WO2019122341A1

WO2019122341A1 - Method for the detection and treatment of colorectal adenomas

Info

Publication number: WO2019122341A1
Application number: PCT/EP2018/086614
Authority: WO
Inventors: Jacob MICALLEF; Marielle HERZOG; Mark ECCLESTON; Jason TERRELL
Original assignee: Belgian Volition Sprl
Priority date: 2017-12-21
Filing date: 2018-12-21
Publication date: 2019-06-27
Also published as: TW201930881A; GB201721569D0

Abstract

The present invention relates to the detection of particular biomarkers in a blood, serum or plasma sample for diagnosing the presence of colorectal polyps or adenomas.

Description

METHOD FOR THE DETECTION AND TREATMENT OF COLORECTAL ADENOMAS

FIELD OF THE INVENTION

The invention relates to a method for the detection and treatment of colorectal polyps or adenomas by means of a blood test.

BACKGROUND OF THE INVENTION

Colorectal Cancer (CRC) is a common disease with a high mortality. The biology of the disease is understood to involve a progression from pre-cancerous adenoma (polyp) with increasing dysplasia from a Low-Risk adenoma, to a Medium-Risk adenoma, to a High-Risk adenoma which may then lead to stage I, II, III and eventually stage IV CRC. Mortality varies greatly depending on whether the disease is detected at a precancerous adenoma stage, an early localized stage of CRC (e.g. CRC stage I) or at a late stage of CRC (e.g. CRC stage IV) when the disease may have spread within the colon or rectum or beyond when treatment is more difficult.

Detection and removal of adenomas is thought to prevent the development of cancer and the prognosis of these patients is excellent. If the disease has progressed to CRC, the 5- year survival rate is >90% for those in whom CRC disease is detected at stage I, but only about 10% for those in whom stage IV CRC metastatic disease is detected. For this reason, many countries have CRC screening programmes to identify individuals with CRC or precancerous adenomas or polyps. CRC incidence is age and sex dependent; being more common in men than women and more common in elderly people. CRC is rare in people below the age of 50 so it is more useful to screen older people. The actual screening age range tested varies in screening programmes in different countries but is typically of the order of 50-74 years.

The prevalence of CRC in the screening population is around 0.6%. However, the prevalence of adenomas is much higher. Around 36% of the screening age population have one or more colorectal adenomas. Of these around 8% are High-Risk or Advanced adenomas and 28% are Low or Medium-Risk or non-advanced adenomas (Imperiale et al. (2014)). Whilst, all or most cases of CRC are thought to develop from High-Risk adenomas, it is clear that not all subjects with a High-Risk adenoma will actually develop CRC.

It is important to remove or treat colorectal adenomas, particularly High-Risk adenomas, because a proportion of patients with High-Risk adenomas will develop CRC if untreated. Currently adenomas are usually detected by colonoscopy. When detected, Medium and High-Risk adenomas are routinely removed surgically on colonoscopy. Effective drug treatments for adenomas are also available including Non-Steroidal-Anti-Inflammatory Drugs (NSAIDs). For example, the enzyme cyclooxygenase 2 (COX-2) is overexpressed in colorectal polyps and COX-2 inhibitor drugs, such as aspirin, Celebrex and Vioxx, are effective in reducing the adenoma burden in people suffering from familial adenomatous polyposis. COX-2 inhibitors reduce both the number and size of polyps. COX-2 inhibitors also reduce the recurrence of adenomas, especially High-Risk adenomas, after surgical removal (Arber (2008)). A problem associated with these drugs is identification of patients with adenomas for treatment, particularly High-Risk adenomas. Some 8% of people of screening age have one or more High-Risk adenomas and are potential candidates for drug treatment. However, High-Risk adenomas can currently only be diagnosed by colonoscopy and, if identified this way, they are removed surgically rendering drug treatment

unnecessary. Thus, a blood test which could identify people with High-Risk adenomas would have great potential as a companion product to a polyp treatment drug such as a COX-2 inhibitor.

