WO2018087129A1 - Colorectal cancer methylation markers - Google Patents

Abstract

A method is provided for determining colorectal cancer, or the prognosis of a colorectal cancer in a subject, said method comprising in a sample from said subject determining the methylation status of a specific gene marker panel.

Description

Colorectal cancer methylation markers Field of invention

The present invention relates to DNA methylation biomarkers for colorectal cancer.

Background of invention

Colorectal cancer (CRC) is one of the most common cancers, reaching annual incidence rates of >1.3 million cases worldwide. Ensuring survival of CRC patients relies on detection at early stages, which can be achieved through population-based screening. The current screening programme in Denmark includes an immunochemical faecal occult blood test (iFOBT) followed by a colonoscopy if iFOBT is positive.

However, global patient adherence to current screening programmes is limited by low completion rates with great differences between ethnic groups. This necessitates the development of other methods for early CRC detection in order to ensure patient survival. Cell-free nucleic acids are currently of great interest as blood-based biomarkers for cancer, as these might be more specific for individual cancer types than protein-based biomarkers.

Methylation of the carbon-5 position of cytosine residues within CpG dinucleotides is a well-established epigenetic mechanism involved in the regulation of gene expression. Most CpG dinucleotides cluster in CpG rich regions in the genome, known as CpG islands (CGI), and these regions are often located within gene regulatory elements. In fact, the promoter region of more than half of all protein encoding genes contain a CGI and the methylation status of this sequence is instrumental in regulating the

transcriptional activity of the gene. Consequently, disruption of the cell's normal methylation pattern can have severe consequences and contribute to neoplastic transformation. Genome-wide studies have shown that aberrant DNA methylation is a common feature in human cancer and hundreds of tumour suppressor genes have been shown to be subject to DNA-methylation mediated silencing.

Hypermethylation of the gene promoter regions induces gene silencing in two ways: (i) inhibition of transcription factor binding, and (ii) binding of methyl-binding domain proteins, which enable chromatin modifications. DNA hypermethylation occurs early and often in the adenoma to carcinoma sequence, promoting it as an ideal blood-based biomarker for CRC. This has led to the development of a commercially available blood test for DNA methylation analysing the promoter for the SEPT9 gene. However, evaluation of the SEPT9 assay in a large-scale cross-sectional study revealed sub- optimal sensitivity and specificity (48.2% and 91.5% respectively). Other case-control studies analysing a larger panel of genes, have revealed more promising results, however, these studies often lacked well-defined control groups.

Summary of invention

The invention relates to methylation biomarkers for colorectal cancer. The invention provides a number of methylation markers, which can be used to distinguish between patients with colorectal cancer and healthy subjects. A plurality of individual methylation biomarkers are identified, which show high sensitivity and specificity.

In one aspect, a method is provided for determining colorectal cancer, the prognosis of a colorectal cancer, and/or monitoring a colorectal cancer in a subject, said method comprising in a sample from said subject determining the methylation status of at least one gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. More preferred, the invention relates to a method of determining colorectal cancer, the prognosis of a colorectal cancer, and/or monitoring a colorectal cancer in a subject, said method comprising in a sample from said subject determining the methylation status for the genes in the group consisting of ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM. Another aspect pertains to a method for assessing whether a human subject is likely to develop colorectal cancer, said method comprising

i. providing a sample from said human subject,

ii. determining in said sample the methylation status of at least one gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

iii. on the basis of said methylation status identifying a human subject that is more likely to develop colorectal cancer. More preferred, a method is provided for assessing whether a human subject is likely to develop colorectal cancer, said method comprising

i. providing a sample from said human subject,

ii. determining in said sample the methylation status for the genes in the group consisting of ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM, and iii. on the basis of said methylation status identifying a human subject that is more likely to develop colorectal cancer.

A third aspect pertains to a method for categorizing or predicting the clinical outcome of a colorectal cancer of a subject, said method comprising in a sample from said subject determining the methylation status of at least one gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. It is preferred that the methylation status is determined for the genes in the group consisting of ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM.

A fourth aspect relates to a method of evaluating the risk for a subject of contracting colorectal cancer, said method comprising in a sample from said subject determining the methylation status of a gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. It is preferred that the methylation status is determined for the genes in the group consisting of ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM. In a fifth aspect, a method is provided of treating a colorectal cancer in a human subject, said method comprising the steps of

i. determining colorectal cancer, or the prognosis of a colorectal cancer in a subject by a method as defined above,

ii. selecting human subjects having colorectal cancer, or a negative or positive prognosis of a colorectal cancer,

iii. subjecting said subjects identified in step ii. to a suitable treatment for colorectal cancer. A kit for determining colorectal cancer, or categorizing or predicting the clinical outcome of a colorectal cancer, or monitoring the treatment of a colorectal cancer, said kit comprising

i. an agent that (a) modifies methylated cytosine residues but not non- methylated cytosine residues; or (b) modifies non-methylated cytosine residues but not methylated cytosine residues; or (c) modifies a nucleic acid sequence in a methylation- dependent manner, and

ii. at least one pair of oligonucleotide primers that specifically hybridizes under amplification conditions to a region of a gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. It is preferred that the kit comprises oligonucleotide primers that specifically hybridize under amplification conditions to a region of the genes in the group consisting of ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM.

In a seventh aspect of the present disclosure provides a use of oligonucleotide primers comprising a sequence, which is a subsequence of a gene loci selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK or the

complement thereof for diagnosing colorectal cancer in any of the methods provided in the present disclosure. It is preferred that the primers comprise a subsequence of a gene loci selected from the group consisting of ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM or the complement thereof. In yet an eighth aspect, a method is provided for identifying therapeutically effective agents for treatment of colorectal cancer, said method comprising

i. providing a colorectal cancer cell line comprising one or more genetic loci selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK,

ii. providing one or more potential therapeutic agents,

iii. treating said colorectal cancer cells by bringing said agents in contact with said colorectal cancer cells,

iv. determining methylation status of said one or more genetic loci v. comparing said methylation status of said treated colorectal cancer cells with the methylation status of said colorectal cancer cells, when untreated, wherein a decreased level of methylation positive alleles is indicative of a therapeutic agent. It is preferred that the genetic loci are the genes in the group consisting of ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM.

Description of Drawings

Figure 1 Number of methylated promoter regions according patient group Figure 2 Stepwise backwards selection according to model number. Note: Logistical regression modelling with stepwise backwards selection. Potential predictor variables are located in the top row. Model number is recorded in the left column. Area under the receiver operating curve (AUROC) according to model number and the P-value according to the removed predictor variable is located in the two rightward columns. Model 12 is marked in darker shade.

Figure 3 Receiver operating characteristic curve for Model 12 for all stage colorectal cancer Figure 4 Receiver operating characteristic curve for Model 12 for stage I and II colorectal cancer

Detailed description of the invention

The present invention relates to methylation biomarkers for use in the diagnosis and treatment of colorectal cancer. Generally, the methylation markers of the invention can be used in methods for identifying subjects, which are at risk of having to colorectal cancer; i.e. subjects having an increased likelihood of having a colorectal cancer. The methylation markers of the invention can also be used in methods for identifying subjects having colorectal cancer, and in this case, the markers allow early diagnosis, Further, the markers provide prognostic information with respect to colorectal cancer, and thus, the markers can be used to identify a subject having colorectal cancer, and the cancer DNA can be tested for predictive prognostic information based on methylation markers provided herein, and thus provide information on which curative and/or ameliorative treatment to the colorectal cancer can be provided. The methylation status of the methylation markers may also be used to monitor a treatment provided for the curing and/or amelioration of colorectal cancer. Additionally, the marker methylation status can be used to monitor relapse of colorectal cancer for subjects previously cured for colorectal cancer.

Thus, aspects of the present invention relates to i) methods for identifying subjects, which have a colorectal cancer, including early stages, such as asymptomatic stages of colorectal cancer, ii) methods for providing prognostic information of a colorectal cancer and/or inferring a suitable treatment based thereupon, iii) methods of monitoring a treatment of a colorectal cancer, and/or monitoring relapse of a colorectal cancer. In order to facilitate the understanding of the invention a number of definitions are provided below.

Definitions

Amplification according to the present invention is the process wherein a plurality of exact copies of one or more gene loci or gene portions (template) is synthesised. In one preferred embodiment of the present invention, amplification of a template comprises the process wherein a template is copied by a nucleic acid polymerase or polymerase homologue, for example a DNA polymerase or an RNA polymerase. For example, templates may be amplified using reverse transcription, the polymerase chain reaction (PCR), ligase chain reaction (LCR), in vivo amplification of cloned DNA, isothermal amplification techniques, and other similar procedures capable of generating a complementing nucleic acid sequence. Amplified copies of a targeted genetic region are sometimes referred to as an amplicon.

The term "PCR bias" as used herein refers to conditions, wherein PCR more efficiently amplifies templates with a specific methylation status. It has been reported that at least some unmethylated nucleic acid templates are more efficiently amplified than methylated nucleic acid template.

A double stranded nucleic acid contains two strands that are complementary in sequence and capable of hybridizing to one another. In general, a gene is defined in terms of its coding strand, but in the context of the present invention, an oligonucleotide primer, which hybridize to a gene as defined by the sequence of its coding strand, also comprise oligonucleotide primers, which hybridize to the complement thereof. A nucleotide is herein defined as a monomer of RNA or DNA. A nucleotide is a ribose or a deoxyribose ring attached to both a base and a phosphate group. Both mono-, di-, and tri-phosphate nucleosides are referred to as nucleotides.

The term oligonucleotide comprises oligonucleotides of both natural and/or non-natural nucleotides, including any combination thereof. The natural and/or non-natural nucleotides may be linked by natural phosphodiester bonds or by non-natural bonds. Preferred oligonucleotides comprise only natural nucleotides linked by phosphodiester bonds. The oligomer or polymer sequences of the present invention are formed from the chemical or enzymatic addition of monomer subunits. The term "oligonucleotide" as used herein includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleotides, ribonucleotides, anomeric forms thereof, peptide nucleic acid monomers (PNAs), locked nucleotide acid monomers (LNA), and the like, capable of specifically binding to a single stranded polynucleotide tag by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to several tens of monomeric units, e.g. 40-60. Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5'→ 3' order from left to right and the "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted. When a double stranded DNA molecule is shown, the nucleotides of the top strand are in 5'→ 3' order from left to right and the nucleotides of the bottom strand are then in 3'→ 5' order from left to right. Usually, oligonucleotides of the invention comprise the four natural nucleotides; however, they may also comprise methylated or non-natural nucleotide analogs.

The term "dinucleotide" as used herein refers to two sequential nucleotides. The dinucleotide may be comprised in an oligonucleotide or a nucleic acid sequence. In particular, the dinucleotide CpG, which denotes a cytosine linked to a guanine by a phosphodiester bond, may be comprised in an oligonucleotide according to the present invention, and also comprised in a targeted gene locus sequence according to the present invention. A CpG dinucleotide is also herein referred to as a CpG site. CpG sites are targets for methylation of the cytosine residue.

Methylation status: the term "methylation status" as used herein, refers to the presence or absence of methylation in a specific nucleic acid region. In particular, the present invention relates to detection of methylated cytosine (5-methylcytosine). A nucleic acid sequence, e.g. a gene locus of the invention, may comprise one or more CpG methylation sites. The nucleic acid sequence of the gene locus may be methylated on all methylation sites (i.e. 100% methylated), or unmethylated on all methylation sites (i.e. 0% methylated). However, the nucleic acid sequence may also be methylated on a subset of its potential methylation sites (CpG-sites). In this latter case, the nucleic acid molecule is heterogeneously methylated.

The gene loci methylation markers of the present invention can be used to infer colorectal cancer based on the relative amount of methylation positive and methylation negative alleles in a sample comprising in a mixture of nucleic acid molecules from a subject. For example, the methylation status of a specific gene locus marker of the present invention may be that at least 50%, such as on at least 60%, such as on at least 70%, for example on at least 80%, such as on at least 90%, such as on at least 95%, for example on at least 99%, such as least 99.9% of the nucleic acid sequence molecules (alleles) in a sample are methylation positive (fully methylated). Gene locus: The term "gene locus" as used herein, such as the gene loci defined by the genes RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK, is meant to include all regions relevant for expression of a given gene, both the coding region and upstream and downstream regions, which may comprise cis-acting activating signals. A gene locus, specifically is meant to include at least 1000 bp upstream and/or downstream of the open reading frame of an encoded gene, such as at least 900 bp, such as at least 800 bp, such as at least 700 bp, such as at least 600 bp, such as at least 500 bp, such as at least 400 bp, such as at least 300 bp, such as at least 200 bp, such as at least 100 bp upstream and/or downstream of the open reading frame of an encoded gene. A gene locus is also meant to include any intronic sequences in the open reading frame. It is also understood that specific subregions of a gene locus specified herein can be of particular importance for the methods described herein. In particular CG-rich regions also known as CpG islands are particularly relevant, because CG-dinucleotides are targets for methylation.

