The present disclosure provides biomarkers, methods and kits for the assessment of whole blood samples, wherein such assessment targets any of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyps. Biomarkers disclosed are polynucleotides originating from genetic loci selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and

LRR 3.

Biomarkers have been identified for assessment and further analysis of colorectal cancer, colorectal adenoma, and colorectal polyps (the latter two are also referred to as precursors of colorectal cancer). Originally, the biomarkers have been identified using an oligonucleotide array-based screening of R A from biopsies and blood from patients with colorectal cancer or colorectal adenoma. The biomarkers have been established to assess changes in blood samples against normal blood. To facilitate a more robust test system suitable for routine screening, the biomarkers have been developed to a real-time PCR format suitable for assessing in vitro different stages selected from colorectal cancer, colorectal adenoma, and colorectal polyp using a blood sample from a patient. The biomarkers of the present disclosure, either alone or combined in a panel of biomarkers may be used for providing a cost effective and rapid procedure for assessment, including risk assessment, early diagnosis, establishing prognosis, monitoring patient treatment, detecting relapse, and discovery of therapeutic intervention of colorectal cancer. In a particular embodiment, the present disclosure relates to a method for assessing an object selected from colorectal cancer, colorectal adenoma, and colorectal polyp, wherein a whole blood sample is assessed, and wherein the sample is derived from an individual; the assessment is based on measuring the level of a polynucleotide originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1 , CSNK1D, C19orf43, and LRR 3 in the sample.

Background of the Invention

The field of art of this disclosure concerns biomarkers for the assessment of colorectal cancer or a precursor thereof. These biomarkers are useful for risk assessment, early detection, establishing prognosis, evaluation of intervention, recurrence of neoplastic tissue, and discovery of therapeutic intervention, and methods of use thereof. In the field of medicine, clinical procedures providing for the risk assessment and early detection of colorectal cancer have been long sought. Currently, colorectal cancer is the second leading cause of cancer-related deaths in the Western world. One picture that has clearly emerged through decades of research into colorectal cancer is that early detection is critical to enhanced survival rates.

The currently accepted methods for colorectal cancer screening include the fecal occult blood test (FOBT), x-ray using double contrast between barium enema and air (DCBE), sigmoidoscopy, and colonoscopy. Sigmoidoscopy is an invasive procedure that visually examines the lower third of the colon using a lighted, flexible endoscope, while a related method, colonoscopy, is a procedure that examines the entire colon. In both cases, biopsy samples can be taken during the procedure.

Further methods include PreGen-Plus™ which is a non-invasive test that isolates and analyzes DNA extracted from a patient's stool sample for alterations associated with the presence of colorectal cancer. ColonSentry™ is also a blood-based colon cancer detection test that measures the expression of seven genes as a basis for estimating the patient's current risk of having colorectal cancer. Another assay is based on the detection of methylated septin-9 DNA (mSEPT9) aiming at early detection of colorectal cancer by analyzing a blood sample. The test is based on the observation of epigenetic change of the septin-9 gene in a number of colorectal tumors. Further methylation markers for detection and prognosis of colon cancers were developed by Oncomethylome. Signature Diagnostics markets an assay called Detector C which is a blood-based screening test for the detection of colorectal cancer. The test uses oligonucleotide chip technology to evaluate the expression of 202 genes that are altered in response to tumor formation and growth.

Concerning accepted methods for screening, none clearly possess what is desired in a screening examination for colorectal cancer, and the methods of the state of the art have certain disadvantages. Particularly, there is a need for other means than colonoscopy to enable detection of colorectal adenoma and colorectal polyps. Accordingly, there is a need in the art for approaches that have value in early detection and treatment of colorectal cancer, colorectal adenoma and colorectal polyps that are cost effective, rapid, and minimally or noninvasive. Additional utility would be realized from an approach that would also serve as the basis for establishing prognosis, monitoring patient treatment, and detecting relapse, as well as the discovery of therapeutic intervention of colorectal cancer.

Summary of the Invention

A first aspect of the teachings herein is the use of a polynucleotide originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6,

MALAT1, CSNK1D, C19orf43, and LRR 3 as a biomarker for and/or in the assessment of one or more selected member(s) of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp. A second aspect of the teachings herein is a panel of biomarkers for assessing any member of the group consisting of colorectal cancer, colorectal adenoma and colorectal polyp, the panel comprising at least two polynucleotides, each polynucleotide corresponding to a different genetic locus, wherein at least one, and particulatly at least two of the polynucleotides originate(s) from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and LRRN3. A third aspect of the teachings herein is a method for measuring the expression levels of a polynucleotide from a biomarker, the biomarker being an indicator for the presence of any member of the group consisting of colorectal cancer, colorectal adenoma and colorectal polyp in a human individual, the method comprising: selecting a biomarker comprising a contiguous polynucleotide included in a member of the group consisting of SEQ ID NOs: 1, 14, 27, 40, 53, 66, 79, 92, 105, a fragment thereof or its complement, and a variant thereof or its complement; obtaining a biological sample from the human individual; isolating cellular RNA from the sample and reverse-transcribing isolated RNA to obtain cDNA; amplifying copies of cDNA for the biomarker originating from the sample; and quantifying the level of cDNA amplified from the sample, thereby measuring the expression level of a polynucleotide from a biomarker. A fourth aspect of the teachings herein is a kit for the assessment of any member of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp, the kit comprising: at least one reagent that is used in analysis of polynucleotide expression level for a biomarker for a member selected from the group consisting of colorectal cancer, colorectal adenoma and colorectal polyp, wherein the biomarker comprises a polynucleotide selected from SEQ ID NOs: 1, 4, 8, 12, 14, 17, 21, 25, 27, 30, 34, 38, 40, 43, 47, 51, 53, 56, 60, 64, 66, 69, 73, 77, 79, 82, 86, 90, 92, 95, 99, 103, 105, 108, 112, a complement therof, a variant thereof, and a fragment thereof; and instructions for using the kit for analyzing the polynucleotide expression level. Detailed Description of the Invention

The following additional definitions and explanations are provided for specific terms, which are used in the written description of the present disclosure.

As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a molecule" also includes a plurality of molecules (i.e. one or more).

The expression "one or more" denotes a single item or a plurality thereof; in an embodiment, "one or more indicates 1 to 50; an embodiment thereof is 1 to 20, other embodiments thereof are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15. The term "polynucleotide" denotes a single-stranded DNA or RNA molecule, or its respective complement, wherein in an embodiment the polynucleotide is a polymer of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleoside monomers. A particular embodiment is a polymer of 15 to 35 nucleoside monomers. Another particular embodiment is a polymer of 35 to 100 nucleoside monomers. Yet, another particular embodiment is a polymer of 50 to 150 nucleoside monomers. Yet, another particular embodiment is a polymer of 150 to 1500 nucleoside monomers. The term "polynucleotide" also encompasses homo- and heteroduplex double- stranded nucleic acid molecules of which each strand comprises 8 or more nucleoside monomers. Further, heteroduplices are encompassed of which one strand comprises one or more non-natural nucleoside analog(s). In a particular embodiment, the strand with the nucleoside analog(s) comprises 8 or more monomers. An example for a non-natural nucleoside analog is a LNA (locked nucleic acid) monomer. Other non-natural nucleoside analogs are known to the art and encompassed herein. Particular embodiments are non-natural nucleoside analogs capable of increasing the melting point of a heteroduplex with RNA or DNA. Being directed to polynucleotides capable of forming hybrids, or being hybridized or in the process of hybridization, the term "duplex" denotes the double helix formed between a RNA or DNA molecule and its complement. A "variant" of a polynucleotide differs from the polynucleotide by one or more mutations while preserving substantial sequence similarity as well as specificity in hybridization processes in the procedures disclosed herein. A mutation can be a point mutation wherein at a given position a first nucleobase is substituted by a second nucleobase or an analog thereof. Further mutations are deletions or insertions. In particular embodiments, a polynucleotide and its variant have a sequence identity of a value selected from 95%, 96%, 97%, 98%, 99%, and a value higher than 95% and below 100%.

A "fragment", and particularly a "fragment of a polynucleotide" is understood to be an incomplete or isolated portion of the polynucleotide, comprising at least a single strand with an undisrupted (contiguous) nucleotide sequence of 8 or more positions, according to the entire polynucleotide. In particular embodiments, according to the entire polynucleotide, the fragment thereof comprises an undisrupted nucleotide sequence of 17, 18, 19, 20, 25, 50, 75, 100, 200, 300, 500, 700, 1000 or more positions.

