice could benefit from suitable follow-up diagnostic possibilities or early treatment options (see Table 8 and Figure 5).

[0358] Table 8: Calculation of diagnostic performance of CEA and anti-p53 biomarkers combined with ANPEP 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, the sensitivity at 90% specificity and the number of true positives (TP). In addition, performance improvement for each metric over CEA and anti-p53 alone is given. N depicts the number of samples tested (cases plus controls, numbers in each group vary depending on the analyses).

Claims

Roche Diagnostics International AG RD39651PC Roche Diagnostics GmbHClaims1. A method for assessing colorectal cancer or a precancerous condition thereof in a subject suspected to suffer from colorectal cancer or the precancerous condition thereof, comprising the steps of a) detecting the presence or absence of an anti-ANPEP antibody (anti- Aminopeptidase N antibody) in a blood, serum or plasma sample from the subject, and b) assessing colorectal cancer or the precancerous condition thereof based on the presence or absence of the anti-ANPEP antibody.

2. The method of claim 1, wherein step a) further comprises detecting the presence or absence or determining the level of at least one further biomarker for colorectal cancer or a precancerous condition thereof in the blood, serum or plasma sample, for example wherein the at least one further biomarker is selected from the group consisting of carcinoembryonic antigen (CEA), of CLEC3B (C-type lectin domain family 3 member B), SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), anti-p53, Ferritin and AREG (Amphiregulin) and wherein colorectal cancer or the precancerous condition thereof is assessed based on the presence or absence of the anti-ANPEP antibody and the detected presence or absence or the determined level of said at least one further biomarker for colorectal cancer or a precancerous condition thereof.

3. The method of claim 2, wherein the assessment in step b) comprises calculating a score for assessing colorectal cancer or the precancerous condition thereof, wherein the score is calculated based on the presence or absence of the anti-ANPEP antibody and the level or presence or absence of the at least one further biomarker, or wherein colorectal cancer or the precancerous condition thereof is assessed based on i) the presence or absence of the anti-ANPEP antibody and ii) the presence or absence of the at least one further biomarker or the comparison of the level of the at least one further biomarker to a reference level for said at least one further biomarker, wherein preferably the reference level for said at least one further biomarker is a predeterminedvalue for the level of said at least one further biomarker which allows for assessing colorectal cancer or a precancerous condition thereof.

4. The method of any one of claims 1 to 3, 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.

5. The method of any one of claims 1 to 4, wherein the assessment of colorectal cancer or the precancerous condition thereof is the diagnosis of colorectal cancer or the precancerous condition thereof.

6. The method of claim 5, wherein the presence of the anti-ANPEP antibody indicates that the subject suffers from colorectal cancer or a precancerous condition thereof.

7. The method of any one of claims 1 to 6, 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 suffering from colorectal cancer or the precancerous condition thereof.

8. The method of any one of claims 1 to 4, 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 4, 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 preferably 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 particular wherein the further diagnostic measure is colonoscopy.

10. The method of any one of claims 1 to 9, wherein the anti-ANPEP antibody is detected by an immunoprecipitation assay, an immunoblot assay, or an immunoassay, forexample wherein the detection of the anti-ANPEP antibody in the sample comprises the step of contacting the sample with an anti-ANPEP detection antigen to allow the formation of a complex comprising the anti-ANPEP detection antigen and the anti- ANPEP antibody present in the sample and the further step of detecting said complex.

11. A computer-implemented method for assessing colorectal cancer or a precancerous condition thereof, comprising a) receiving, at a processing unit, information on the presence or absence of an anti-ANPEP antibody in a sample from a subject, and, optionally information for at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), SOX9 (SRY-box transcription factor 9), of CLEC3B (C-type lectin domain family 3 member B), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), anti-p53, Ferritin and AREG (Amphiregulin) in said sample, wherein the information on the at least one further biomarker is information on the presence or absence of the biomarker or at least one value for the level of said at least one further biomarker, and b) assessing, preferably by the processing unit, colorectal cancer or the precancerous condition thereof based on the information on the presence or absence of an anti-ANPEP antibody and, optionally, on the information for the at least one further biomarker received in step a).

12. Use of i. an anti-ANPEP antibody, or one or more detection agents for said anti- ANPEP antibody, and optionally ii. at least one biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), CLEC3B (C-type lectin domain family 3 member B), anti-p53, 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.

13. A device for assessing colorectal cancer or a precancerous condition thereof, said device comprising: a) at least one detection unit for detecting an anti-ANPEP antibody and, optionally, for detecting or determining the level of at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), CLEC3B (C-type lectin domain family 3 member B), Anti-p53, 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 detection unit comprising at least one detection agent for the anti-ANPEP antibody and, optionally, at least one detection agent for the biomarker CEA, at least one detection agent for the biomarker CLEC3B, at least one detection agent for the 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 detection unit, said evaluation unit comprising a data processor comprising instructions for assessing colorectal cancer or a precancerous condition thereof based on the presence or absence of the anti-ANPEP antibody and, optionally, based on the presence or absence or the level of at least one further biomarker.

14. A kit for assessing colorectal cancer or a precancerous condition thereof, said kit comprising at least one detection agent for anti-ANPEP antibody and at least one detection agent for at least one further biomarker for colorectal cancer or a precancerous condition thereof, for example at least one further biomarker selected from the group consisting of carcinoembryonic antigen (CEA), CLEC3B, Anti-p53 SOX9 (SRY-box transcription factor 9), DNER (Delta and Notch-like epidermal growth factor-related receptor), CFB (Complement factor B), Ferritin and AREG (Amphiregulin).

15. A method for biomarker detection 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 which specifically bind to an anti-ANPEP antibody to allow the formation of a complex comprising the anti-ANPEP antibody present in the sample and the one or more detection agents, and ii) detecting said complex.

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