Method of determining colorectal cancer

A number of methods are provided herein for analysing a human subject with respect to colorectal cancer. In particular, methods are provided for determining colorectal cancer in a human subject, methods for determining the prognosis of a colorectal cancer in a subject and/or inferring a suitable treatment, methods for categorizing or staging a colorectal cancer of a human subject, methods for monitoring a colorectal cancer, such as monitoring the treatment of a colorectal cancer and/or relapse of a colorectal cancer. The methylation biomarkers for colorectal cancer are described in more detailed herein below. Generally, the one or more methylation biomarkers for colorectal cancer according to the methods herein are selected from a gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

Thus in one aspect, a method is provided for determining colorectal cancer, the prognosis of a colorectal cancer, and/or monitoring a colorectal cancer in a subject, said method comprising in a sample from said subject determining the methylation status of at least one gene including regulatory sequences of said gene, wherein said gene locus is selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

In another aspect, a method is provided for categorizing or predicting the clinical outcome of a colorectal cancer of a subject, said method comprising in a sample from said subject determining the methylation status of at least one gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. In another aspect, a method is provided for evaluating the risk for a human subject of developing colorectal cancer, or for monitoring relapse of a colorectal cancer, said method comprising in a sample from said subject determining the methylation status of a gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

A further aspect relates to a method for assessing whether a human subject is likely to develop colorectal cancer, said method comprising

i) providing a sample from said human subject,

ii) determining in said sample the methylation status of at least one gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK,

iii) on the basis of said methylation status identifying a human subject that is more likely to develop colorectal cancer.

The methods thus involve determining the methylation status of one or more gene loci as defined herein. Methylation status is preferably determined for multiple gene loci, for example methylation status for at least two gene loci are determined, such as at least three gene loci, such as at least four gene loci, or five or more gene loci. The plurality of gene loci is preferably selected from the marker gene loci provided herein, i.e. a gene loci selected from the group consisting RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

In one embodiment, the methylation status is determined in two or more, such as all, of the, gene loci selected from the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 ,

HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT and CDKN2A, or the group consisting of - RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2 and MGMT, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3 and TFPI2, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC and PHACTR3, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF and APC, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 and HLTF, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 and WIF1 , or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD and HIC1 , or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 and THBD, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM and SFRP1 , or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2 and VIM, or the group consisting of - RARB, ALX4, NPTX2, SEPT9, BMP3 and SDC2, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9 and BMP3, or the group consisting of

- RARB, ALX4, NPTX2 and SEPT9, or the group consisting of

- RARB, ALX4 and NPTX2, or the group consisting of

- RARB and ALX4.

In a preferred embodiment, the methylation status is determined in two or more, such as all, of the gene loci

- RARB, ALX4, NPTX2 and SEPT9, such as in

- RARB, ALX4, NPTX2 and SEPT9, or in

- RARB, ALX4 and NPTX2, or in

- RARB, NPTX2 and SEPT9, or in

- RARB, ALX4, and SEPT9, or in

- RARB and SEPT9, or in

- RARB and ALX4, or in - RARB, and NPTX2, or in

- ALX4, NPTX2 and SEPT9, or in

- ALX4 and NPTX2, or in

- ALX4 and SEPT9, or in

- NPTX2 and SEPT9.

In another preferred embodiment, the methylation status is determined in RARB, ALX4, NPTX2, SEPT9 and BMP3. In another preferred embodiment, the methylation status is determined in RARB, ALX4, NPTX2, SEPT9, BMP3 and SDC2. In another preferred embodiment, the methylation status is determined in RARB, ALX4, NPTX2, SEPT9, BMP3 and SDC2. In a most preferred embodiment, the methylation status is determined in RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2 and VIM. In another preferred embodiment, the methylation status is determined in RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM and SFRP1. In another preferred embodiment, the methylation status is determined in RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 and THBD. In another preferred embodiment, the methylation status is determined in RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD and HIC1 .

Generally, increased levels of methylation positive alleles of the respective marker gene locus relative to methylation levels of a predetermined control sample of non- cancer origin is indicative of the presence of a colorectal cancer, higher likelihood of developing cancer, decreased overall survival, negative outcome, different stage cancer, different grade cancer, and/or higher risk of contracting cancer.

Thus, the provided methods may preferably comprise steps of comparing the methylation status of the respective gene locus determined for a subject with a predetermined methylation status for the corresponding gene of a reference sample obtained from a healthy subject and/or a subject with a different stage of cancer. The predetermined status is preferably determined in a sample obtained from other subjects, which do not have colorectal cancer. The predetermined methylation status differs between the different methylation markers of the invention. In one embodiment, for any gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK, any level of methylation positive alleles above 1 % is indicative of a colorectal cancer, higher likelihood of developing cancer, decreased overall survival, negative outcome, different stage cancer, different grade cancer, and/or higher risk of contracting cancer for a human subject.

Thus, a level of methylation positive alleles above 1 %, such as above 2%, 3%, 4%, 5%, such as above 10%, such as above 15%, such as above 20%, such as above 25%, such as above 30%, such as above 35%, such as above 40%, such as above 45%, such as above 50%, such as above 55%, such as above 60%, such as above 65%, such as preferably above 65.3%, such as above 70%, such as above 75%, such as above 80%, such as above 85%, such as above 90%, such as above 95%, such as above 96%, 97%, 98%, or 99%, such as 100% is indicative of colorectal cancer, increased risk of colorectal cancer, the prognosis of colorectal cancer, and/or relapse of colorectal cancer, and thus indicates that a given treatment being monitored is inefficient.

It is appreciated that the entire genetic region of the target loci need not be methylated. Thus, in one embodiment, a target loci wherein at least 1 % of the targeted genetic region is methylated in indicative of colorectal cancer, increased risk of colorectal cancer, the prognosis of colorectal cancer, and/or relapse of colorectal cancer, and thus indicates that a given treatment being monitored is inefficient. However, in other embodiments, a methylation level of the target loci above 2%, such as above 3%, 4%, 5%, such as above 10%, such as above 15%, such as above 20%, such as above 25%, such as above 30%, such as above 35%, such as above 40%, such as above 45%, such as above 50%, such as above 55%, such as above 60%, such as above 65%, such as preferably above 65.3%, such as above 70%, such as above 75%, such as above 80%, such as above 85%, such as above 90%, such as above 95%, such as above 96%, 97%, 98%, or 99%, such as 100% is indicative of colorectal cancer, increased risk of colorectal cancer, the prognosis of colorectal cancer, and/or relapse of colorectal cancer, and thus indicates that a given treatment being monitored is inefficient. The methylation status of the marker regions identified is in a particularly preferred embodiment, combined with information on sex and age.

For example, where sex=female is indicative of colorectal cancer, increased risk of colorectal cancer, the prognosis of colorectal cancer, and/or relapse of colorectal cancer, and thus indicates that a given treatment being monitored is inefficient.

Also, age above 50 years, such as above 51 years, such as above 52 years, such as above 53, such as above 54 years, such as above 55, such as above 56 years, such as above 57 years, such as above 58 years, such as above 59 years, such as above 60 years, such as above 61 years, such as above 62 years, such as above 63 years, such as above 64 years, such as above 65 years, such as above 66 years, such as above 67 years, such as above 68 years, such as above 69 years, such as above 70 years, such as above 71 years, such as above 72 years, such as above 73 years, such as above 74 years, such as above 75 years, such as above 76 years, such as above 77 years, such as above 78 years, such as above 79 years, such as above 80, is indicative of colorectal cancer, increased risk of colorectal cancer, the prognosis of colorectal cancer, and/or relapse of colorectal cancer, and thus indicates that a given treatment being monitored is inefficient. In a preferred embodiment, age above 66 years, is indicative of colorectal cancer, increased risk of colorectal cancer, the prognosis of colorectal cancer, and/or relapse of colorectal cancer, and thus indicates that a given treatment being monitored is inefficient.

Thus, preferably, a combination of female sex and age above 66 years is specifically indicative of colorectal cancer, increased risk of colorectal cancer, the prognosis of colorectal cancer, and/or relapse of colorectal cancer, and thus indicates that a given treatment being monitored is inefficient. The combination of female sex and age above 66 years is preferably used together with predictive methylation markers as defined elsewhere herein, such as preferably methylation of one or more, such as all of the gene loci ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM. In one embodiment, sex and/or age are used as co-variables to determine, which marker loci to select for determination of methylation status. Method for treatment of colorectal cancer

Aspects provided herein also relates to methods for determining colorectal cancer or the prognosis of a colorectal cancer in a subject and/or inferring a suitable treatment, as well as for monitoring a colorectal cancer, and in particular monitoring the treatment of a colorectal cancer and/or monitoring relapse of a colorectal cancer.

So in one aspect, a method is provided for treatment of colorectal cancer in a human subject, the method comprises the steps of

i. determining colorectal cancer, or the prognosis of a colorectal cancer in a subject by a method of the present invention, as defined elsewhere herein,

ii. selecting human subjects having colorectal cancer, or a relapse or increased risk of relapse, of a colorectal cancer,

iii. subjecting said subjects identified in step ii. to a suitable treatment for colorectal cancer.

The step of determining colorectal cancer by a method of the present invention allows early detection of colorectal cancer, and therefore allows treatment of the cancer to be initiated before developing into later stages and/or before forming metastases. Early detection may for example allow less serious types of therapeutic interventions, such as minimal invasive procedures. In one embodiment, the selected human subject is subjected to a treatment selected form surgery, chemotherapy, immunotherapy and/or radiotherapy, however, in a preferred embodiment, the treatment is radiotherapy. In one embodiment, the treatment is a combination of surgery, chemotherapy and radiotherapy, for example surgery followed by chemotherapy and/or radiotherapy. In one embodiment, the preferred treatments are 5-FU calciumfolinat, Capecitabin,

FOLFOX, XELOX treatments. The treatment thus can encompass administering one or more of Calciumfolinat, Capecitabin and/or Oxaliplatin. Preferred administration regimes are indicated in the table below. Table B. Preferred administration regimes

The methylation markers also allow monitoring relapse of colorectal cancer, as well as offering a personalized treatment of colorectal cancer by surveillance and quality of control of the treatment offered, thereby allowing terminating ineffective treatments and offering alternative treatments. Thus, in another aspect, the invention provides a method for personalized treatment of a colorectal cancer of a human subject, said method comprising

i) in a sample from said human subject, determining the methylation status of one or two or more gene loci selected from the group consisting of RARB, ALX4, NPTX2,

SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK

ii) providing a treatment of colorectal cancer to said human subject,

iii) after a sufficient amount of time having provided the treatment, in a sample from said human subject, determining the methylation status of said gene loci selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK, iv) comparing the methylation status of said gene loci before and after treatment, and v) if methylation of said genetic loci is similar to the methylation before treatment, terminating said provided treatment and preferably offering an alternative treatment, or vi) if methylation of said genetic loci is reduced relative to the methylation before treatment, continuing said provided treatment or even terminating the treatment. Methylation biomarkers for colorectal cancer

As described herein above, a number of different methods are provided herein for evaluating colorectal cancer in a human subject based on methylation status of specific gene loci. The invention also provides specific oligonucleotide primers and kits for use in determining methylation status of specific gene loci, which are methylation biomarkers for colorectal cancer according to the present invention. These gene loci include RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. Generally, in the methods of the invention, the methylation status is determined for two or more gene loci selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. In specific one embodiment, methylation status is determined in the RARB gene locus and at least one additional gene locus selected from the group consisting of ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. In specific one embodiment, methylation status is determined in the ALX4 gene locus and at least one additional gene locus selected from the group consisting of RARB, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. In specific one embodiment, methylation status is determined in the NPTX2 gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC,

PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. In specific one embodiment, methylation status is determined in the SEPT9 gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC,

PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. In specific one embodiment, methylation status is determined in the BMP3 gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

In specific one embodiment, methylation status is determined in the SDC2 gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

In specific one embodiment, methylation status is determined in the VIM gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

In specific one embodiment, methylation status is determined in the SFRP1 gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, THBD, HIC1 , WIF1 , HLTF, APC,

PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

In specific one embodiment, methylation status is determined in the THBD gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

In specific one embodiment, methylation status is determined in the HIC1 gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

In specific one embodiment, methylation status is determined in the WIF1 gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. In specific one embodiment, methylation status is determined in the HLTF gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

In specific one embodiment, methylation status is determined in the APC gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

In specific one embodiment, methylation status is determined in the PHACTR3 gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, TFPI2, MGMT, CDKN2A and PPENK.