A polynucleotide or a nucleotide sequence "originating from" a genetic locus is understood to include a nucleic acid molecule (DNA or R A) which results from transcription of the genetic locus, i.e. a transcript, or from reverse-transcription of a transcript. Taking a more operational perspective, the term polynucleotide "originating from" a genetic locus encompasses a nucleic acid which is obtainable as a result of a sample preparation process in combination with one or more process(es) of treatment, that is treatment of the processed sample and/or treatment of the sample prior to applying the sample preparation process. Accordingly, the nucleic acid obtainable as a result of the processes is double-stranded or single - stranded, and its sequence is comprised in or complementary to the sequence of any of the complements of the respective genetic locus.

The term "genetic locus" denotes a genomic region which is transcribed in a tumor cell and/or a non-tumor cell. A transcribed region of a gene can comprise any of non-coding leader, non-coding trailer, intron and exon sequences. However the term "genetic locus" further includes any transcribed region of a pseudogene, any transcribed region with established or putative regulatory function, and any other transcribed region. A transcribed region, generally, is genomic DNA which is a template for a DNA-dependent RNA polymerase at least once or more times in the lifetime of a tumor cell and/or a non-tumor cell. A "biomarker" is a biological molecule which indicates a particular disease state or another physiological state of an organism. Thus, the term biomarker includes a substance whose detection indicates a particular disease state. A biomarker may also indicate a change in gene or protein expression that correlates with the risk or progression of a disease, or with the susceptibility of the disease to a given treatment. Biomarkers are characteristic biological properties that can be detected and measured in parts of the body like the blood or tissue. They may indicate either normal or diseased processes in the body. Biomarkers can be specific cells, molecules, or genes, gene products, enzymes, or hormones. The term "biomarker" is encompassed in the broader term "marker" which denotes an object to indicate a position or a status or which serves as a standard of comparison or as an indication of what may be expected. A panel of biomarkers is a selection of two or more biomarkers.

A "marker of cancer" and in particular a "marker of colorectal cancer" in the sense of the present disclosure is any marker that, either alone or in combination with one or more further biomarker(s), adds relevant information in the assessment of colorectal cancer. Likewise, a "marker of colorectal adenoma" and a "marker of colorectal polyp" adds relevant information in the assessment of the respective neoplastic potential precursor of colorectal cancer. Any such information is particularly considered relevant or of additive value if at a given specificity the sensitivity, or if at a given sensitivity the specificity, respectively, for the assessment of colorectal cancer, colorectal adenoma or colorectal polyp can be improved by combining the marker with another biomarker or with a diagnostic result, or by including the marker into a panel of biomarkers. In an embodiment of any such assessment, the improvement in sensitivity or specificity, respectively, is statistically significant at a level of significance of p = .05, .02, .01 or lower.

The term "sample" as used herein refers to a biological sample obtained for the purpose of evaluation in vitro. In the embodied methods of the present disclosure, the sample or patient sample is whole blood, e.g. peripheral blood; however, further samples are possible such as plasma, serum, stool and tissue. It is understood that any such evaluation is made in vitro, which implies that the sample material is physically separated from the patient's body in the process of such evaluation. The patient sample is discarded afterwards. The patient sample is solely used for the in vitro diagnostic method as disclosed, and the material of the patient sample is not transferred back into the patient's body.

The expressions "assessment of an object or "assess" or "assessing" an object, wherein the object is selected from colorectal cancer, colorectal adenoma, and colorectal polyp, are used to indicate that the disclosed subject-matter will (alone or together with other markers or variables, e.g., the classification criteria set forth by the AJCC or the Dukes stage classification, see below) e.g., aid the physician to establish or confirm the absence or presence of colorectal cancer, colorectal adenoma, and colorectal polyp, or aid the physician in the prognosis, the detection of recurrence (follow-up of patients after surgery) and/or the monitoring of treatment, especially of chemotherapy. Thus, "assessing" and "assessment" encompass the use of technical items and execution of procedural steps which yield the results on which the physician establishes or confirms the presence or absence of an object selected from colorectal cancer, colorectal adenoma, and colorectal polyp.

A "colorectal polyp" is understood as being a clump of cells on the inside (the lining) of the colon or the rectum. A colorectal polyp may give rise to colorectal cancer. Colorectal polyps are conventionally divided into two groups - non-neoplastic polyps and neoplastic polyps. Neoplastic polyps are also known as adenomatous polyps or adenomas. Non-neoplastic polyps include juvenile, hyperplastic, inflammatory, and lymphoid polyps. Non-neoplastic polyps have not been considered precursors of cancer while the neoplastic polyps bear the risk of being precursors of colorectal cancer. The gastroenterologist uses a colonoscopy to find and remove polyps and adenomas to prevent them from acquiring genetic changes that will lead to an invasive adenocarcinoma. In medical practice it is desired to have alternative means to detect a polyp of the colon. A particular desire is to have a biomarker indicating the presence of a polyp.

An "adenoma" is a benign tumor of glandular origin which can grow from many organs including the colon and/or the rectum ("colorectal adenoma"). Although these growths are benign, over time they may progress to become malignant, at which point they are referred to as "adenocarcinomas". Most colorectal adenomas are polypoid. Large flat and depressed colorectal adenomas may be more likely to be severely dysplastic and give rise to malignancies. Colorectal adenomas are found commonly at colonoscopy. They are removed because of their tendency to become malignant and to lead to colorectal cancer. In medical practice it is desired to have alternative means to detect colon adenoma. A particular desire is to have a biomarker indicating the presence of colon adenoma.

Colorectal polyp tissue (= colorectal polyp) and colorectal adenoma are understood as each representing a precursor stage of colorectal cancer. In this regard, a precursor stage poses the risk of developing further into colorectal cancer, i.e. a malignant cancer. "Adenocarcinoma" is defined as a malignant tumor that grows on the glandular epithelial cells of an internal organ. "Colorectal cancer" is a cancer of the colon and/or the rectum, a malignant tumor arising from the inner wall of the large intestine. The majority of colorectal cancer is of the adenocarcinoma type. Colorectal cancer staging is performed for diagnostic and research purposes, and to determine the best method of treatment. The systems for staging colorectal cancers depend on the extent of local invasion, the degree of lymph node involvement and whether there is distant metastasis.

A common staging system is the TNM (for tumors/nodes/metastases) system, from the American Joint Committee on Cancer (AJCC), AJCC Cancer Staging Manual (Sixth ed.). Springer- Verlag New York, Inc.. (2002).

An alternative staging system for colorectal cancer is the Dukes classification. It identifies stages as

It is a goal here to provide further and/or alternative and/or improved means to detect and assess carcinoma of the colon and/or the rectum, including adenocarcinoma. A particular goal of the disclosure is to provide a biomarker indicating the presence of colorectal cancer at an early stage. A further goal of the disclosure is to provide a biomarker indicating the presence of a precursor of colorectal cancer, i.e. the presence of polyp or adenoma tissue in the colon or the rectum.

The starting point of the present disclosure was a search for biomarkers that have value in detection and treatment of colorectal cancer. Biomarkers for cancer in general and colorectal cancer in particular have several potential uses in the management of patient care. Ideally, they would be used for risk assessment, for early diagnosis, for establishing prognosis, for monitoring treatment, and for detecting relapse. Additionally, such markers could play a valuable role in developing therapeutic interventions.

It is further advantageous for the sampling methods used in conjunction with biomarker analysis to be minimally invasive or non-invasive. Examples of such sampling methods are directed to sampling whole blood, plasma, serum, stool, swabs, and the like. Non-invasive and minimally invasive methods increase patient compliance, and generally reduce cost.

Clinically, the two criteria that are important for assessing the effectiveness of biomarkers are specificity and sensitivity. "Specificity" of the biomarker is a percentage of correctly analysed healthy subjects of all healthy subjects or the probability for the negative diagnosis of healthy subjects. "Sensitivity" ist the percentage of correctly analysed diseased patients of all diseased patients or the probability for a positive diagnosis of diseased patients. Ideally, biomarkers would have 100% clinical specificity and 100% clinical sensitivity. To date, no single biomarker has been identified that has an acceptably high degree of specificity and sensitivity required to be effective in for the broad range of needs in patient care management. However, from the clinical perspective, single serum biomarkers, such as alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) have proven to provide value in some aspects of patient care management.