In specific one embodiment, methylation status is determined in the TFPI2 gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, MGMT, CDKN2A and PPENK.

In specific one embodiment, methylation status is determined in the MGMT gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, CDKN2A and PPENK.

In specific one embodiment, methylation status is determined in the CDKN2A gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT and PPENK.

In specific one embodiment, methylation status is determined in the PPENK gene locus and at least one additional gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT and CDKN2A.

DNA sequences of specific gene loci are provided herein below. In a preferred embodiment, the methylation status is determined in a gene locus identified by SEQ ID NO: 150-180 or the complement thereof.

In a preferred embodiment, the methylation status is determined by a method comprising amplifying a gene locus of the invention using at least one primer selected from the group consisting of SEQ ID NO: 1 -120. Methylation status is preferably determined for a gene mentioned in table A and/or table 7 using the respective primers identified in table A or table 7; i.e.

ALX4: inner primer pair SEQ ID NO: 1 or 2 and/or outer primer pair SEQ ID NO: 61 and 62;

APC: inner primer pair SEQ ID NO: 3 or 4 and/or outer primer pair SEQ ID NO: 63 and 64;

NPTX2: inner primer pair SEQ ID NO: 21 or 22 and/or outer primer pair SEQ ID NO: 81 and 82;

RARB: inner primer pair SEQ ID NO: 33 or 34 and/or outer primer pair SEQ ID NO: 93 and 94;

SEPT9: inner primer pair SEQ ID NO: 39 or 40 and/or outer primer pair SEQ ID NO: 99 and 100;

etc .;

Sample

In the methods provided herein, the methylation status is determined for one or more gene loci in a sample from a human subject. The sample comprises biological material, in particular genetic material comprising nucleic acid molecules. The nucleic acid molecules may be extracted from the sample prior to the analysis. The sample may be obtained or provided from any human source. In one embodiment, determination of methylation status of a gene locus or genetic region of the invention is performed on samples selected from the group consisting of colon tissue, hematopoietic tissue, stem cells, including cancer stem cell, and body fluids, such as blood, stool, urine or sputum.

It is well-known that tumour DNA may leak to the blood stream or other bodily fluids. In preferred embodiments the sample is or comprises a body fluid, such as blood, stool, urine or sputum. In a preferred embodiment, the sample is a blood sample. In particular, it is preferred that the sample is a blood or plasma sample. Body fluids are retrievable by less invasive methods than tissue samples, which must be obtained surgically for example by biopsies. Blood samples are also generally preferred over stool samples.

The provided sample is in one embodiment a formalin-fixed paraffin-embedded (ffpe) sample, for example an ffpe sample, wherein prestages to colorectal cancer can be seen. In particular, the sample used for predetermining methylation status can be an ffpe sample. Many ffpe samples may be provided, which can give rise to statistically strong predetermined values with respect to evaluation of colorectal cancer risk, categorizing or staging a colorectal cancer of a human subject, methods for monitoring a colorectal cancer, such as monitoring the treatment of a colorectal cancer and/or relapse of a colorectal cancer.

The nucleic acid to be analysed for the presence of methylated CpG may be extracted from the samples by a variety of techniques such as that described by Maniatis, et al (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp 280, 281 , 1982). However, the sample may be used directly.

Any nucleic acid, in purified or nonpurified form, can be utilized as the starting nucleic acid or acids, provided it contains, or is suspected of containing, the specific nucleic acid sequence containing the methylation target site (e.g., CpG). The specific nucleic acid sequence which is to be amplified may be a part of a larger molecule or is present initially as a discrete molecule. The nucleic acid sequence to be amplified need not to be present in a pure form, it may for example be a fraction of a complex mixture of other DNA molecules, and/or RNA. In one example, the nucleic acid sequence is a fraction of a genomic nucleic acid preparation.

Extremely low amounts of nucleic acid may be used as target sequence according to the provided methods. It is appreciated by the person skilled in the art that in practical terms no upper limit for the amount of nucleic acid to be analysed exists. The problem that the skilled person may encounter is that the amount of sample to be analysed is limited. Therefore, it is beneficial that the method can be performed on a small amount of sample and thus a limited amount of nucleic acid in said sample. The present methods allow the detection of only very few nucleic acid copies. The amount of the nucleic acid to be analysed is in one embodiment at least 0.01 ng, such as 0.1 ng, such as 0.5 ng, for example 1 ng, such as at least 10 ng, for example at least 25 ng, such as at least 50 ng, for example at least 75 ng, such as at least 100 ng, for example at least 125 ng, such as at least 150 ng, for example at least 200 ng, such as at least 225 ng, for example at least 250 ng, such as at least 275 ng, for example at least 300 ng, 400 ng, for example at least 500 ng, such as at least 600 ng, for example at least 700 ng, such as at least 800, ng, for example at least 900 ng or such as at least 1000 ng.

In one preferred embodiment the amount of nucleic acid as the starting material for the method of the present invention is approximately 50 ng, alternatively 100 ng or 200 ng.

Methylation status

The methods of the present invention for determining colorectal cancer in a human subject, methods for determining the prognosis of a colorectal cancer in a subject and/or inferring a suitable (personalized) treatment, methods for categorizing or staging a colorectal cancer of a human subject and methods for monitoring a colorectal cancer, all include a step of providing or obtaining a sample from the human subject, and in that sample determining the methylation status of at least one genetic locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK, as well as subregions thereof, in particular GC-rich subregions, such as those delineated by the respective primer pairs identified in table A and/or table 7.

Methylation status of the target gene loci or genetic regions of the present invention may be determined by any suitable method available to the skilled person for detecting methylation status. However, in a preferred embodiment, methylation status is determined by a quantitative method, such as a semi-quantitative method, which is capable of detecting levels of methylation positive alleles and/or methylation negative alleles in a population of target molecules present in a sample. For example, the quantitative method is preferably capable of detecting different levels of methylation positive alleles of a given target locus sequence, such as detecting whether 0%, less than 1 %, more that 1 %, such as approximately 10%, 25%, 50%, 75% or 100% of the alleles of a given marker locus are methylation positive. A semi- quantitative method provides categorical data, such as the level of 1 -10% methylated templates or 10-50% methylated templates. Some techniques in the art merely detect the presence of one or more methylation positive and/or methylation negative alleles of a given target sequence without providing quantitative data, and without providing information on the relative levels of methylation positive and methylation negative alleles. However, preferred methods of the present invention provide a quantitative measure of the relative level of methylation positive alleles of a specific target region.

The term "methylation status" as used herein, refers to the extent to which a nucleic acid region and/or in particular a CpG methylation site is methylated or unmethylated, which may be expressed as the methylation level of a given sample. The methylation status of a single CpG methylation site can be either methylated or unmethylated. A nucleic acid sequence comprising multiple potential methylation (CpG) sites, may be methylated on only a subset of those CpG sites. Such nucleic acid molecules/alleles are heterogeneous methylated. The term "methylation status", thus, refers to whether a nucleic acid sequence is methylation positive (methylated on all CpG sites), is methylation negative (all CpG sites of the sequence is unnmethylated), or is

heterogeneous methylated (a subset of CpG sites of the sequence is methylated.

The methods for inferring colorectal cancer of the present invention, thus determine methylation status of specific methylation markers by determining whether a specific methylation marker in a sample obtained or provided from a subject is methylation positive, methylation negative or heterogeneously methylated, as well as detecting the relative level of methylated alleles of a given locus. The methods may also include detecting marker sequences with low methylation, which defines methylation of less than 1 % of the alleles of a sample. Methylation status may be determined by any suitable method available to those of skill in the art.

The methods provided herein for determining colorectal cancer, prognosis of a colorectal cancer and/or inferring a suitable (personalized) treatment as well as methods for categorizing or staging a colorectal cancer are not necessarily limited to specific methylation detection methods. It is preferred that the methylation detection method employed in convenient, fast, reliable and low-cost.

For example, the method may be selected from the group consisting of Methylation- Sensitive High Resolution Melting (MS-HRM), Methylation - Sensitive Melting Curve Analysis (MS-MCA), EpiTyper, Methylation-Specific PCR (MSP), DNA methylation specific qPCR (qMSP), Pyrosequencing, Methyl Light, Amplicon bisulfite sequencing (AmpliconBS), Enrichment bisulfite sequencing (EnrichmentBS), Whole genome bisulfite sequencing (BS-Seq), HELP assays, and Methyl Sensitive Southern Blotting, and determination may involve Methylated DNA immunoprecipitation (MeDIP). In one embodiment, the method is selected from the group consisting of AmpliconBS 1 , AmpliconBS 2, AmpliconBS 3, AmpliconBS 4, EnrichmentBS 1 , EnrichmentBS 2, EpiTyper 1 , EpiTyper 3, Infinium, Pyroseq 1 , Pyroseq 1 (replicate), Pyroseq 2, Pyroseq 3, Pyroseq 4, Pyroseq 5, MethyLight, MS-HRM, MS-MCA, qMSP (preamp), qMSP (standard), DNA-methylation-specific amplification by qPCR, HPLC-MS, Immunoquant, Pyroseq AluYb8, Bisulfite pyrosequencing using primers that amplify AluYb8 repetitive DNA, Pyroseq D4Z4, Pyroseq LINE1 , Pyroseq NBL2 and ClonalBS; cf. Bock et al, 2016, nature biotech. 34 (7). In another embodiment, the method is selected from the group consisting of High- performance liquid chromatography (HPLC), High-performance capillary

electrophoresis (HPCE), Sssl assay , Gene specific Methylation-specific PCR (MSP- PCR), Methyl-sensitive restriction enzyme PCR (MSRE-PCR), MethyLight,

Pyrosequencing , Methylation-sensitive single nucleotide Primer extension (MS- SNuPE), Combined bisulfite restriction analysis (COBRA), Methylation sensitive-high resolution melting (MS-HRM), Methylation-specific multiplex ligationdependent probe amplification (MS-MLPA), Mass ARRAY EpiTYPER , Restriction landmark genomic scanning (RLGS), Differential methylation hybridization (DMH), Methylated DNA immunoprecipitation and microarray chip (MeDIPchip), Bead arrays (lllumina) Bisulfite, Whole-genome bisulfite sequencing, Single molecule real time (SMRT) sequencing and MethylCap sequencing; cf. Syedmoradi et al, 2016, Royal Soc of Chem. (DOI: 10.1039/c6an01649a).

In one embodiment of methods of the present invention for determining colorectal cancer in a human subject, for determining the prognosis of a colorectal cancer in a subject and/or inferring a suitable treatment, for categorizing or staging a colorectal cancer of a human subject, and/or for monitoring a colorectal cancer, such as monitoring the treatment of a colorectal cancer and/or relapse of a colorectal cancer, the methylation status is determined by the use of methylation-sensitive restriction enzymes. Many restriction enzymes are sensitive to the DNA methylation states. Cleavage can be blocked or impaired when a particular base in the recognition site is modified. For example, the MspJI family of restriction enzymes has been found to be dependent on methylation and hydroxymethylation for cleavage to occur. These enzymes excise ~ 32 base pair fragments containing a centrally located 5-hmC or 5- mC modified residue that can be extracted and sequenced. Due to the known position of this epigenetic modification, bisulfite conversion is not required prior to downstream analysis.

Methylation-sensitive enzymes are well-known in the art and include:

Aatll, Accll, Aor13HI, Aor51 HI, BspT104l, BssHII, Cfrl OI, Clal Cpol, Eco52l, Haell,

Hapll, Hhal, Mlul, Nael, NotI, Nrul, Nsbl, PmaCI, Psp1406l, Pvul, Sacll, Sail, Smal and SnaBI.

The digested nucleic acid sample is subsequently analysed by for example gel electrophoresis.

So, in one embodiment of the methods of the invention, methylation status is determined by a method comprising the steps of

i) providing a sample, such as a blood sample or a blood or plasma sample from said subject comprising nucleic acid material comprising said gene,

ii) processing said nucleic acid sequence using one or more methylation- sensitive restriction endonuclease enzymes,

iii) optionally, amplifying said processed nucleic acid sequence in order to obtain an amplification product, and

iv) analyzing said processed nucleic acid sequence or said amplification product for the presence of processed and/or unprocessed nucleic acid sequences, thereby inferring the presence of methylated and/or unmethylated nucleic acid sequences.

In a preferred embodiment of methods of the present invention, the methodology employed for determining methylation status is determined by a method, which comprises at least the steps of modifying the DNA with an agent which targets either methylated or unmethylated sequences, amplifying the DNA, and analysing the amplification products. For example, amplification product is analysed by detecting the presence or absence of amplification product, wherein the presence of amplification product indicates that the target nucleic acid has not been cleaved by the restriction enzymes, and wherein the absence of amplification product indicates that the target nucleic acid has been cleaved by the restriction enzymes.

Thus, generally, in the methods of the invention, methylation status is determined by a method comprising the steps of

i) providing a sample, such as a blood sample or a blood or plasma sample from said subject comprising nucleic acid material comprising a gene locus of the invention,

ii) modifying said nucleic acid material using an agent, which modifies nucleic acid sequences in a methylation-dependent manner,

iii) amplifying at least one portion of said gene locus using primers, which span or comprise at least one CpG dinucleotide in said gene locus in order to obtain an amplification product, and

iv) analysing said amplification product for the presence of modified and/or unmodified cytosine residues, wherein the presence of modified cytosine residues are indicative of methylated cytosine residues.

For example, the method comprises the steps of

i) providing a sample, such as a blood sample, from said subject comprising nucleic acid material comprising said gene locus,

ii) modifying said nucleic acid using an agent which modifies unmethylated cytosine,

iii) amplifying at least one portion of said gene locus using primers, which span or comprise at least one CpG dinucleotide in said gene locus in order to obtain an amplification product, and

iv) analysing said amplification product.

The amplification product can be analysed for nucleic acid substitutions resulting from conversion of modified cytosine residues, preferably wherein the presence of converted cytosine residues are indicative of unmethylated cytosine residues, and presence of unconverted cytosine residues is indicative of methylated cytosine residues. Typically, unmethylated cytosine is converted to thymidine after bisulphite treatment and amplification, while methylated cytosine is left unchanged after same treatment.

In a preferred embodiment, the amplification product is analysed by melting curve analysis.

The amplification product, the amplicon, is in a preferred embodiment a genetic region of a gene of the invention, wherein said region is delineated by the primer pairs identified in table A and/or table 7 for each respective gene; i.e.

ALX4: inner primer pair SEQ ID NO: 1 or 2 and/or outer primer pair SEQ ID NO: 61 and 62;

APC: inner primer pair SEQ ID NO: 3 or 4 and/or outer primer pair SEQ ID NO: 63 and 64;

NPTX2: inner primer pair SEQ ID NO: 21 or 22 and/or outer primer pair SEQ ID NO: 81 and 82;

RARB: inner primer pair SEQ ID NO: 33 or 34 and/or outer primer pair SEQ ID NO: 93 and 94; SEPT9: inner primer pair SEQ ID NO: 39 or 40 and/or outer primer pair SEQ ID NO: 99 and 100;

etc .;

Modification of DNA

The method for determining methylation status in the present invention preferably comprise a step of modifying the nucleic acids comprised in the sample, or extracted from the sample, using an agent which specifically modifies unmethylated cytosine in the nucleic acid. As used herein the term "modifies" refers the specific modification of either an unmethylated cytosine or a methylated cytosine, for example the specific conversion of an unmethylated cytosine to another nucleotide which will distinguish the modified unmethylated cytosine from a methylated cytosine. In one preferred embodiment, an agent modifies unmethylated cytosine to uracil. Such an agent may be any agent conferring said conversion, wherein unmethylated cytosine is modified, but not methylated cytosine. In one preferred embodiment the agent for modifying unmethylated cytosine is sodium bisulfite. Sodium bisulfite (NaHS0₃) reacts readily with the 5,6-double bond of cytosine, but only poorly with methylated cytosine. The cytosine reacts with the bisulfite ion, forming a reaction intermediate in the form of a sulfonated cytosine which is prone to deamination, eventually resulting in a sulfonated uracil. Uracil can subsequently be formed under alkaline conditions which removes the sulfonate group.

During a nucleic acid amplification process, uracil will by the Taq polymerase be recognised as a thymidine. The product upon PCR amplification of a Sodium bisulfite modified nucleic acid contains cytosine at the position where a methylated cytosine (5- methylcytosine) occurred in the starting template DNA of the sample. Moreover, the product upon PCR amplification of a Sodium bisulfite modified nucleic acid contains thymidine at the position where an unmethylated cytosine (5-methylcytosine) occurred in the starting template DNA of the sample. Thus, an unmethylated cytosine is converted into a thymidine residue upon amplification of a bisulfite modified nucleic acid.

In a preferred embodiment of the present invention, the nucleic acids are modified using an agent which modifies unmethylated cytosine in the nucleic acid. In a specific embodiment, such an agent is a bisulfite, hydrogen sulfite, and/or disulfite reagent, for example sodium bisulfite.

However, in another embodiment, an agent is used, which specifically modifies methylated cytosine in the nucleic acid and does not modify unmethylated cytosine.

Amplifying step

After modification of the nucleic acids of the sample, the specific genetic region selected for determination of methylation status is preferably amplified in order to generate and thereby obtain multiple copies (amplicons) of the respective genetic regions, which can allow its further analysis with respect to methylation status. The amplification is preferably preformed using at least one oligonucleotide primer, which targets the specific genetic region comprising methylation markers for colorectal cancer according to the present invention. Most preferably amplification is performed using two oligonucleotide primers, which delineates the analysed region. The skilled person may use his common general knowledge in designing suitable primers. However, in a preferred embodiment, at least one, and preferably two or four methylation-specific oligonucleotide primers are employed for amplification of the modified nucleic acid. The amplifying step is a polymerisation reaction wherein an agent for polymerisation is involved, effecting an oligonucleotide primer extension. The agent for polymerization may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes. Enzymes that are suitable for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, polymerase muteins, reverse transcriptase, and other enzymes, including heat-stable enzymes (i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation also known as Taq polymerases). Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each locus nucleic acid strand. Generally, the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths. There may be agents for polymerization, however, which initiate synthesis at the 5' end and proceed in the other direction, using the same process as described above.

A preferred method for amplifying the modified nucleic acid by means of at least one oligonucleotide primer pair as shown in table A and/or table 7 is by the polymerase chain reaction (PCR), as described herein and as is commonly used by those skilled in the art. PCR amplification requires a set of oligonucleotide primers, one forward primer and one reverse primer. The forward primer can be a methylation-specific primer. The reverse primer is in another embodiment a methylation-specific primer. However, both reverse and forward primer may be methylation-specific oligonucleotide primers. The amplification product (amplicon) may be of any length, however in one preferred embodiment, the amplification product comprise between 15 and 1000 nucleotides, such as between 15 and 500 nucleotides, such as between 50 and 150 nucleotides, preferably between 70 and 120 nucleotides. In a preferred embodiment, the amplicon is delineated by the primers identified in table 7 for each respective gene, cf. herein above.

The PCR reaction is characterised by three steps a) melting a nucleic acid template, b) annealing at least one methylation-independent oligonucleotide primer to said nucleic acid template, and c) elongating said at least one methylation-independent

oligonucleotide primer. The general characteristics of PCR amplification are well-known to those of skill in the art. The melting of a CpG-containing nucleic acid template may also be referred to as strand separation. Melting is necessary where the target nucleic acid contains two complementary strands bound together by hydrogen bonds. This strand separation can be accomplished using various suitable denaturing conditions, including physical, chemical, or enzymatic means. The is typically accomplished by increasing

temperature toa melting temperature is typically between 80 and 90 degrees Celsius for a few seconds.

Separated strands are used as a template for the synthesis of additional nucleic acid strands. The annealing temperature depends on the primers used and is typically between 40 and 75 degrees Celsius

The optimal annealing temperature can be calculated by standard algorithms, as known to people skilled within the art. In one embodiment, the optimal primer annealing temperature (Tm) is calculated as: Tm = 4(G + C) + 2(A + T), wherein G, C, A, T designates the number of the respective nucleotides. In another embodiment, the optimal primer annealing temperature (Tm) is calculated as:

Tm = 64.9°C + 41 °C x (number of G's and C's in the primer - 16.4)/N, where N is the length of the primer. However, the annealing temperature should be empirically determined in respect of each specific primer.

The oligonucleotide primers annealed to the template is elongated to form an amplification product. Elongation occurs in a buffered aqueous solution, preferably at a pH of 7-9. The two oligonucleotide primers are added to the reaction mixture in a molar excess of primer: template especially when the template is genomic DNA which will ensure an improved efficiency. Deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP are added to the reaction mixture, either separately or together with the primers. An appropriate agent for effecting the primer extension reaction, referred to and described elsewhere herein as an agent for polymerization is added to the reaction mixture. It is appreciated by a person skilled in the art that for PCR the agent for polymerisation preferable is a heat-stable polymerase enzyme, such as Taq polymerase.

The PCR method comprises incubating the nucleic acid at a cycle of different specific temperatures in order to control the steps of amplification. The amplification buffer and polymerase required for PCR are well known to people of skill within the art.

The PCR reaction mixture is incubated sequentially at the melting temperature, the annealing temperature and the elongating temperature, respectively, for a number of cycles. The PCR reaction may run between 10 and 70 cycles. Typically, the PCR reaction run between 25 and 55 cycles, such as at least 25, at least 30, at least 35, at least 40, preferably at least 45, at least 50 or at least 55 cycles.

PCR can be performed on a PCR machine, which is also known as a thermal cycler. Specifically, the thermal cycler may be coupled to a fluorometer, thus allowing the monitoring of the nucleic acid amplification in real time by use of intercalating fluorescent dyes, or other fluorescent probes. Applicable dyes according to the present invention include any DNA intercalating dye. Suitable dyes include ethidium bromide, EvaGreen, LC Green, Syto9, SYBR Green, SensiMix HRM™ kit dye, however many dies are available for this same purpose.

Real-time PCR allows for easy performance of quantitative PCR (qPCR), which is usually aided by algorithms comprised in the software, which is usually supplied with the PCR machines.

The fluorometer can furthermore be equipped with software that will allow interpretation of the results. Such software for data analyses may also be supplied with the kit of the present invention. Another variant of the PCR technique, multiplex PCR, enables the simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. PCR according to the present invention comprise all known variants of the PCR technique known to people of skill within the art. Thus, the PCR technology comprise real-time PCR, qPCR, multiplex PCR.

Oligonucleotide primers

The oligonucleotide primer employed for amplification of modified nucleic acid is preferably a methylation-specific primer. The term "methylation-specific primer" refers to an oligonucleotide primer, which span one or more CpG sites and which specifically hybridizes to nucleic acid alleles, where methylated Cytosine has been protected from deamination and convention to Uracil. These primers will therefore only provide an amplification product if the targeted region was methylated.

Methylation-specific primers will support amplification of either unmethylated nucleic acid regions or methylated regions, because the sequence of the primers are design to match either the modified targets or unmodified targets.

The oligonucleotide primers of the present invention are capable of being employed in amplification reactions, wherein the primers are used in amplification of template DNA originating from either a methylation positive strand. The preferred methylation-specific primers comprise at least one CpG dinucleotide. Accordingly, in a methylation positive and bisulfite modified nucleic acid target sequence, the primer sequence will anneal to the nucleic acid template with a perfect match, wherein all of the nucleotides in a consecutive region of the primer forms base pairs with a complementary region in the nucleic acid target. The design of oligonucleotide primers suitable for nucleic acid amplification techniques, such as PCR, is known to people skilled within the art. The design of such primers involves analysis of the primer's melting temperatures and ability to form duplexes, hairpins or other secondary structures. Both the sequence and the length of the oligonucleotide primers are relevant in this context. The oligonucleotide primers according to the present invention comprise between 10 and 200 consecutive nucleotides, such as at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 180 or at least 200 nucleotides. In a specific embodiment, the oligonucleotide primers comprise between 15 and 60 consecutive nucleotides, such as 15, 16, 17, 18, 19, 20, preferably 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, such as 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, alternatively at least 41 , at least 42, at least 44, at least 46, at least 48, at least 50, at least 52, at least 54, at least 56, at least 58, or at least 60 consecutive nucleotides.