For example, elevated serum levels of CEA were first discovered in patients with adenocarcinoma of the colon. Elevated levels can be found in a variety of benign and malignant conditions other than colorectal cancer. Additionally, the production of CEA by early localized tumors of the colon is in the normal range. Therefore

CEA lacks both the sensitivity and specificity required to be of value for risk assessment or early diagnosis. Further, elevated levels of CEA correlate poorly with colon tumor differentiation and stage, rendering CEA as a biomarker for prognosis of colorectal cancer of limited value. The two areas for which CEA has proven helpful clinically in managing patient care are in evaluating the effectiveness of treatment, and for detecting relapse. Illustrative of this, numerous studies have found that there is high correlation between elevated serum levels of CEA preceding clinical detection of recurrence of colorectal cancer. This has proven to be of value in managing the care of high-risk patents with second-look surgical procedures based on rising levels of CEA.

Currently, investigations across numerous areas of oncology research, including colorectal cancer, ovarian cancer, breast cancer, and head and neck cancer, are finding increased sensitivity and specificity in panels of markers. It is now generally held that many mutations must take place before normal cell processes are altered, resulting in a disease, such as cancer. Still, given the complexity of biological systems, discovery of panels useful in providing value in patient care management for colorectal cancer is in the nascent stage.

To date, a greater understanding of the biology of colorectal cancer has been gained through the research on adenomous polyposis coli (APC), p53, and Ki-ras genes, as well as the corresponding proteins, and related pathways involved regulation thereof. However, there is a distinct difference between research on a specific a gene, its expression, protein product, and regulation, and understanding what genes are critical to include in a panel used to for the analysis of colorectal cancer that is useful in the management of patient care for the disease. Previously, panels have been suggested for colorectal cancer, which are comprised of specific point mutations of the APC, p53, and Ki-ras, as well as BAT-26. Further nucleic acid biomarker panels for colorectal cancer are known to the art from documents including US 2005/0014165, WO 2009/015299 Al, and WO 2010/053539 A2.

It is highly desired to detect colorectal cancer at an early stage and/or to detect a precursor stage of colorectal cancer such as colorectal adenoma and colorectal polyp. What is disclosed herein is based on discovery studies with mRNA isolated from biopsy and blood samples from patients using an oligonucleotide array-based screening approach. Out of a large number of candidate sequences, a panel of biomarkers has been established to assess changes in human tissue from surgical and biopsy samples and from blood samples against control tissue and blood, respectively. Clinical samples from patients were used to identify significant genomic alterations as correlates of the emergence and progression of disease. To this end, the Affymetrix® platform of oligonucleotide arrays and analysis means served in the analysis of tumor RNA expression. From these human subject studies, a plurality of biomarkers is disclosed herein.

The biomarkers can be of medical use when used as single markers. However, their technical advantage in the assessment of colorectal cancer, precursor and early stages of colorectal cancer is increased when a panel of these biomarkers is used. Further, what is disclosed are methods for measuring gene expression levels based on individual markers and on a panel of markers. Additionally, another aspect of what is disclosed are kits which provide the reagents and instructions for measuring gene and protein expression levels based on the panel. The panel, methods and kits are useful in the management of patient care for colorectal cancer. Additionally, the panel, methods and kits are believed useful as the basis for discovery of therapeutic interventions for colorectal cancer.

Measuring gene expression means to quantify the level at which a particular gene is expressed within a cell, tissue or organism. While the amount of a final gene product may be indicative for the level of expression of the respective gene, measurement of gene expression in line with the present disclosure is typically made by detecting and quantifying mRNA, and infer gene expression level. Thus, the level of a given mRNA in a given sample is used as a biomarker.

An advantageous and frequently applied approach for measuring mRNA level is reverse transcription quantitative polymerase chain reaction (RT-qPCR), i.e. the combination of reverse transcription (RT) and quantitative PCR (qPCR), i.e. PCR which provides quantitative detection of the respective target nucleic acid. The RT step first generates a DNA template from the mRNA by reverse transcription, which is called cDNA. The cDNA serves as a template in the subsequent step of qPCR. In some qPCR embodiments fluorescence of a detection probe changes as the DNA amplification process progresses. Other qPCR embodiments make use of an intercalating dye which interacts with double-stranded DNA and which is capable of indicating newly formed DNA. In a calibrated system including controls qPCR can produce an absolute measurement such as number of copies of mRNA present in the sample. The qPCR method is very sensitive as detection of a single mRNA molecule is possible.

Using DNA microarrays highly parallel analysis can be performed. Arrays of different capture probes can be used to detect and/or quantify transcript levels for many genes at once (expression profiling). Recent advances in microarray technology allow for the quantitation, even on a single array, of transcript levels for every known gene in several organism's genomes, including the human genome.

Table 1 gives a first overview of the marker genes and genetic loci; nucleotide sequences originating from these loci are used as biomarkers, according to the present disclosure. A transcript of each marker gene or genetic locus alone advantageously serves as an indicator in the assessment of colorectal cancer or a precursor thereof. In another embodiment, a combination of two or more biomarkers as disclosed herein forms the basis for assessing colorectal cancer or a precursor thereof with enhanced specificity and sensitivity, and therefore provides enhanced management of patient care for colorectal cancer.

It is to be understood that fragments and variants of the biomarkers described in Table 1 and in the sequence listings are also useful biomarkers, either alone or in a panel used for the analysis of colorectal cancer or a precursor thereof. What is meant by fragment is any incomplete or isolated portion of a polynucleotide in the sequence listing. It is recognized that almost daily, new discoveries are announced for gene variants, particularly for those genes under intense study, such as genes implicated in diseases like cancer. Therefore, the sequence listings given are exemplary of what is now reported for a gene, but it is recognized that for the purpose of an analytical methodology, variants of the gene, and fragments of the genetic loci are also included. In a particular embodiment, the disclosure incorporates any nucleotide sequence which is in the range of 80% to 100% identical to a nucleotide sequence disclosed herein or the complement thereof. In another embodiment, the identity is in the range of 90% to 100%, in yet another embodiment the identity is in the range of 95% and 100%.

Table 1

Genomic locations are indicated by "Chromosome No.:start position-end position as defined by Ensembl release 63 - June 2011 © Wellcome Trust Sanger Institute

(WTSI) / EMBL-European Bioinformatics Institute (EBI); http://www.ensembl.org

The following presents some details concerning the marker genes found in the present study of biomarkers.

ASGR2 [synonyms: ASGP-R2 | ASGPR2 | CLEC4H2 | FLJ60040 | HBXBP | HL- 2 I gxHOMSA103126] encodes a subunit of the asialoglycoprotem receptor. This receptor is a transmembrane protein that plays a critical role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine residues. The asialoglycoprotem receptor may facilitate hepatic infection by multiple viruses including hepatitis B, and is also a target for liver-specific drug delivery. The asialoglycoprotem receptor is a hetero-oligomeric protein composed of major and minor subunits, which are encoded by different genes. The protein encoded by this gene is the less abundant minor subunit. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. CLIPl [synonyms: CLIP | CLIP- 170 | CLIP 170 | CYLN1 | MGC131604 |

RESTIN I RSN | gxHOMSA6696 | CAP-GLY domain containing linker protein 1] is a protein that mediates interactions between organelles and microtubules. CLIPl and its known effectors IQGAP1 and Cdc42 are present in the lamellar body- enriched fraction. Lamellar bodies are tubulovesicular secretory organelles of epithelial cells related to lysosomes. Confocal microscopy analysis of skin cryosections has shown that CLIPl is expressed in differentiated keratinocytes and can be observed co-localized with Cdc42 and with the known lamellar body protein cathepsin D. CLIPl also co-localizes with Rab7. It is hypothesized that CLIPl is involved together with Cdc42 and/or Rab7 in the intracellular trafficking of lamellar bodies.

ARHGAP18 [synonyms: FLJ25728 | MGC126757 | MGC138145 | MacGAP | bA307O14.2 | gxHOMSA121255] is the Rho GTPase activating protein 18 and belongs to a family of Rho GTPase-activating proteins that modulate cell signaling. ARHGAP18 gene products belong to the human RhoGAP family with approximately 80 RhoGAP proteins known to be encoded in the human genome. The RhoGAPs, GTPase-activating proteins have the ability to modulate Rho- mediated signaling pathways by controlling the balance between active and inactive Rho proteins. Rho proteins belong to the Ras superfamily that is composed of over 50 members divided into 6 families, including Ras, Sar, Rho, Ran, Rab and Arf They participate in an array of physiological processes, such as cell migration, intercellular adhesion, cytokinesis, proliferation, differentiation and apoptosis. Rho GTPases are important regulators of the actin cytoskeleton and consequently influence the shape and migration of cells. GTPases of the Rho family are strong regulators of signaling pathways that link growth factors and/or their receptors to adhesions and associated structures. One signaling pathway mediated by Ras is initiated by the EGF receptor (EGFR), leading to cell proliferation. EGFR signaling can induce mitosis, proliferation, cell motility, differentiation and protein secretion.