The methods employed for determining the methylation status of a nucleic acid according to the present invention, preferably comprise amplification of a modified nucleic acid by use of a methylation independent oligonucleotide primer. In one embodiment, the oligonucleotide primers of the present invention are able to hybridize to a nucleic acid sequence comprising CpG islands. In a preferred embodiment, at least one of the oligonucleotide primers comprises at least one CpG dinucleotide. In another embodiment, the oligonucleotide primers comprise 2, alternatively 3, 4, 5, 6, 7, 8, 9 or 10 CpG dinucleotides. In even further embodiments, the oligonucleotide primers comprise at least 10 CpG dinucleotides. In one preferred embodiment the at least one methylation-independent oligonucleotide primer comprises one CpG dinucleotide at the 5 '-end of the primer.

The primers of the present invention may in one preferred embodiment comprise at least one CpG site and anneal with a higher efficiency to a methylated than to an unmethylated template upon modification of unmethylated cytosine. The primers of the present invention comprise at least one CpG site. However, the primers comprise also for example two CpG sites. The at least one CpG site is positioned in the 3' end of the primer, for example within 10 nucleotides of the 3' end of the primer, within 9 nucleotides of the 3' end of the primer, within 8 nucleotides of the 3' end of the primer, within 7 nucleotides of the 3' end of the primer, within 6 nucleotides of the 3' end of the primer, within 5 nucleotides of the 3' end of the primer, within 4 nucleotides of the 3' end of the primer or within 3 nucleotides of the 3' end of the primer. In a preferred embodiment the CpG site is introduced as the two 3' terminal nucleotides of the primer. Specific hybridization typically is accomplished by a primer having at least 10, for example at least 12, such as at least 14, for example at least 16, such as at least 18, for example at least 20, such as at least 22, for example at least 24, such as at least 26, for example at least 28, or such as at least 30 contiguous nucleotides, which are complementary to the target template, such as a gene loci selected from the group consisting of SEQ ID NO: 151 -180. Often the primer will be 100% identical to the target template or close to 100% identical. However, the primer may also be 98% identical to the target template or for example at least 97%, such as at least 96%, for example at least 95%, such as at least 94%, for example at least 93%, such as at least 92%, for example at least 91 %, such as at least 90%, for example at least 89%, such as at least 88%, for example at least 87%, such as at least 86%, for example at least 85%, such as at least 84%, for example at least 83%, such as at least 82%, for example at least 81 %, such as at least 80%, for example at least 79%, such as at least 78%, for example at least 77%, such as at least 76%, for example at least 75%, such as at least 74%, for example at least 73%, such as at least 72%, for example at least 71 %, such as at least 70%, for example at least 68%, such as at least 66%, for example at least 64%, such as at least 62% or for example at least 60% identical to the target template, such as a gene loci selected from the group consisting of SEQ ID NO: 151 -180. If there is a sufficient region of complementary nucleotides, e.g., at least 10, such as at least 12, for example at least 15, such as at least 18, or for example at least 20, for example at least 30, such as at least 40, for example at least 50, such as at least 60, for example at least 70 nucleotides, then the primer may also contain additional nucleotide residues that do not interfere with hybridization but may be useful for other

manipulations. Examples of such other residues may be sites for restriction

endonuclease cleavage, for ligand binding or for factor binding or linkers.

The oligonucleotide primer of the present invention is designed to hybridize to nucleic acids in a sample. Importantly, the nucleic acids in that sample are treated with an agent, which modifies unmethylated cytosine in said nucleic acid. Thereby, any unmethylated Cytosine of CpG dinucleotides comprised in the nucleic acid are converted to Uracil as explained elsewhere herein. Consequently, in primers comprising a CpG dinucleotide, designed to hybridize with the complementary CpG dinucleotide of the nucleic acid of the sample, the CpG dinucleotide will predominantly hybridize to the methylated CpG dinucleotide fraction of the nucleic acid. In the unmethylated fraction of CpG dinucleotides comprised in the nucleic acid of the sample, Cytosine are modified to uracil which does not hybridize with the CpG dinucleotide of the oligonucleotide primer or at least hybridize with much lower affinity. The oligonucleotide primers are designed to comprise sufficient nucleotides for specific hybridization to the relevant target nucleic acid sequence. In some embodiments the oligonucleotide primers also comprise one or more CpG dinucleotides, as specified elsewhere herein. These CpG dinucleotides only hybridize with the originally methylated alleles of the nucleic acids.

The presence of one or more mismatches between the primer and template affects the optimal annealing temperature of said oligonucleotide primer for use in amplification reactions. The more hybridizing nucleotides comprised on the oligonucleotide primers, the higher is the optimal annealing temperature. Consequently, amplification of methylated alleles of nucleic acids by CpG-containing oligonucleotide primers according to the present invention is favoured by increased annealing temperature. Conversely, amplification of unmethylated alleles is favoured by decreased annealing temperature. A PCR bias towards amplification of unmethylated alleles of a nucleic acid template can be reversed by amplification of the target nucleic acid template at a relatively higher annealing temperature, which favours oligonucleotide primer binding and priming of the methylated allele.

Besides annealing temperature, other factors also affect hybridisation to a target sequence of the oligonucleotide primer. At highly stringent conditions, hybridization between perfect matching primer and target sequences are favoured, such as hybridization between a methylation-specific primer and a methylated target sequence upon cytosine modification. Less stringent conditions will tend to favour oligonucleotide primer binding, priming and amplification of the unmethylated allele upon modification and conversion. Modulation of temperature is one way of adjusting the stringency of hybridization, but the stringency of hybridization may also be modulated by adjusting buffer composition, and/or salt concentrations in the hybridization mixture, which is known to those of skill within the art. Any such method of modulating hybridization stringency to balance the PCR bias towards amplification of unmethylated template may be applied. However, modulation of temperature is preferred. In one embodiment, the oligonucleotide primer of the present invention is selected from the group consisting of SEQ ID NO: 1 -120. Methylation status is preferably determined for a gene mentioned in table A and/or table 7 using the respective primers identified in table A and/or table 7; i.e.

ALX4: inner primer pair SEQ ID NO: 1 or 2 and/or outer primer pair SEQ ID NO: 61 and 62;

APC: inner primer pair SEQ ID NO: 3 or 4 and/or outer primer pair SEQ ID NO: 63 and 64;

NPTX2: inner primer pair SEQ ID NO: 21 or 22 and/or outer primer pair SEQ ID NO: 81 and 82;

RARB: inner primer pair SEQ ID NO: 33 or 34 and/or outer primer pair SEQ ID NO: 93 and 94;

SEPT9: inner primer pair SEQ ID NO: 39 or 40 and/or outer primer pair SEQ ID NO: 99 and 100;

etc .;

The oligonucleotide primers provided herein comprise or hybridize to a target sequence provided herein, i.e. a target sequence comprising a sequence or subsequence of a gene loci selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK, or the complement thereof. In one embodiment, the

oligonucleotide primer specifically comprises or hybridizes to regions within 1 kb of the coding region of the relevant gene loci. In a preferred embodiment, the oligonucleotide primer comprises or hybridizes to a promoter region located within 1000 kb upstream of the coding region of the relevant gene. Thus, in one embodiment, the oligonucleotide primers comprise or hybridize to a target nucleic acid sequence of a gene loci selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK, or the complement thereof and in particular to a region located within 1000 kb upstream of the coding region of the relevant gene. In one embodiment, the oligonucleotide primers hybridize to a target nucleic acid sequence or comprise or consist of 5-50, such as 5-30, such as 10-20 consecutive nucleotides of a subsequence of a target nucleic acid sequence in a gene loci selected from the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT and CDKN2A, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2 and MGMT, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3 and TFPI2, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC and PHACTR3, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF and APC, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 and HLTF, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 and WIF1 , or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD and HIC1 , or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 and THBD, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM and SFRP1 , or the group

consisting of

- RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2 and VIM, or the group consisting of - RARB, ALX4, NPTX2, SEPT9, BMP3 and SDC2, or the group consisting of

- RARB, ALX4, NPTX2, SEPT9 and BMP3, or the group consisting of

- RARB, ALX4, NPTX2 and SEPT9, or the group consisting of

- RARB, ALX4 and NPTX2, or the group consisting of

- RARB and ALX4. In a preferred embodiment, the oligonucleotide primers hybridize to a target nucleic acid sequence or comprise or consist of 5-50, such as 5-30, such as 10-20 consecutive nucleotides of a subsequence of a target nucleic acid sequence in a gene loci selected from the group consisting of

- RARB, ALX4, NPTX2 and SEPT9, such as in

- RARB, ALX4, NPTX2 and SEPT9, or in

- RARB, ALX4 and NPTX2, or in

- RARB, NPTX2 and SEPT9, or in

- RARB, ALX4, and SEPT9, or in

- RARB and SEPT9, or in

- RARB and ALX4, or in

- RARB, and NPTX2, or in

- ALX4, NPTX2 and SEPT9, or in

- ALX4 and NPTX2, or in

- ALX4 and SEPT9, or in

- NPTX2 and SEPT9.

In a preferred embodiment of the present invention the at least one oligonucleotide primer hybridizes to a target nucleic acid sequence selected from the group consisting of SEQ ID NO: 151 -180, or the complement thereof.

In one embodiment, an oligonucleotide primer of the present invention specifically comprises or consists of 5-50, such as 5-30, such as 10-20 consecutive nucleotides of a subsequence of a gene loci selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK, or the complement thereof.

In particular, the present invention relates to oligonucleotide primer pairs, which delineates at least one CpG dinucleotide in a gene locus of the invention. The term "delineate" as used in this context is meant to indicated the at least one CpG site is located in the nucleic acid region between the primer pairs; i.e. the amplified nucleic acid region comprise at least one CpG dinucleotide. The term "comprising" as used in connection with "primers comprising at least one CpG dinucleotide is meant to specify that the oligonucleotide primer itself comprise a CpG site.

In a preferred embodiment, the at least one oligonucleotide primer comprises or consists of 5-50, such as 5-30, such as 10-20 consecutive nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 151 -180.

Thus, in the methods of the present invention for determining or prognosing colorectal cancer, categorizing or predicting colorectal cancer, or evaluating the risk of contracting a colorectal cancer, methylation status is preferably determined by amplifying at least one portion of a gene loci selected from RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK, using at least one primer pair selected from the nucleic acid sequences set forth in table 7, i.e.

ALX4: inner primer pair SEQ ID NO: 1 or 2 and/or outer primer pair SEQ ID NO: 61 and 62;

APC: inner primer pair SEQ ID NO: 3 or 4 and/or outer primer pair SEQ ID NO: 63 and 64; NPTX2: inner primer pair SEQ ID NO: 21 or 22 and/or outer primer pair SEQ ID NO: 81 and 82;

RARB: inner primer pair SEQ ID NO: 33 or 34 and/or outer primer pair SEQ ID NO: 93 and 94;

SEPT9: inner primer pair SEQ ID NO: 39 or 40 and/or outer primer pair SEQ ID NO: 99 and 100;

etc .; Detection of an amplification product can be performed by hybridizing the amplification product to an oligonucleotide probe, as described below. In a preferred embodiment, methylation status is determined by amplifying at least one portion of the respective at least one gene loci, and further employing at least one oligonucleotide probe which hybridizes to an amplification product. In a preferred embodiment, the oligonucleotide probe comprises 10-100 consecutive nucleic acids selected from the group of sequences consisting SEQ ID NO: 151 -180 and/or the complement thereof. The probe is preferably selected from the group consisting of SEQ ID Nos: 121 -150; cf. in table A and/or table 7

One aspect of the invention also relates to the use of oligonucleotide primers of the present invention for determining or prognosing a colorectal cancer, categorizing or predicting colorectal cancer, or evaluating the risk of contracting a colorectal cancer. Thus, in one aspect, the present invention provides a use of oligonucleotide primers comprising a subsequence of a loci selected from the group consisting RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK or the complement thereof for diagnosing colorectal cancer in a method of the invention as defined elsewhere herein. In a preferred embodiment of the use of the invention, the primers are selected from the group set forth in table A and/or table 7 (SEQ ID NO: SEQ ID NO: 1 -120). In a preferred embodiment, the oligonucleotide primers comprise a sequence selected from the group consisting of SEQ ID NO: 151 -180 and/or the complement thereof. In a preferred embodiment, the oligonucleotide primers comprising a subsequence selected from a gene loci selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

Analysis of amplified CpG-containing nucleic acids

As explained above, the nucleic acid (target) sample is preferably subjected to an agent that converts an unmethylated cytosine to another nucleotide which will distinguish the unmethylated from the methylated cytosine. In a preferred embodiment the agent modifies unmethylated cytosine to uracil. The modifying agent can be sodium bisulphite. During the amplification process uracil will be converted to thymidine. Thus, after conversion of unmethylated cytosines to uracils in the nucleic acid (target) sample, the subsequent PCR amplification converts uracils to thymine. As a consequence of the sodium bisulfite and PCR-mediated specific conversion of unmethylated cytosines to thymines, G:C base pairs are converted to A:T base pairs at positions, where the cytosine was methylated. The difference in nucleic acid sequence at previously methylated (methylation positive) or unmethylated (methylation negative) cytosines allows for the analysis of methylation status in a sample. This analysis can comprise identifying cytosine residues, which have been converted to thymidine after amplification, as unmethylated cytosine residues, and identifying cytosine residues, which has not been converted under as methylated cytosine residues.

By this method, analysis of the amplified nucleic acid after treatment with a modifying agent such as sodium bisulphite and subsequent PCR amplification can reveal the methylation status of the target nucleic acid sequence. Thus, in one embodiment, the method for determining methylation status of a nucleic acid comprises a step of analyzing the amplified nucleic acids. The present invention is not necessarily limited to specific methylation detection methods; any method available to the skilled person can in theory be employed. It is preferred that the methylation detection method employed in convenient, fast, reliable and low-cost. For example, the subsequent analysis can be selected from the group consisting of melting curve analysis, high resolution melting analysis, nucleic acid sequencing, primer extension, denaturing gradient gel electrophoresis, southern blotting, restriction enzyme digestion, methylation-sensitive single-strand conformation analysis (MS-SSCA) and denaturing high performance liquid chromatography (DHPLC).

In one embodiment, the methylation status of the amplified containing nucleic acid is determined by any method selected from the group consisting of Methylation-Specific PCR (MSP), Whole genome bisulfite sequencing (BS-Seq), HELP assays, ChlP-on- chip assays, Restriction landmark genomic scanning, Methylated DNA

immunoprecipitation (MeDIP), Pyrosequencing of bisulfite treated DNA, Molecular break light assays, and Methyl Sensitive Southern Blotting. In a preferred embodiment, Methylation-Specific PCR (MSP) is used. In another embodiment, the methylation status of the amplified containing nucleic acid is determined by a method selected from the group consisting methylation specific PCR, bisulfite sequencing, COBRA, melting curve analysis, or DNA methylation arrays. In a preferred embodiment of the present invention, the analysis of the amplified nucleic acid region is melting curve analysis. In another preferred embodiment of the present invention, the analysis of the amplified nucleic acid is high resolution melting analysis (HRM).

Kit

One aspect of the present invention relates to a kit for the detection of methylation status of a nucleic acid in a sample. A kit will typically comprise both a forward and a reverse primer to be used in the amplifying step of the present invention. The forward primer, the reverse primer or both may be a methylation-specific oligonucleotide primer as described herein. Thus, one aspect of the invention relates to a kit for determining colorectal cancer, or categorizing or predicting the clinical outcome of a colorectal cancer, or monitoring the treatment of a colorectal cancer, and/or monitoring relapse of a previously treated colorectal cancer.

The kit of the invention comprise

i. an agent that (a) modifies methylated cytosine residues but not non- methylated cytosine residues; or (b) modifies non-methylated cytosine residues but not methylated cytosine residues; or (c) modifies a nucleic acid sequence in a methylation- dependent manner,

ii. and at least one pair of oligonucleotide primers that specifically hybridizes under amplification conditions to a region of a gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK. The agent is preferably a methylation-dependent endonuclease as described elsewhere herein, and/or an agent capable of modifying non-methylated cytosine residues but not methylated cytosine residues, such as a bisulphite compound as decribed elsewhere heren, for example sodium bisulphite. Generally, the kit preferably comprises at least one oligonucleotide primer of probe of the present invention, as defined herein above. In a preferred embodiment, the kit comprises at least one oligonucleotide primer selected from the group consisting of SEQ ID NO: 1 -120. In a more preferred embodiment, the kit comprises at least one primer pair selected from in table A and/or table 7; i.e. ALX4: inner primer pair SEQ ID NO: 1 or 2 and/or outer primer pair SEQ ID NO: 61 and 62;

APC: inner primer pair SEQ ID NO: 3 or 4 and/or outer primer pair SEQ ID NO: 63 and 64;...

NPTX2: inner primer pair SEQ ID NO: 21 or 22 and/or outer primer pair SEQ ID NO: 81 and 82;...

RARB: inner primer pair SEQ ID NO: 33 or 34 and/or outer primer pair SEQ ID NO: 93 and 94;...

SEPT9: inner primer pair SEQ ID NO: 39 or 40 and/or outer primer pair SEQ ID NO: 99 and 100; etc...

The kit may also comprise one or more reference sample, in particular reference samples comprising a nucleic acid sequence selected from a gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 . WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

For example, the at least one reference sample comprises 100% methylation positive reference nucleic acid, and/or 100% methylation negative reference nucleic acid. In a preferred embodiment the kit comprises at least two reference samples, wherein one of said reference samples comprises 100% methylation positive reference nucleic acid and a second reference sample comprises 100% methylation negative reference nucleic acid. The methylation positive and methylation negative reference nucleic acids may be mixed, by a person employing the kit, in ratios that are suitable for the detection of methylation in a particular sample. It is understood that reference samples in different ratios of methylation positive to methylation negative CpG-containing nucleic acids may be comprised in the kit. For example the kit may comprise at least one reference sample comprising 50% methylated and 50% non-methylated nucleic acid alleles of the respective genetic locus marker.

In particular, the nucleic acid comprised on the reference sample of the kit is preferably methylated (methylation positive) or non-methylated (methylation negative), and the kit preferably comprise two or more reference samples with different methylation status; i.e. different levels of methylation positive and methylation negative alleles. Thus, the specific nucleic acid alleles (e.g. alleles of the gene locus ALX4) of the reference sample may be unmethylated (0% methylated), 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% methylated. The kit may thus comprise one reference sample with a nucleic acid sequence as defined above, which is

unmethylated; and another reference sample with the same nucleic acid sequence, which is 100% methylated; and one or more samples comprising the same nucleic acid sequence having different levels of intermediate methylation status, e.g. 10%, 50% and/or 90% methylation.

The kit preferably comprises the following combinations of one or more reference samples and primer pairs, which corresponds to the primers and target gene loci set out in in table A and/or table 7; i.e.:

Reference sequence comprising gene locus ALX4: inner primer pair SEQ ID NO: 1 or 2 and/or outer primer pair SEQ ID NO: 61 and 62;

Reference sequence comprising gene locus APC: inner primer pair SEQ ID NO: 3 or 4 and/or outer primer pair SEQ ID NO: 63 and 64;...

Reference sequence comprising gene locus NPTX2: inner primer pair SEQ ID NO: 21 or 22 and/or outer primer pair SEQ ID NO: 81 and 82;...

Reference sequence comprising gene locus RARB: inner primer pair SEQ ID NO: 33 or 34 and/or outer primer pair SEQ ID NO: 93 and 94;...

Reference sequence comprising gene locus SEPT9: inner primer pair SEQ ID NO: 39 or 40 and/or outer primer pair SEQ ID NO: 99 and 100; etc...

The kit may also comprise at least one probe. Probes of the invention are defined herein above, and in a preferred embodiment, the kit comprise at least one

oligonucleotide probe comprising 10-100 consecutive nucleic acids selected from the group of sequences consisting of SEQ ID NO: 151-180. Thus, the kit of the invention preferably comprise at least one oligonucleotide probe which hybridizes to a nucleic acid sequence selected from the group consisting SEQ ID NO: 151 -180 and/or the complement thereof. The kit of the invention preferably comprise at least one oligonucleotide probe which hybridizes to an amplification product generated by a primer pair set out in table 7. In one embodiment, the probe is selected from the group consisting of SEQ ID NO: 121 - 150. The kit may also comprise additional reagents used in the amplifying step of the detection method as disclosed herein. Thus, the kit may further comprise

deoxyribonucleoside triphosphates, DNA polymerase enzyme and/or nucleic acid amplification buffer. In another embodiment the kit further comprises an agent that modifies unmethylated cytosine nucleotides. Such an agent may for example be bisulfite, hydrogen sulfite, and/or disulfite reagent.

The kit may also comprise other components suitable for detection of methylation status. For example, the kit may comprise a methylation-sensitive restriction enzyme.

The kit may in preferred embodiments further comprise instructions for the

performance of the detection method of the kit and for the interpretation of the results. The instructions for performing the method of the kit comprises for example information of particular annealing temperatures to be used for the at least one methylation- independent primers, as well as for example information on PCR cycling parameters. The kit may further comprise instructions for the interpretation of the results obtained by the method. For example how to interpret the amplified products subsequently analysed by melting curve analysis or methods as described elsewhere herein.

Information of the interpretation of melting curve analysis is described elsewhere herein.

The kit may in preferred embodiments further comprise software comprising an algorithm for calculation of primer annealing temperature and interpretation of results. Preferred embodiments for the CpG-containing nucleic acid for which the methylation It is appreciated that the kit may be used for evaluating a colorectal cancer in a human subject based on methylation status of specific genes as specified elsewhere herein.

Examples Example 1

The present example shows the performance of a broad selection of hypermethylated DNA promoter regions as blood based biomarkers for detection of colorectal cancer (CRC). A case-control study was conducted using plasma samples from 193 CRC patients and 102 colonoscopy-verified healthy controls. Using methylation specific polymerase chain reaction, we evaluated 30 DNA promoter regions previously analysed in stool or blood samples of CRC patients. The data was analysed using a multivariable logistic regression model with stepwise backwards selection. Using leave-pair-out cross validation, we calculated the optimism corrected area under the receiver operating characteristics curve (AUROC) for all stage as well as early stage CRC.

None of the analysed DNA promoter regions alone reached an overall sensitivity above 30% at a reasonable specificity. However, the combined information from a

combination of specific hypermethylated regions was found to highly relevant. For example, seven hypermethylated promoter regions (ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9, and VIM) were particularly informative, and even more when combined with the covariates sex (female) and age (>66 years), which reached an optimism corrected AUROC of 0.86 for all stage CRC and 0.85 for early stage CRC. Overall sensitivity for CRC detection was 90.7% at 72.5% specificity using a cut point value of 0.5. The clinical sensitivity of the model was 88.7% at 73.5% specificity for stage I and II CRC. Thus, the present example shows that individual hypermethylated DNA promoter regions appear to have limited value as CRC screening markers. However, panels of hypermethylated promoter regions show great promise as a model for CRC detection.

Results

Participants

Review of the CRC patients led to the exclusion of eleven patients: seven with benign disease or non-colorectal cancer, three did not have any residual cancer after endoscopic resection, one patient initially refused surgery, and one patient did not provide informed consent. Five additional patients were excluded, because the reference gene could not be amplified during PCR analysis. Review of the control patients lead to the exclusion of five patients with inflammatory bowel disease and 27 patients diagnosed with any kind of neoplastic disease in the years following inclusion. This left blood samples from 193 CRC patients and 102 controls without CRC. The remaining control group included 33 patients with resectable adenomas, none of which were of high-grade dysplasia. Patient characteristics are provided in Table 1. The distribution of CRC patients according to the TNM classification is provided in Table 2. Model development

The hypermethylation status of all the promoter regions is provided in Table 3. The median number of hypermethylated promoters were 4 (range[1 ,1 1 ]) in the control group and 5 (range[0,28]) in the CRC group (Figure 1 ). The difference was not significant (p=0.212).