SLC16A6 [synonyms: MCT6 | MCT7 | gxHOMSA15027] encodes the solute carrier family 16, member 6 (monocarboxylic acid transporter 7). The monocarboxylate cotransporter (MCT) family comprises at least 14 members. Members of the family catalyse proton-linked transport of metabolically important monocarboxylates such as lactate, pyruvate and ketone bodies. Lactic acid transport across the plasma membrane is fundamental for the metabolism of and pH regulation of all cells, removing lactic acid produced by glycolysis and allowing uptake by those cells utilizing it for gluconeogenesis or as a respiratory fuel. MALATl [synonyms: HCN | MALAT-1 | NCRNA00047 | NEAT2 | PRO 1073 | PR02853] is originally known as the metastasis associated lung adenocarcinoma transcript 1 and appears to be non-protein coding. Recent evidence shows that MALATl has a role in the regulation of serine/arginine splicing factors. These factors are known to regulate alternative splicing of pre-mRNA, in a tissue- and/or cell type-specific manner.

CSNKID [synonyms: HCKID | gxHOMSA9978] encodes casein kinase 1, delta. This gene is a member of the casein kinase I (CKI) gene family whose members have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair. The encoded protein is highly similar to the mouse and rat CKI delta homologs. Two transcript variants encoding different isoforms have been found for this gene.

C19orf43 [synonyms: MGC2803 | fSAP18 | gxHOMSAl 15028] is the chromosome 19 open reading frame 43. LRRN3 [synonyms: FIGLER5 | FLJ11129 | NLRR-3 | NLRR3 | gxHOMSA68022] encodes a protein termed leucine rich repeat neuronal 3, also known as fibronectin type III, immunoglobulin and leucine rich repeat domains 5. LRRN3 is expressed at very high levels in humans, about 2.3 times the average gene. It is most highly expressed in brain, heart, and testes tissues. It is also slightly expressed in kidney, muscle, pharynx, placental, and thymus tissue. The highest expression of the LRRN3 gene for the developmental stages is the fetal stage, but it is also expressed in the infant, juvenile, and adult stages, as can be seen in the EST profile.

Surprisingly, it has been found that sequences derived from a genetic locus according to the present disclosure and as given by the entries ## 1-8 of Table 1 are helpful to assess colorectal cancer or a precursor thereof. An embodiment of what is disclosed is therefore a biomarker for the assessment of any member of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp, the assessment including detection of any of these members, wherein the biomarker origins from a gene or a genetic locus selected from the group consisting of ASGR2, CLIPI, ARHGAP18, SLC16A6, MALATl, CSNKID, C19orf43, and LRRN3. Thus, an embodiment is the use of (i) a partial or complete transcribed sequence or (ii) a fragment - of a gene or a genetic locus selected from the group consisting of ASGR2, CLIPI, ARHGAP18, SLC16A6, MALATl, CSNKID, C19orf43, and LRRN3, the use being that of a biomarker in the assessment of a member of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp.

Quantitative analysis of the respective markers revealed that in the diseased state (i.e. when a member of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp is present in the patient of whom a sample is assayed) the level of a marker is either higher or lower than in the non-diseased state. Table 2 indicates the deviation for each maker locus.

Table 2

δ "diseased state" indicates the presence of neoplastic tissue, either colorectal cancer or a precursor thereof.

A particular embodiment is the use of any of the above markers in the assessment of an early stage of colorectal cancer, particularly a stage selected from Tis NO MO, Tl-4 NO MO, Dukes A, and Dukes B. A further embodiment is a method for assessing in vitro an object selected from colorectal cancer, colorectal adenoma, and colorectal polyp, the method comprising measuring in a whole blood sample the concentration and/or activity of a biomarker, wherein the biomarker is a polynucleotide originating from a genetic locus selected from the group consisting of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALATl, CSNKID, C19orf43, and LRRN3, or a fragment of any of the aforementioned polynucleotides. In a particular embodiment, the biomarker is a transcript originating from a genetic locus selected from the group consisting of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALATl, CSNKID, C19orf43, and LRRN3, or a fragment of any of the aforementioned transcripts. Another embodiment of what is disclosed is a panel of biomarkers with the specificity and sensitivity required for managing patient care for colorectal cancer. In Tables 1 and 2, entries ## 1-8 refer to the biomarkers. Encompassed are the transcripts and the polynucleotide sequences therof, of the respective genes and genetic loci. The surprising advantage in the use of any of these biomarkers is obtained when a marker listed in Table 1 is used alone or in a panel of biomarkers. A particular embodiment is a panel of biomarkers comprising two or more of the aforementioned polynucleotides.

As previously mentioned, there is still a need for biomarkers for colorectal cancer as well as precursors thereof, the biomarkers having the specificity and sensitivity required to be effective for all aspects of patient care management. Therefore, the selection of an effective set of biomarkers is differentiating in providing the basis for effective determination of colorectal cancer.

In another embodiment of this disclosure, expression levels of polynucleotides for the biomarkers indicated in SEQ ID NOs: 1, 14, 27, 40, 53, 66, 79, 92, and 105 and particularly SEQ ID NOs :4, 8, 12, 17, 21, 25, 30, 34, 38, 43, 47, 51, 56, 60, 64, 69, 73, 77, 82, 86, 90, 95, 99, 103, 108, and 112, are used in the determination of colorectal cancer or a precursor thereof. Such analysis of polynucleotide expression levels is frequently referred to in the art as gene expression profiling. In gene expression profiling, mRNA of a sample is first reverse-transcribed in a quantitative manner into a complementary DNA (cDNA). That is to say, the relative amount of cDNA derived from a given RNA species and resulting from the process of reverse transcription corresponds to the relative amount of the RNA species relative to all RNAs which are reverse-transcribed in the process. In a subsequent step, the cDNA serves as template for qPCR using specific oligonucleotides as primers and a quantitative detection method for the DNA which is newly generated after each amplification round. The deduced level of original target mRNA in the sample, also in comparison to other samples, is a leading indicator of a biological state; as disclosed herein, the level of target mRNA is used as an indicator of colorectal cancer or a precursor thereof in comparison to healthy individuals or individuals with none of the above neoplastic growths. Pairs of primers advantageously used in qPCR, as a subsequent step after the reverse transcription process and for sequences derived from each gene locus are listed in entries 1-8 of Tables 1 and 2. Since the combination of RT and qPCR amplifies copies of cDNA proportional to the original level of the corresponding mRNA in a sample, it has become a standard method that allows the analysis of even low levels of mRNA present in a biological sample. Compared to a normal/healthy/reference sample target sequences (polynucleotides) derived from certain genetic loci may either be up-regulated or down-regulated in any particular biological state, and hence mRNA levels shift accordingly.

FIGURES

Figures 1 and 2 show box plots and corresponding ROC diagrams reflecting results with regards to biomarkers for assessing precursors of colorectal cancer, i.e. adenoma and colorectal polyp tissue. The basis of each assessmenrt was the level of RNA transcribed from the genetic loci as indicated. Each assessment comprised assaying in vitro whole blood samples obtained from individuals. In the box plots each box contains the middle 50% of the respective data. The middle line is the median, whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the box. Data points in the box plots are indicative of the relative amount of target cDNA polynucleotide amplified (y-axis). Data are shown for quantitative target sequence determination normalized ("norm.") against the level of a housekeeping gene (SDHA, one out of several commonly chosen alternatives of the state of the art). The labeling of the box-and- whisker plots indicates the healthy control ("normal"), adenoma with a size of 1 cm or smaller ("adn-<=lcm"), adenoma with a size larger than 1 cm ("adn->lcm"), and adenoma without information on size ("adn-na"). In each "adn" group results from samples obtained from patients with colorectal polyp tissue were comprised, too.

Figure 1 A LRRN3 (209841. s.at) norm. box plot

Figure 1 B LRRN3 (209841. s.at) norm. ROC

Figure 2 A MALAT1 (224568.x.a) norm. box plot

Figure 2 B MALAT1 (224568.x.a) norm. ROC

In a similar way, Figures 3 to 18 reflect the results of patients with colorectal cancer. The labeling of the box-and-whisker plots indicate the healthy control ("normal"), TNM stages [Tl NO M0] and [T2 NO M0] grouped in "crc-1", stages [T3 NO M0] and [T4 NO M0] grouped in "crc-2", and stages [Tl-2 Nl M0], [T3-4

Nl M0] and [any T, N2 M0] grouped in "crc-3". Data are shown for quantitative target sequence determination normalized ("norm.") against the level of a housekeeping gene (SDHA, one out of several commonly chosen alternatives of the state of the art) and without normalization ("raw"). As above, each ROC diagram refers to the data set of the previous Figure depicting the respective box plot.

Figure 3 A ASGR2 (206130.s.at) norm. box plot

Figure 3 B ASGR2 (206130.s.at) norm. ROC

Figure 4 A ASGR2 (206130.s.at) raw box plot

Figure 4 B ASGR2 (206130.s.at) raw ROC

Figure 5 A CLIPl (1558924.s.at) norm. box plot

Figure 5 B CLIPl (1558924.s.at) norm. ROC

Figure 6 A CLIPl (1558924.s.at) raw box plot

Figure 6 B CLIPl (1558924.s.at) raw ROC

Figure 7 A ARHGAP18 (225173. at) norm. box plot

Figure 7 B ARHGAP18 (225173. at) norm. ROC

Figure 8 A ARHGAP18 (225173. at) raw box plot

Figure 8 B ARHGAP18 (225173. at) raw ROC

Figure 9 A SLC16A6 (207038.at) norm. box plot

Figure 9 B SLC16A6 (207038.at) norm. ROC

Figure 10 A SLC16A6 (207038. at) raw box plot

Figure 10 B SLC16A6 (207038. at) raw ROC

Figure 11 A MALAT1 (224568.x.at) norm. box plot

Figure 11 B MALAT1 (224568.x.at) norm. ROC

Figure 12 A MALAT1 (224568.x.at) raw box plot

Figure 12 B MALAT1 (224568.x.at) raw ROC

Figure 13 A MALAT1 (223940.x.at) norm. box plot

Figure 13 B MALAT1 (223940.x.at) norm. ROC

Figure 14 A MALAT1 (223940.x.at) raw box plot

Figure 14 B MALAT1 (223940.x.at) raw ROC

Figure 15 A CSNK1D (1569263.at) norm. box plot

Figure 15 B CSNK1D (1569263.at) norm. ROC

Figure 16 A CSNK1D (1569263.at) raw box plot

Figure 16 B CSNK1D (1569263.at) raw ROC

Figure 17 A C19orf43 (230213. at) norm. box plot

Figure 17 B C19orf43 (230213. at) norm. ROC

Figure 18 A C19orf43 (230213. at) raw box plot

Figure 18 B C19orf43 (230213. at) raw ROC Methods and kits for determination of any of the polynucleotides shown, and expression profiling for a panel of molecular markers are also contemplated as part of the present disclosure. As used herein, the term "determination" encompasses qualitative and quantitative determination. In one embodiment, a method for gene expression profiling comprises measuring the level of one or more a polynucleotide(s) originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1 , CSNK1D, C19orf43, and LRR 3. Accordingly, methods are included which facilitate the determination of a level of mRNA directly, or of a level of cDNA, for one or more biomarker(s) disclosed herein. Such a method requires the use of primers, enzymes, and other reagents for the preparation, detection, and quantitation of mRNAs/cDNAs. The method of creating cDNA from mRNA in a sample is referred to as reverse transcription (RT). The primer pairs of SEQ. ID NOs: 2 and 3, 6 and 7, 10 and 11, 15 and 16, 19 and 20, 23 and 24, 28 and 29, 32 and 33, 36 and 37, 41 and 42, 45 and 46, 49 and 50, 54 and 55, 58 and 59,

62 and 63, 67 and 68, 71 and 72, 75 and 76, 80 and 81, 84 and 85, 88 and 89, 93 and 94, 97 and 98, 101 and 102, 106 and 107, and 110 and 111 are particularly suited for use in gene expression profiling using RT-qPCR based on the claimed panel. However, use of a primer pair for the assessment of a single biomarker is also an intended use. In addition to the primers, reagents such as one including a desoxynucleoside triphosphate mixture having all four desoxynucleoside triphosphates (e.g. dATP, dGTP, dCTP, and dTTP), one having the reverse transcriptase enzyme, and one having a thermostable DNA polymerase are required for RT-qPCR. Additionally buffers, inhibitors and activators are also required for the RT-qPCR process. Once the cDNA has been sufficiently amplified to a specified end point, the cDNA sample must be prepared for detection and quantitation. Though a number of detection schemes are contemplated, as will be discussed in more detail below, one method contemplated for detection of polynucleotides is fluorescence spectroscopy, and therefore chromophores that are suited for fluorescence spectroscopy are desirable for labeling polynucleotides.

One example of such a fluorescent label is the intercalating dye SYBR Green, though numerous related chromophores capable of forming complexes with newly formed nucleic acids by way of intercalating exist, and are known in the art.

In another embodiment, the hydrolysis probe principle is used. It is also known as the TaqMan® format. It relies on the 5 ^' -3 ^' exonuclease activity of Taq polymerase to cleave a dual-labeled probe (hydrolysis probe) during hybridization to the complementary target sequence and fluorophore-based detection. As in other real-time PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR; however, the hydrolysis (TaqMan®) probe significantly increases the specificity of the detection. A hydrolysis probes consists of a fluorophore covalently attached to the 5 '-end of the oligonucleotide probe and a quencher at the 3 '-end. Several different combinations of fluorophores and quenchers are known to the art and commercially available. In an intact hydrolysis probe the quencher molecule absorbs the fluorescence emitted by the fluorophore. As long as the fluorophore and the quencher remain in proximity, the quencher inhibit the fluorescence signal and therefore prevents its detection.

Hydrolysis (TaqMan®) probes are designed such that they anneal within a DNA region amplified by a specific set of primers. As the Taq polymerase extends the primer and synthesizes the nascent strand, the 5' to 3' exonuclease activity of the polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore to be detected. Hence, the amount of fluorescence detected by way of spectroscopy in a real-time PCR thermal cycler is dependent on the fluorophore released, hence the amount of DNA template present in the PCR.

Examples of detection modes contemplated for the disclosed methods include, but are not limited to spectroscopic techniques, such as fluorescence and UV-Vis spectroscopy, scintillation counting, and mass spectroscopy. Complementary to these modes of detection, examples of labels for the purpose of detection and quantitation used in these methods include, but are not limited to chromophoric labels, scintillation labels, and mass labels. The expression levels of polynucleotides and polypeptides measured using these methods may be normalized to a control established for the purpose of the targeted determination. These methods are believed useful in providing determinations as the basis of effective management of patient care for colorectal cancer. These methods may also be used in the discovery of therapeutic interventions for colorectal cancer. Additionally, not only blood samples, biopsy samples from sigmoidoscopy, colonoscopy, or surgery may be analyzed by these methods, but biological samples from non-invasive or minimally evasive collection methods are indicated for these methods, as well. It is further contemplated in what is disclosed to provide kits having the reagents and procedures that facilitate the ready implementation of the methods, and provide consistency and quality control thereby.

In another embodiment, a kit for gene expression analysis in the assessment of colorectal cancer or a precursor thereof, wherein the kit comprises the reagents and instructions necessary for the detection and quantitative analysis of a polynucleotide originating from any one of the genetic loci selected from the group consisting of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and LRR 3. Yet another embodiment is a kit for gene expression profiling of a panel comprising ASGR2, CLIPl, ARHGAP18, CR2, SLC16A6, MALAT1, CSNK1D, C19orf43, CD200, and LRRN3. Thus, for example, the reagents may include primers, enzymes, and other reagents for the preparation, detection, and quantitation of cDNAs for the claimed panel of biomarkers, or individual biomarkers. As discussed above, the method of creating cDNA from mRNA in a sample is referred to as the combination of reverse transcription and quantitative polymerase chain reaction (RT-qPCR). The primer pairs of SEQ. ID NOs: 2 and 3, 6 and 7, 10 and 11, 15 and 16, 19 and 20, 23 and 24, 28 and 29, 32 and 33, 36 and 37, 41 and 42, 45 and 46, 49 and 50, 54 and 55, 58 and 59, 62 and 63, 67 and 68, 71 and 72, 75 and 76, 80 and 81, 84 and 85, 88 and 89, 93 and 94, 97 and 98, 101 and 102, 106 and 107, and 110 and 111 are particularly suited for use in gene expression profiling using RT-qPCR based on the claimed panel. These primer pairs were specifically designed, selected, and tested accordingly. In addition to the primers, reagents such as one including a desoxynucleoside triphosphate mixture having all four desoxynucleoside triphosphates (e.g. dATP, dGTP, dCTP, and dTTP), one having the reverse transcriptase enzyme, and one having a thermostable DNA polymerase are required for RT-qPCR. Additionally buffers, inhibitors and activators used for the RT-qPCR process are suitable reagents for inclusion in the kit embodiment. In another embodiment, the reverse transcriptase and the thermostable DNA polymerase are provided together in a mixture, to enable one-step RT-qPCR. In a variation of the embodiment, the thermostable DNA polymerase aditionally comprises as a further enzymatic activity that of a reverse transcriptase.