The initial univariate screening left 19 of the potential predictor variables along with sex and age>66 for further analysis. The logistic regression with stepwise backwards selection is visualised in Figure 2. Model 12 was considered the most applicable, because it contained a limited number of genes and the model did not differ from the model produced by Penalised regression using Firth's method.

None of the prediction models showed a significant lack of model fit at the 0.05 significance level.

Model performance

Model 12 which included seven hypermethylated gene promoter regions (ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9, and VIM) and the covariates: sex(female) and age>66 had the ability to distinguish CRC patients from patients without CRC with an optimism- corrected AUROC of 0.860 (optimism = 0.027) (Figure 3). There was a significant interaction between the prediction markers RARB and VIM (p<0.001 ). However, inclusion of the interaction in the model only provided modest information (optimism corrected AUROC = 0.869). The interaction was therefore omitted from the final model. In order to characterise Model 12's performance for early stage cancers, we evaluated the model by removing the patients with stage three and four CRC, and controls with adenomas. This rendered an optimism-corrected AUROC of 0.853 (optimism = 0.025) (Figure 4).

The sensitivity, specificity, positive and negative predictive values of Model 12 for CRC detection in all stages and early stages only, were calculated from the ROC analysis using a 0.5 cut point value. This cut point value led to the most accurate classification of the cancer patients and the controls. The values are provided in Table 4.

We also assessed the putative effect of PCR cycle numbers on the results of the assay, between CRC patients and controls. We classified the promoter regions into groups according to their cycle threshold values (0, 0-25, 25-30, and >30). The distribution is shown in Supplementary table 3. It shows that there is a difference in cycle value between CRC patients and controls. However, because of limited power, the effect of this difference could not be evaluated in the multivariable logistic regression model. Therefore, hypermethylation status was treated as a dichotomised variable.

Discussion

Through this study, we analysed a panel of 30 genes previously found to be hypermethylated in patients with CRC and other neoplasia. Through multivariable logistic regression, we selected seven gene promoter regions (ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9, and VIM), which could distinguish early stage CRC patients from a colonoscopy verified healthy control population.

Circulating DNA as biomarkers for CRC and cancer in general is an area of great interest. DNA hypermethylation biomarkers are already implemented in commercially available kits for CRC detection. Cologuard^® (Exact Sciences Corporation, Madison, Wl, USA) is currently the first and only FDA approved stool DNA CRC screening test, utilising hypermethylated BMP3 and NDRG4 along with occult blood and KRAS mutation to detect early stage CRC. Epi proColon^® (Epigenomics AG Corporation, Berlin, Germany) is the only blood based DNA hypermethylation screening test for CRC. The test is based on SEPT9 hypermethylation, and even though there are promising results, there are still some drawbacks. In a large-scale cross-sectional study of 1 .516 patients scheduled for colonoscopy, the assay alone only reached a sensitivity of 24.4% at 95.1 % specificity, showing the apparent lack of sensitivity as an individual CRC biomarker. However, SEPT9 was incorporated in our prediction model for CRC detection, and proved promising as part of a diagnostic biomarker panel. Knockout of SEPT9 in mice is lethal, and the septin gene is involved in many cellular processes including cellular motility and cytoskeletal integrity. We examined the SEPT9 v2 promoter, which is methylated in -90% colorectal malignant and premalignant lesions.²¹ Our study shows, that hypermethylation of SEPT9 measured in plasma is more frequent in CRC patients, but a sufficient sensitivity is only achieved when SEPT9 is included as part of a larger panel of hypermethylation biomarkers.

ALX4 hypermethylation was the superior individual marker for the differentiation of CRC patients from the control population. However, with a sensitivity of only 28.5% at 99.0% specificity, the need for at multigene hypermethylation panel in order to raise sensitivity is apparent. ALX4 has been proposed as a blood-based biomarker for CRC in several previous studies, with sensitivities ranging from 46.6% to 83.3%. It was therefore not surprising that it reaches our diagnostic prediction model. However, remarkably NEUROG1 did not. One study showed, that hypermethylation of

NEUROG1 was more sensitive for the detection of early stage CRC, when compared to ALX4 hypermethylation. Our study could not reproduce this result, as the NEUROG1 promoter was hypermethylated in both CRC and control patients at a frequency of approximately 20%.

Interestingly, RARB hypermethylation was a protective factor, more frequently hypermethylated in healthy controls (69.6%), compared to patients with CRC (25.0%). This is in contrast with the fact that RARB has been found to be preferentially hypermethylated in early stage rectal tumours along with high-grade cervical lesions and high-risk prostate cancer. However, there is some controversy as to the direct effect of RARB inactivation and the impact on carcinogenesis. In mammary tumours, it is proposed that inactivation (e.g. as a result of DNA hypermethylation) could lead to an inhibition of the WNT1 pathway, and therefore be a potential therapeutic target in the treatment of breast cancer. Since T3 tumours represent the bulk of our CRC cohort, along with the fact that RARB methylation is lost through the progression of rectal tumours, our results could indicate that RARB hypomethylation is a biomarker for late stage CRC.

We also analysed the hypermethylation frequencies of BMP3 and NDRG4 - the two genes implemented in the Cologuard^® (Exact Sciences Corporation, Madison, Wl, USA) screening assay. BMP3 reached our gene panel for CRC detection, and while NDRG4 was only hypermethylated in CRC patients, it was only positive in 9.3% of cases and thus, not implemented in the diagnostic model. Whether this difference is due to the media of choice (blood vs. stool) or other factors remains to be elucidated. However, the use of stool could provide increased DNA for analysis as colonic epithelial cells undergo anoikis. It should, however, be remembered, that a substantial degree of the DNA released in this manner undergoes degradation, which could lead to a decreased sensitivity for right-sided tumours. Degradation after sample collection has been proposed as another problem for the analysis of stool DNA hypermethylation, but in vitro studies have shown, that using the correct stool homogenization buffer makes degradation due to prolonged pre-analysis storage before analysis a non-significant issue. Moreover, the limited sensitivity of NDRG4 in plasma could be due to some of the factors listed in the section below. Limitations

There are some limitations to our analytical setup.

The gene promoter regions were initially selected based on a systematic literature search. We used this approach to evaluate proven CRC hypermethylation biomarkers, previously analysed in tumour remote media like blood and stool samples. However, these studies were carried out over the better part of fifteen years. Sequencing methods have since improved, providing new knowledge of complex genetic and epigenetic disruptions in colorectal carcinogenesis, and the field of epigenetic research is growing rapidly. Another approach for marker selection could have been from "The cancer genome atlas" where the molecular characterization of different cancer types is expanding. However, these data are developed through CRC tissue studies, and have never been evaluated in blood samples. Another approach for the initial marker selection could be through an epigenome-wide association study on the resected tumours. However, we did not have such analytical capability in our study setting. Whether other DNA hypermethylation biomarkers are more efficient as cell-free DNA based biomarkers for CRC remains to be elucidated.

Even though our panel of promoter regions resemble those analysed by others, there seems to be a rather large gap in both marker selection and performance. Lee et al found, that APC, MGMT, RASSF2A, and WIF1 had a sensitivity of 86.5% with a specificity of 92.1 %. We evaluated three of these gene promoter regions, through our multivariate logistic regression. None reached the final model. The marker SDC2 was found through an epigenome wide association study and evaluated in serum samples, revealing a sensitivity of 92.3% for stage I CRC at a specificity of 95.2%. Our results on SDC2 are more modest with a sensitivity and specificity of 24.0% and 94.1 % respectively. The differences in marker performance may be due to differences in ethnicity and choice of control population. The control population in the study by Lee et al. was only specified as Asian patients undergoing a regular health check, whereas our population is from an age-matched Caucasian patient cohort referred for colonoscopy with CRC symptoms. Moreover, methylation specific PCR only enables the analysis of one site in each of the promoter regions, meaning that any discordance in the choice of primers and probes could lead to different results. Furthermore, we used a rather small amount of plasma (<1 ml) for our analysis whereas the new kit for SEPT9 analysis (Epi proColon^® 2.0 [Epigenomics AG Corporation, Berlin, Germany]) isolates DNA from -5 ml of plasma, which renders more DNA for analysis and possible replicate analysis. This problem is stressed by the fact, that the number of cell-free tumour DNA fragments per 5 ml could be less than 10 for stage I CRC, making the amount of plasma used for methylation detection critical. Therefore, comparison of results across studies should be done with some caution.

This study was exploratory in order to construct a biomarker panel of hypermethylated DNA promoter regions for CRC detection. The selection of the prediction markers was made on the entire cohort, in order to construct the most robust model. To evaluate the inherit problem of overfitting, we conducted internal validation using "leave pair out cross validation". However, before the model can be used in a clinical setting, external validation in an independent cohort must be conducted.

Strengths

The major strength of our study is the use of a cohort of CRC patients included prospectively and consecutively and a well-defined age-matched control group. Other studies have employed less than optimal control groups, often not described in more detail than "healthy controls". We have used patients who, albeit having symptoms, are an excellent representation of the screening cohort. The results acquired through our study, could very well be a reliable estimate of the methylation frequencies among CRC patients and healthy controls.

Another major strength of our study is the method used for DNA extraction and bisulphite conversion for methylation analysis. Previously, the amount of DNA degradation through the bisulphite treatment was between 84 and 96%.³⁸ Through an optimised conversion step, we were able to treat the DNA with bisulphite, with a recovery as high as 60%, enabling us to detect hypermethylated DNA fragments in the limited amounts of plasma available in our study.

The development of the prediction model is in full accordance with current guidelines for the development of biomarkers for outcome detection.

Conclusion

This example shows that it is indeed possible to differentiate between CRC patients and patients referred for colonoscopy suspected for CRC, when only sparse amounts of plasma are available. A panel of seven hypermethylated gene promoter regions {ALX4, BMP3, NPTX2, RARB2, SDC2, SEPT9, and VIM) with the covariates:

sex(female) and age>66 was specifically efficient for distinguishing CRC patients from patients suspected for, but without CRC. Importantly, the model also had the ability of detecting early stage CRC with a similar performance. The example shows that single hypermethylated DNA biomarker are less efficient for use in a diagnostic setting for CRC, whereas a panel of markers may be of greater clinical interest. Methods

Study population for the development of the prediction model

This is a cross-sectional study analysing the utility of DNA hypermethylation in the plasma of patients suspect for CRC. Between 2003 and 2005, CRC patients were prospectively and consecutively included to evaluate the correlation between coagulation status and CRC at The Department of Gastrointestinal Surgery, Aalborg University Hospital. The inclusion of the patients is described elsewhere. Blood samples were available from 210 patients diagnosed with, or suspect for CRC. We classified patient tumours according to the tumour, node, and metastasis (TNM) system, and staging was in accordance with the American Joint Committee on Cancer staging system (AJCC) 7^th Edition. During the same study period, blood samples from 134 patients referred for colonoscopy with symptoms of, but without CRC were also collected. These patients were of a similar age as the CRC patients, and constituted the control group.

Written informed consent was obtained from all patients, and the study was approved by The North Denmark Region Committee on Health Research Ethics (N-20040067). Outcome and predictor variables

We aimed to establish a multivariable prediction model for CRC detection using a panel of 30 promoter regions, previously evaluated in stool or blood, as biomarkers for CRC with varying sensitivities. We defined the outcome variable as patients with/without CRC.

Along with the covariates sex and age, these 30 hypermethylated promoter regions were the potential predictor variables. A list of the gene names, and their know function is provided in Supplementary table 1.

Blood sampling

Blood samples from the CRC patients were obtained at the time of diagnosis and before any kind of treatment. Blood samples from the control group were all obtained prior to colonoscopy. Blood sampling was conducted in accordance with The European Concerted Action on Thrombosis (ECAT) procedures. All blood samples were centrifuged at 4 °C for 20 minutes at 4000 rpm and the plasma samples were immediately stored at -80 °C for future use.

Hypermethylation analysis

The method for DNA extraction and methylation analysis is based on a rapid bisulfite- treatment protocol described by Pedersen et al, 2012.