One method contemplated for detection of polynucleotides is fluorescence spectroscopy, and therefore fluorophores that are suited for fluorescence spectroscopy are desirable for labeling polynucleotides and may also be included in reagents of the kit embodiment. Alternatively, labeled hydrolysis probes which can be used in the TaqMan® detection format may be included in the kits. Such hydrolysis probes may comprise LNA nucleosides to facilitate provision of shorter hydrolysis probes.

Further detection formats are known to the art and can be applied to practice the quantitation of biomarkers as disclosed herein.

Instructions included with the kit embodiment for gene expression profiling teach the user the following steps: to obtain a biological sample; to isolate cellular RNA from the sample; to synthesize cDNA from the isolated RNA; to amplify copies of cDNA from the sample for the biomarker(s), and the panel for which the reagents are provided; and to quantify levels of cDNA amplified from the sample. Various samples including whole blood from a variety of procedures may be used. The instructions for obtaining a biological sample typically instruct the user to obtain a sample of blood (or another sample material as indicated elsewhere herein), optionally to stabilize the sample material, and to subject the sample to a nucleic acid preparation procedure. The instructions may also include the step of comparing the cDNA levels quantified to a control.

Additionally, consumable labware required for sample collection, preparation, and analysis may be provided with the kits.

The following items further provide aspects of the disclosure, and embodiments to practice the teachings provided herein.

1. Use of a polynucleotide originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and LRRN3 as a biomarker for and/or in the assessment of neoplastic tissue selected from any of colorectal cancer and a precursor stage of colorectal cancer (a precursor stage being selected from any of colorectal adenoma and colorectal polyp), wherein in a specific embodiment the biomarker is the level of an RNA transcribed from the genetic locus, and in another specific embodiment the sample is whole blood.

2. The use according to item 1, wherein the assessment comprises assaying in vitro (ex-vivo) a sample obtained from a patient (a human individual), particularly a blood sample. The use according to any of the items 1 and 2, wherein assessment of one or more selected member(s) comprises assaying a polynucleotide selected from RNA, cDNA, and a complement thereof. The use according to any of the items 1-3, wherein the assessment includes quantification of the polynucleotide. The use according to any of the items 1-3, wherein the assessment includes comparing the presence and/or the level of the polynucleotide in the patient sample with a control. The use according to any of the items 1-5, wherein colorectal polyp and/or colorectal adenoma are detected. The use according to any of the items 1-5, wherein colorectal cancer is assessed at a stage selected from any of Tis NO MO, T 1-4 NO MO, Dukes A, and Dukes B. The use according to any of the items 1-5, wherein colorectal cancer is assessed at a stage more advanced than any of T4 NO MO, and Dukes B. The use according to any of the items 1 to 8, wherein the assessment is detection of (or the establishment of, or the confirmation of) the presence or absence of a member of the group selected from colorectal cancer, colorectal adenoma, and colorectal polyp. The use according to any of the items 5 to 9, wherein, when compared to a sample from a healthy control, particularly a healthy control individual, the level of the polynucleotide is increased in the sample obtained from the human individual with colorectal neoplastic tissue, and the biomarker is selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, and C19orf43. The use according to item 10, wherein the colorectal neoplastic tissue is selected from any of colorectal cancer, colorectal adenoma, and colorectal polyp. The use according to item 10, wherein the colorectal neoplastic tissue is selected from any of colorectal cancer and colorectal adenoma. 13. The use according to item 10, wherein the colorectal neoplastic tissue is colorectal cancer.

14. The use according to any of the items 5 to 9, wherein, when compared to a sample from a healthy control, particularly a healthy control individual, the level of the polynucleotide is decreased in the sample obtained from the human individual with colorectal neoplastic tissue, and the biomarker is LRR 3.

15. The use according to item 14, wherein the colorectal neoplastic tissue is selected from any of colorectal cancer, colorectal adenoma, and colorectal polyp.

16. The use according to item 14, wherein the colorectal neoplastic tissue is colorectal polyp.

17. The use of any according to any of the the items 1 to 16, wherein the biomarker is combined with one or more further biomarker(s). 18. The use according to item 17, wherein the one or more further biomarker(s) is/are one or more polynucleotide(s) originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and LRRN3.

19. The use according to any of the items 1-18, wherein the polynucleotide originating from the genetic locus ASGR2 is selected from SEQ ID NO: l, the complement of SEQ ID NO: l, a fragment thereof or its complement, and a variant thereof or its complement. 0. The use according to any of the items 1-18, wherein the polynucleotide originating from the genetic locus CLIPl is selected from SEQ ID NO: 14, the complement of SEQ ID NO: 14, a fragment thereof or its complement, and a variant thereof or its complement. 1. The use according to any of the items 1-18, wherein the polynucleotide originating from the genetic locus ARHGAP18 is selected from SEQ ID NO:27, the complement of SEQ ID NO:27, a fragment thereof or its complement, and a variant thereof or its complement. 22. The use according to any of the items 1-18, wherein the polynucleotide originating from the genetic locus SLC16A6 is selected from SEQ ID NO:40, the complement of SEQ ID NO:40, a fragment thereof or its complement, and a variant thereof or its complement. 23. The use according to any of the items 1-18, wherein the polynucleotide originating from the genetic locus MALATl is selected from SEQ ID NO:53, the complement of SEQ ID NO:53, a fragment thereof or its complement, and a variant thereof or its complement.

24. The use according to any of the items 1-18, wherein the polynucleotide originating from the genetic locus MALATl is selected from SEQ ID NO: 66, the complement of SEQ ID NO:66, a fragment thereof or its complement, and a variant thereof or its complement.

25. The use according to any of the items 1-18, wherein the polynucleotide originating from the genetic locus CSNKID is selected from SEQ ID NO:79, the complement of SEQ ID NO:79, a fragment thereof or its complement, and a variant thereof or its complement.

26. The use according to any of the items 1-18, wherein the polynucleotide originating from the genetic locus C19orf43 is selected from SEQ ID NO:92, the complement of SEQ ID NO:92, a fragment thereof or its complement, and a variant thereof or its complement.

27. The use according to any of the items 1-18, wherein the polynucleotide originating from the genetic locus LRRN3 is selected from SEQ ID NO: 105, the complement of SEQ ID NO: 105, a fragment thereof or its complement, and a variant thereof or its complement. 28. A panel of biomarkers for assessing, particularly assessing in a whole blood sample, any member of the group consisting of colorectal cancer, colorectal adenoma and colorectal polyp, the panel comprising at least two polynucleotides, each polynucleotide corresponding to a different genetic locus, wherein at least one, and particulatly at least two of the polynucleotides originate(s) from a genetic locus selected from any of

ASGR2, CLIPl, ARHGAP18, SLC16A6, MALATl, CSNKID, C19orf43, and LRRN3. 29. The panel according to item 28, wherein the at least one, and particularly the at least two polynucleotide(s) is/are selected from any of SEQ ID NOs: 1, 14, 27, 40, 53, 66, 79, 92, 105, a fragment thereof or its complement, and a variant thereof or its complement. 30. The panel according to any of the items 28 and 29, wherein the at least one, and particularly the at least two polynucleotide(s) is/are selected from any of SEQ ID NOs: 4, 8, 12, 17, 21, 25, 30, 34, 38, 43, 47, 51, 56, 60, 64, 69, 73, 77, 82, 86, 90, 95, 99, 103, 108, and 112.

31. The panel according to any of items 28 to 30, wherein the panel is selected for analysis of polynucleotide expression levels for any member of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp.

32. The panel of item 31, wherein the polynucleotide expression levels are or reflect the expression levels of mRNAs.

33. The panel of item 32, wherein the polynucleotide expression levels are the levels of cDNAs produced by the process of reverse transcription of mRNAs.

34. The panel of any of items 28 to 33, wherein at least one of the polynucleotides is a fragment of a polynucleotide originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and LRRN3, or a variant thereof. 35. The panel of any of items 28 to 33, wherein at least one of the polynucleotides is a variant of a polynucleotide originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and LRRN3, or a fragment thereof.

36. The panel of any of items 28 to 35, wherein the panel is used for the management of patient care in any of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp.

37. The panel of any of items 28 to 36, wherein the management of patient care includes one or more of risk assessment, early diagnosis, establishing prognosis, monitoring patient treatment, and detecting relapse. 38. The panel of any of items 28 to 37, wherein the panel is used in discovery of therapeutic intervention of any of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp.

39. A method for measuring the expression levels of a polynucleotide from a biomarker, the biomarker being an indicator for the presence of any member of the group consisting of colorectal cancer, colorectal adenoma and colorectal polyp in a human individual, the method comprising: selecting a biomarker comprising a contiguous polynucleotide included in a member of the group consisting of SEQ ID NOs: 1, 14, 27, 40, 53, 66, 79, 92, 105, a fragment thereof or its complement, and a variant thereof or its complement; obtaining a biological sample from the human individual; in vitro (ex vivo) isolating cellular RNA from the sample and reverse-transcribing isolated RNA to obtain cDNA; amplifying copies of cDNA for the biomarker originating from the sample; and quantifying the level of cDNA amplified from the sample, thereby measuring the expression level of the polynucleotide from a biomarker, wherein in a specific embodiment the biological sample is whole blood.

40. The method according to item 39, wherein the expression levels of two or more polynucleotides from two or more biomarkers are measured. 41. The method according to item 40, wherein the method includes the step of selecting two or more biomarkers, each comprising a contiguous polynucleotide included in a member of the group consisting of SEQ ID NOs: 1, 14, 27, 40, 53, 66, 79, 92, 105, a fragment thereof, a complement thereof, and a variant thereof. 42. The method according to any of the items 39 to 41, wherein the method includes the step of selecting a biomarker comprising at least one polynucleotide from SEQ ID NOs: 4, 8, 12, 17, 21, 25, 30, 34, 38, 43, 47, 51, 56, 60, 64, 69, 73, 77, 82, 86, 90, 95, 99, 103, 108, and 112.

43. The method according to item 42, wherein the step of amplifying copies of cDNA further comprises at least two pairs of primers chosen from the primer pairs of SEQ. ID NOs: 2 and 3, 6 and 7, 10 and 11, 15 and 16, 19 and 20, 23 and 24, 28 and 29, 32 and 33, 36 and 37, 41 and 42, 45 and 46, 49 and 50, 54 and 55, 58 and 59, 62 and 63, 67 and 68, 71 and 72, 75 and 76, 80 and 81, 84 and 85, 88 and 89, 93 and 94, 97 and 98, 101 and 102, 106 and 107, and 110 and 111.

44. The method according to item 43, wherein the step of reverse-transcribing isolated RNA to obtain cDNA comprises using enzymes and reagents for the preparation of cDNAs.

45. The method according to any of the items 39 to 44, wherein the step of quantifying the level of cDNA further comprises labeling cDNA.

46. The method according to item 45, wherein labeling cDNA includes at least one fluorophore capable of interacting with double stranded nucleic acid by way of intercalation.

47. The method according to item 45, wherein labeling cDNA includes hybridization of a dual-labeled (fluorophore and quencher) hydrolysis probe to a the cDNA.

48. The method according to any of items 39 to 47, wherein the cDNA level(s) determined for the sample is/are compared to a control.

49. The method according to item 48, wherein the comparison is used in the management of patient care in any of colorectal cancer, colorectal adenoma and colorectal polyp.

50. The method according to item 49, wherein the management of patient care includes one or more of risk assessment, early diagnosis, establishing prognosis, monitoring patient treatment, and detecting relapse.

51. The method according to item 48, wherein the comparison is used in discovery of therapeutic intervention of any of colorectal cancer, colorectal adenoma and colorectal polyp. 52. The method according to any of the items 39 to 51, wherein the step of obtaining a biological sample from the human individual is by obtaining a sample of whole blood.

53. The method according to any of the items 39 to 51, wherein the step of obtaining a biological sample is by obtaining a sample of colorectal cells. 54. The method according to any of the items 39 to 51, wherein the step of obtaining a biological sample is by obtaining a sample of stool.

55. A kit for the assessment of any member of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp, the kit comprising: at least one reagent that is used in analysis of polynucleotide expression level for a biomarker for a member selected from the group consisting of colorectal cancer, colorectal adenoma and colorectal polyp, wherein the biomarker comprises a polynucleotide selected from SEQ ID NOs: 1, 4, 8, 12, 14, 17, 21, 25, 27, 30, 34, 38, 40, 43, 47, 51, 53, 56, 60, 64, 66, 69, 73, 77, 79, 82, 86, 90, 92, 95, 99, 103, 105, 108, 112, a complement therof, a variant thereof, and a fragment thereof; and instructions for using the kit for analyzing the polynucleotide expression level.

56. The kit according to item 55, comprising two or more reagents for use in analysis of polynucleotide expression levels for a plurality of biomarkers. 57. The kit according to item 56, wherein the plurality of biomarkers comprises two or more polynucleotides selected from 4, 8, 12, 17, 21, 25, 30, 34, 38, 43, 47, 51, 56, 60, 64, 69, 73, 77, 82, 86, 90, 95, 99, 103, 108, and 112.

58. The kit according to any of the items 55-57, wherein the polynucleotide expression levels is/are m NA(s) level(s). 59. The kit according to any of the items 55-57, wherein the polynucleotide expression level(s) is/are cDNA(s).

60. The kit according to any of the items 55-59, wherein the reagent comprises one or more pair(s) of primers chosen from the primer pairs of SEQ. ID NOs: 2 and 3, 6 and 7, 10 and 11, 15 and 16, 19 and 20, 23 and 24, 28 and 29, 32 and 33, 36 and 37, 41 and 42, 45 and 46, 49 and 50, 54 and 55, 58 and 59, 62 and 63, 67 and 68, 71 and 72, 75 and 76, 80 and 81, 84 and 85, 88 and 89, 93 and 94, 97 and 98, 101 and 102, 106 and 107, and 110 and 111.

61. The kit according to item 60, further comprising reagents for the preparation of cDNA. 62. The kit according to any of items 55-59, comprising a reagent that is capable of detecting and quantifying polynucleotides. 63. The kit according to item 62, wherein the reagent includes at least one chromophore, particularly a fluorophore capable of interacting with double- stranded nucleic acid by way of intercalation.

64. The kit according to item 62, wherein the reagent includes at least one (dual- labeled) TaqMan® hydrolysis probe.

65. The kit according to any of the items 55-64, further comprising consumable labware for at least one member of the group consisting of of sample collection, sample preparation, and sample analysis, wherein in a specific embodiment the consumable labware for sample preparation comprises materials and reagents for preparing RNA from whole blood.

The following examples, figures and sequence listing are provided to aid the understanding of the present disclosure, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the disclosure. Example 1

Colon biopsy and blood samples

After informed consent of untreated patients, colon biopsy specimen were taken before colonoscopy and put immediately in RNA later solution, then fresh frozen in less than 3 minutes. In parellel 9 ml of peripheral blood samples were taken into Paxgene Blood RNA Tubes (Qiagen Inc, Germantown, US), before colonoscopy.

The biopsy specimen and the blood samples were stored at -80°C. In each case formalin fixed paraffin embedded specimen were collected und used for immunhistochemical analysis (FFPE).

From peripheral blood samples total RNA was extracted (Paxgene protocol) and Affymetrix microarray analysis was done from peripheral blood samples of 9 patients with early stage colorectal adenocarcinoma (colorectal cancer) (Dukes A, B), 11 with advanced stage colorectal cancer (Dukes C, D), 11 with >1 cm size polyps, 11 with active inflammatory bowel disease (IBD) and of 11 healthy normal controls. Furthermore, an independent set of 45 blood samples (20 colorectal cancer, 10 adenoma, 5 IBD and 10 healthy) was also involved in the TaqMan® real-time PCR analysis. The samples with adenomas were either villous ones (including all villous adenomas >0 cm) or adenomas larger than 1 cm about with tubular phenotype only. The diagnostic groups and the number of patients of each group are represented in Table 3 :

Table 3

Example 2

Microarray analysis of blood samples

Total RNA was extracted using Paxgene Blood RNA Kit (Qiagen) according to the manufacturer's instructions. The isolated RNA samples were concentrated using GeneChip Blood RNA concentration Kit (Affymetrix Inc., Santa Clara, US). Quantity and quality of the RNA were tested by measuring of the absorbance and capillary gelelectrophoresis using 2100Bioanalyzer and RNA 6000 Pico Kit (Agilent Inc, Santa Clara, US). Biotinylated cRNA probes were synthesized from 5 μg total RNA and fragmented according to the Affymetrix description using One-cycle Target Labeling and Control Kit (Affymetrix) and Globin Reduction PNA oligomers (Applied Biosystems, Foster City, US) according to the "Globin

Reduction Protocol" from Affymetrix. Globin reduction was done in order to reduce high amounts of globin transcripts in the cellular RNA purified from whole blood samples: during the reverse transcription step of the probe synthesis, globin-specific PNA oligonucleotides were added, which reduced the amount of cDNA generated from globin mRNA.