According to the manufacturer's instructions, we extracted DNA from 400-1000 μΙ of EDTA-plasma using the easyMAG™ platform (NucliSens^® [bioMerieux SA, France]). The plasma samples were eluted in 35 μΙ elution buffer (NucliSens^® [bioMerieux SA, France]). Subsequently, we deaminated 30 μΙ eluate in 60 μΙ deamination solution for 10 min at 90 °C, followed by a purification step using the easyMAG™ platform

(NucliSens^® [bioMerieux SA, France]). The DNA isolates were eluted in 25 μΙ 10 mM KOH and then subjected to a nested polymerase chain reaction (PCR) using outer primers for the initial amplification, and inner primers and probes for the second round analysis. All primer and probe sequences were evaluated to be hypermethylation specific (MethPrimer^® [The Li Lab, Peking, China]) and designed in accordance

(Beacon Designer^® [PREMIER Biosoft International, Palo Alto, CA]).¹⁶ All primer and probe sequences along with amplicon sizes are available in Supplementary table 2. Statistical analysis

We handled the outcome variable (CRC/no CRC) and the potential predictor variables: hypermethylated DNA promoter regions (hypermethylated/not hypermethylated), sex (male/female), and age (age>66/age<66) as dichotomous variables. This rendered the data suitable for logistic regression modelling. We used all the patients in the model development process and the subsequent cross-validation.

First, we calculated the median and range of the number of hypermethylated promoter regions according to the population groups. We compared the two patient groups using the non-parametric Wilcoxon-Mann-Whitney test. Secondly, using logistical regression, we performed a univariate screening of potential predictors according to the outcome variable at a significance level of p=0.3. Thirdly, we constructed a multivariable logistic regression model, using stepwise backwards selection. Receiver operating

characteristic (ROC) curves are presented and the area under the ROC curve

(AUROC) calculated. Interactions were evaluated between all the potential predictor variables included in the final model. All models were evaluated using Hosmer- Lemeshow's goodness of fit test. Fourthly, we computed a penalised regression model using Firth's method with backwards selection, to assess the impact of separation issues on the model building process. Lastly, we performed internal validation using "Leave pair out cross validation" computing the optimism corrected AUROC in order to evaluate the predictive performance of the final prediction model.

We used STATA^® V.13.1 (StataCorp LP, TX, USA) for all statistical analyses.

Note: All values were calculated using the receiver operating characteristics (R C) curve analysis at a 0.5 cut point value. The positive predictive values (PPV) and negative predictive values (NPV) are calculated from the prevalence of colorectal cancer (CRC) in the Danish population above 60 years of age (-25.000 cases in -1.4 million people)

-) TAAAC AC G C ACTAAC C AAC G 52 CCAAACCCCATCTCATCG 1 12

TATTTTTTAGGTTTCGTTTCGG

+) TATTTTTTAGGTTTCGTTTCGGC 53 1 13

TFPI2 (86) C (72)

-) CGACTTTCTACTCCAAACG 54 AAAC G AC C C G AATAC C C G 1 14 +) GAGGTTTTCGCGTTAGAGAC 55 ATATTTATCGCGTTTTCGTTC 1 15

VIM (143) AC G AAC CTAATAAAC AT AACTA (102)

-) AC G AAC CTAATAAAC ATAACTAC G 56 1 16

CG

+) CGACGCGTTTAGTC 57 GTTG AG G GAGTTGTAG C 1 17

WIF1 (1 1 1 ) (76)

-) TACCGAAAAAACTCCTCG 58 TACCGAAAAAACTCCTCG 1 18

+) CGTGGAATAGTTGTTTGC 59 CGTGGAATAGTTGTTTGC 1 19 WNT5A (153) (135)

-) CGAACCTAAACTCCCG 60 TTAAAACAAAACTAAAATACG 120

Note. The primer sequences for the methylation specific polymerase chain reaction with the individual amplicon sizes represented as number of base pairs in brackets to the right of the outer/inner primers respectively. (+) Forward primer, (-) Reverse primer.

Probes

Table 8

Characteristics of gene specific probes

Gene Loci Probe sequence SEQ ID NO

ALX4 CGCGATTGTCGGTCGTCGTTAAAGTATCGCG 121

APC CGCGATCGTTGGATGCGGAATCGCG 122

BMP3 CGTCGAGCGGGTGAGGTTCGCGTATCGACG 123

BNC1 CGCGATCGTATTTACGGGAGTCGGAGTTTGATCGCG 124

BRCA1 CGATCGGCGGCGTGAGCGTACG 125

HIC1 CGCGACGGTCGTCGTTCGGGTTCGCG 126

HLTF CGCGATCGATTGGATTCGCGGCGAGATCGCG 127

MGMT CGCGATCGTATCGTTTGCGATTTATCGCG 128

MLH1 CGCGATCTCGTCCAACCGCCGAATATCGCG 129

NDRG4 CGCGATCGCGGTTCGTTCGGGATTAGTTGATCDCG 130

NPTX2 CGCGATCGGTGCGGTTGTGAGACGGTGATCGCG 131

NEUROG1 CGCGATGCCCGACCGATCTCCTAAATCGCG 132

OSMR CGCGATCTTCGGACGGCGTTCGGATCGCG 133

Items

The below items represent preferred embodiments of the present invention.

1 . A method of determining colorectal cancer, the prognosis of a colorectal cancer, and/or monitoring a colorectal cancer in a subject, said method comprising in a sample from said subject determining the methylation status of at least one gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

2. A method for assessing whether a human subject is likely to develop colorectal cancer, said method comprising

i. providing a sample from said human subject,

ii. determining in said sample the methylation status of at least one gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

iii. on the basis of said methylation status identifying a human subject that is more likely to develop colorectal cancer.

3. The method according to any of the preceding items, wherein said method comprises determining the presence of metastatic colorectal cancer.

4. The method according to any of the preceding items, wherein said methylation status is determined for the genes in the group consisting of ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM.

5. The method according to any of the preceding items, wherein information of sex and age is included.

6. The method according to any of the preceding items, wherein said sample is a body fluid sample, such as a blood sample.

7. The method according to any of the preceding items, wherein said methylation status is determined by a method comprising the steps of

i) providing a sample, such as a blood sample, from said subject comprising nucleic acid material comprising said gene locus,

ii) modifying said nucleic acid using an agent which modifies unmethylated cytosine, iii) amplifying at least one portion of said gene locus using primers, which span or comprise at least one CpG dinucleotide in said gene locus in order to obtain an amplification product, and

iv) analysing said amplification product.

8. The method according to item 7, wherein said amplification product is analysed for nucleic acid substitutions resulting from conversion of modified cytosine residues, wherein the presence of converted cytosine residues are indicative of unmethylated cytosine residues, and presence of unconverted cytosine residues is indicative of methylated cytosine residues.

9. The method according to any of the preceding items, wherein said methylation status is determined by amplifying at least one portion of said gene locus using at least one primer pair selected from the group consisting of SEQ ID NOs: 1 - 120.

10. The method according to any of the preceding items, wherein said oligonucleotide probe hybridizes to a sequence selected from the group consisting of SEQ ID NO: 151 -180 and/or the complement thereof, wherein said sequence may comprise one or more C to T substitutions.

1 1 . The method according to any of the preceding items, wherein said oligonucleotide probe comprises 10-100 consecutive nucleic acids selected from the group of sequences consisting SEQ ID NO: 151 -180 and/or the complement thereof, wherein said sequence may comprise one or more C to T substitutions.

12. A kit for determining colorectal cancer, or categorizing or predicting the clinical outcome of a colorectal cancer, or monitoring the treatment of a colorectal cancer, said kit comprising

i. an agent that (a) modifies methylated cytosine residues but not non- methylated cytosine residues; or (b) modifies non-methylated cytosine residues but not methylated cytosine residues; or (c) modifies a nucleic acid sequence in a methylation- dependent manner,

ii. and at least one pair of oligonucleotide primers that specifically hybridizes under amplification conditions to a region of a gene locus selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK.

13. The kit according to item 12, wherein said kit comprise at least one oligonucleotide probe comprising 10-100 consecutive nucleic acids selected from the group of sequences consisting SEQ ID NO: 151 -180 and/or the complement thereof, wherein said sequence may comprise one or more C to T substitutions.

14. The kit according to any of items 12 to 13, wherein said kit comprise at least one oligonucleotide probe which hybridizes to a sequence selected from the group consisting of SEQ ID NO: 151 -180 and/or the complement thereof, wherein said sequence may comprise one or more C to T substitutions..

15. Use of oligonucleotide primers comprising a sequence, which is a subsequence of a gene loci selected from the group consisting of RARB, ALX4, NPTX2, SEPT9, BMP3, SDC2, VIM, SFRP1 , THBD, HIC1 , WIF1 , HLTF, APC, PHACTR3, TFPI2, MGMT, CDKN2A and PPENK or the complement thereof for diagnosing colorectal cancer in a method of any of the preceding items.

Claims

Claims

1 . A method of determining colorectal cancer, the prognosis of a colorectal cancer, and/or monitoring a colorectal cancer in a subject, said method comprising in a sample from said subject determining the methylation status for the genes in the group consisting of ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM.

2. A method for assessing whether a human subject is likely to develop colorectal cancer, said method comprising

i. providing a sample from said human subject,

ii. determining in said sample the methylation status for the genes in the group consisting of ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM, and

iii. on the basis of said methylation status identifying a human subject that is more likely to develop colorectal cancer.

3. The method according to any of the preceding claims, wherein said method comprises determining the presence of metastatic colorectal cancer.

4. The method according to any of the preceding claims, wherein information of sex and age is included.

5. The method according to any of the preceding claims, wherein said sample is a body fluid sample, such as a blood sample.

6. The method according to any of the preceding claims, wherein said methylation status is determined by a method comprising the steps of

i) providing a sample, such as a blood sample, from said subject

comprising nucleic acid material comprising said gene locus, ii) modifying said nucleic acid using an agent which modifies unmethylated cytosine,

iii) amplifying at least one portion of said gene locus using primers, which span or comprise at least one CpG dinucleotide in said gene locus in order to obtain an amplification product, and iv) analysing said amplification product.

7. The method according to claim 6, wherein said amplification product is

analysed for nucleic acid substitutions resulting from conversion of modified cytosine residues, wherein the presence of converted cytosine residues are indicative of unmethylated cytosine residues, and presence of unconverted cytosine residues is indicative of methylated cytosine residues.

8 .. The method according to any of the preceding claims, wherein said methylation status is determined by amplifying at least one portion of said gene locus using at least one primer pair selected from the group consisting of SEQ ID NOs: 1 , 2, 5, 6, 21 , 22, 33, 34, 37, 38, 39, 40, 55, 56, 61 , 62, 65, 66, 81 , 82, 93, 94, 97, 98, 99, 100, 1 15 and 1 16.

9 .he method according to a e preceding claims, wherein said

oligonucleotide probe hybr o a sequence selected from the group consisting of SEQ ID NO: 121 , 123, 131 , 137, 139, 140 and 148 and/or the complement thereof, wherein said sequence may comprise one or more C to T substitutions.

10. The method according to any of the preceding claims, wherein said

oligonucleotide probe comprises 10-100 consecutive nucleic acids selected from the group of sequences consisting SEQ ID NO: 121 , 123, 131 , 137, 139, 140 and 148 and/or the complement thereof, wherein said sequence may comprise one or more C to T substitutions.1

1 1. A kit for determining colorectal cancer, or categorizing or predicting the clinical outcome of a colorectal cancer, or monitoring the treatment of a colorectal cancer, said kit comprising

i. an agent that (a) modifies methylated cytosine residues but not non- methylated cytosine residues; or (b) modifies non-methylated cytosine residues but not methylated cytosine residues; or (c) modifies a nucleic acid sequence in a methylation-dependent manner,

ii. and oligonucleotide primers that specifically hybridizes under amplification conditions to a region of the genes in the group consisting of ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM.

12 . The kit according to claim 1 1 , wherein said kit comprise at least one

oligonucleotide probe comprising 10-100 consecutive nucleic acids selected from the group of sequences consisting SEQ ID NO: 121 , 123, 131 , 137, 139, 140 and 148 and/or the complement thereof, wherein said sequence may comprise one or more C to T substitutions.

13. The kit according to any of claims 1 1 to 12, wherein said kit comprise at least one oligonucleotide probe which hybridizes to a sequence selected from the group consisting of SEQ ID NO: 121 , 123, 131 , 137, 139, 140 and 148 and/or the complement thereof, wherein said sequence may comprise one or more C to T substitutions.

14. Use of oligonucleotide primers comprising a sequence, which is a subsequence of a gene loci selected from the group consisting of ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9 and VIM or the complement thereof for diagnosing colorectal cancer in a method of any of the preceding claims 1 to 9.