Ten μg of each fragmented cRNA sample was hybridized into HGU133 Plus2.0 array (Affymetrix) at 45°C for 16 hours. The slides were washed and stained using Fluidics Station 450 and antibody amplification staining method according to the manufacturer's instructions. The fluorescent signals were detected by a GeneChip Scanner 3000.

Statistical evaluation of mR A expression profiles: Quality control analyses were performed according to the suggestions of The Tumor Analysis Best Practices

Working Group {supra). Scanned images were inspected for artifacts, percentage of present calls (>25%) and control of the R A degradation were evaluated. Based on the evaluation criteria all biopsy measurements fulfilled the minimal quality requirements. The Affymetrix expression arrays were pre-processed by gcRMA with quantile normalization and median polish summerization. To identify differentially expressed features Significance Analysis of Microarrays was used. The nearest shrunken centroid method (PAM) was applied for sample classification from gene expression data . The pre-processing and analysis was performed using R-environment with Bioconductor libraries. Tabel 4 lists microarray target sequences for the capture of cDNAs of the genes / genomic loci ASGR2, CLIPl, ARHGAP18, CR2, SLC16A6, MALAT1, CSNK1D, C19orf43, CD200, and LRRN3, which proved to qualify as biomarkers for the assessment of colorectal cancer.

Table 4

# Marker gene Disclosed in SEQ ID NO:

designation

1 ASGR2 1

2 CLIPl 14

3 ARHGAP18 27

4 SLC16A6 40

5 MALAT1 53, 66

6 CSNK1D 79

7 C19orf43 92

8 LRRN3 105 Example 3

Analysis of mRNA from blood samples using RT-qPCR and the TaqMan® real-time format

The Hydrolysis Probe or TaqMan® chemistry relies on the 5 -3' exonuclease activity of Taq polymerase, which degrades a hybridized non-extendible DNA probe during the extension step of the PCR. This probe is designed to hybridize to a region within the amplicon and is duel labeled with a reporter dye and a quenching dye. The close proximity of the quencher suppresses the fluorescence of the reporter dye. Once the exonuclease activity of Taq polymerase degrades the probe, the fluorescence of the reporter increases at a rate that is proportional to the amount of template present.

TaqMan® real-time PCR was used to measure the expression of 94 selected genes in 80 blood samples (35 samples which were also tested by microarrays and 45 independent samples) using an Applied Biosystems Micro Fluidic Card System. The measurements were performed using an ABI PRISM® 7900HT Sequence

Detection System as described in the products User Guide. For data analysis the SDS 2.2 software was used. The extracted delta Ct values (which represent the expression normalized to the GAPDH expression) were grouped according to the histologic groups. Then the Student's t-test was performed to compare the expression values between groups.

Example 4

Re-Design of RT-qPCR assays with LNA detection probes as an embodiment of the TaqMan® real-time format

In order to provide assays for screening with enhanced throughput, RT-PCR assays were designed making use of LNA-based hydrolysis probes of the "Universal

Probe Library" (= UPL; Roche Applied Science, Roche Diagnostics GmbH, Mannheim, Germany. A UPL probe is a short hydrolysis probes. Such a probe is labeled at the 5' end with fluorescein (FAM) and at the 3' end with a dark quencher dye. The length of a UPL probe is just 8-9 nucleotides and comprises a selected sequence. In order to maintain the specificity and melting temperature (Tm) that hybridizing qPCR probes require, Locked Nucleic Acids (LNA) are incorporated into the sequence of each UPL probe. LNA's are DNA nucleotide analogues with increased binding strengths compared to standard DNA nucleotides. The basis for the design of primers and probes as listed in Table N were the sequences targeted by the capture probes of the Affymetrix hybridization microarrays of Example 2 and listed in Table 4.

Table 5 gene Forward Reverse Amplicon UPL UPL designation (left) (right) sequence; probe probe No.

primer; primer; sequence; (Roche

SEQ ID SEQ ID SEQ ID SEQ ID Applied

NO: NO: NO: NO: Science)

ASGR2 2 3 4 5 68

ASGR2 6 7 8 9 158

ASGR2 10 11 12 13 60

CLIPl 15 16 17 18 44

CLIPl 19 20 21 22 121

CLIPl 23 24 25 26 40

ARHGAP18 28 29 30 31 40

ARHGAP18 32 33 34 35 1

ARHGAP18 36 37 38 39 88

SLC16A6 41 42 43 44 19

SLC16A6 45 46 47 48 16

SLC16A6 49 50 51 52 13

MALAT1 54 55 56 57 48

MALAT1 58 59 60 61 2

MALAT1 62 63 64 65 103

MALAT1 67 68 69 70 26

MALAT1 71 72 73 74 60

MALAT1 75 76 77 78 2

CSNK1D 80 81 82 83 7

CSNK1D 84 85 86 87 53

CSNK1D 88 89 90 91 133

C19orf43 93 94 95 96 21

C19orf43 97 98 99 100 43

C19orf43 101 102 103 104 89

LR N3 106 107 108 109 68

LR N3 110 111 112 113 132 Example 5

Assay validation, biostatistics approach

Accuracy of a diagnostic method is best described by its receiver-operating characteristics (ROC) (see especially Zweig, M. H., and Campbell, G., Clin. Chem. 39 (1993) 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 laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease or benign versus malignant disease.

In each case, the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds. On the y-axis is sensitivity, or the true-positive fraction [defined as

(number of true-positive test results)/(number of true-positive + 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 [defined as (number of false-positive results)/(number of true-negative + number of false-positive 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 disease in the sample. Each point on the ROC plot represents a sensitivity/ 1 -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 corner 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. One way to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number. Such an overall parameter e.g. is the so-called "total error" or alternatively the "area under the curve = AUC". The most common global measure is the area under the ROC plot. By convention, this area is always > 0.5 (if it is not, one can reverse the decision rule to make it so). Values range between 1.0 (perfect separation of the test values of the two groups) and 0.5 (no apparent distributional difference between the two groups of test values). The area does not depend only on a particular portion of the plot such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the entire plot. This is a quantitative, descriptive expression of how close the ROC plot is to the perfect one (area = 1.0).

ROC plots are shown in the Figures. In addition, box plots are shown. Each box contains the middle 50% of the respective data. The middle line is the median, whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the box.

Example 6

RT-PCR Analysis of patient blood samples with re-designed RT-PCR assays

After informed consent of patients, colon biopsy specimen were taken before colonoscopy and put immediately in RNAlater RNA stabilization reagent (Qiagen) solution, then fresh frozen in less than 3 minutes. In parellel 9 ml of peripheral blood samples were taken into Paxgene Blood RNA Tubes (Qiagen Inc, Germantown, US), before colonoscopy. The biopsy specimen and the blood samples were stored at -80°C. In each case formalin fixed paraffin embedded specimen were collected und used for immunhistochemical analysis (FFPE). From peripheral blood samples total RNA was extracted (Paxgene protocol,

Qiagen). Table 6 lists the groups of patients from which blood samples were obtained. Groups I and II together served as control. Table 6

Assays as described in Example 4 were performed in a 384-microwell plate format. In each column there were 15 samples and one negative control. In each microplate row 11 marker assays were performed in duplicate. In each row an SDHA

(succinate dehydrogenase complex, subunit A, flavoprotein) housekeeping gene assay was performed in duplicate, as a control and for normalization. SDHA was selected as reference by comparing several standard housekeeping genes on normal blood and selecting the most stable gene closest to the median of all biomarker candidates.

Replicate measurements were averaged. Biomarker measurements were normalized to the expression of the SDHA housekeeping gene which was determined and measured simultaneously.