CRC screening programmes enable CRC detection earlier than would otherwise be the case, leading to earlier treatment and many years of saved life. Earlier CRC detection also saves money and resources for healthcare providers by reducing the need for expensive late stage cancer drug therapies and hospitalizations. Ideally CRC screening programmes would also detect precancerous colorectal polyps or adenomas which may progress to CRC if left untreated, but can be removed before cancer develops if detected. The prognosis for such patients is good and removal of precancerous polyps on a population basis may lead to a decrease in CRC incidence and prevalence.

The methods commonly used for CRC detection and/or screening all suffer from major drawbacks. The primary CRC screening method employed in the USA is colonoscopy. Colonoscopy essentially involves visually examining the colon using a scope which traverses the descending, transverse and ascending colon to find any cancerous or potentially pre- cancerous lesions. The primary advantage of colonoscopy is its accuracy of detection which is of the order of 95% clinical sensitivity for CRC as well as very high detection rates for precancerous adenomas all with very high clinical specificity. This accuracy makes colonoscopy the gold standard for CRC detection, especially as low-grade lesions and adenomas can be removed at the time of their detection. However, colonoscopy suffers from a number of limitations as a frontline CRC detection or screening method. Colonoscopy is a highly invasive procedure requiring a surgical admission. The procedure is usually performed under anaesthesia, requires preparation of the bowel by the patient in advance, causes injury to the patient in some cases (for example, tearing of the bowel) and is expensive (>$1000). CRC is detected in approximately 0.5% of screening colonoscopies and High-Risk adenomas in a further 8%, so the majority of people screened are subjected to a surgical procedure for little benefit. Due to these disadvantages, patient compliance with colonoscopy is low and many people of screening age do not undergo the procedure. For these reasons, colonoscopy is not used as a frontline CRC detection or screening method in most countries of the world.

Some healthcare providers employ a related method called sigmoidoscopy in which a shorter scope is used to examine the descending colon only. Although this method misses two thirds of the colon, it does examine the area where cancers are most commonly observed. The disadvantages of sigmoidoscopy are similar to those of colonoscopy and it is not commonly used as a frontline test for similar reasons. Virtual colonoscopy, or computerized tomography (CT) colonography, is also used. This procedure employs a combination of x-rays and computer technology to create images of the rectum and colon to detect colorectal tumours and polyps.

The most commonly used CRC detection and screening methods involve a two-stage procedure in which the population of screening age is first screened with a non-invasive frontline fecal test to identify a subgroup of the screening population in whom there is a higher risk of CRC. People who test positive in the fecal test are referred for a follow-up colonoscopy. About 5% of these people will typically be found to have CRC. The result of fecal screening is negative for the majority of people, so the two-stage method prevents unnecessary colonoscopies on most people with no lesion.

The principle underlying fecal tests for CRC is the detection of bleeding into the colon or rectum. In simple terms, when the colon or rectum is partially blocked by an intruding cancerous or precancerous adenoma growth, movement of the stool past the blockage is likely to cause injury and bleeding. This bleeding is detected by testing the fecal sample for the presence of hemoglobin. As the degree of bleeding may vary greatly from day to day, the test may need to be performed several times on separate days.

All current fecal CRC tests are designed to detect fecal hemoglobin. The guaiac fecal occult blood test (FOBT or gFOBT) is a chemical test method for hemoglobin in which the patient or operator typically smears a small amount of feces on to an alpha-guaiaconic acid coated paper or other substrate. If blood is present in the feces, addition of hydrogen peroxide to the paper produces a rapid colour change through the oxidation of alpha-guaiaconic acid to a blue coloured quinone in a reaction catalyzed by heme (a component of hemoglobin). The consumption of meat (and hence heme) as well as some vegetables, that contain other catalyst molecules that behave like heme in the test, can cause false positive results.

Similarly, some substances, including vitamin C can lead to false negative results so dietary restriction is often advised prior to the test. Guaiac FOBT tests can have high clinical specificity depending on the cut-off used and have approximately 60% sensitivity for detection of CRC. Detection of precancerous polyps or adenomas is poor. Chemical FOBT methods were the method of choice in the past and, although still widely used, are being displaced by fecal immunochemical test methods.

Fecal immunochemical test (FIT) methods for CRC detection (also called iFOBT or FOBTi) are essentially immunoassay tests for human hemoglobin in fecal samples. FIT methods are less susceptible to false positive and negative results due to interference of dietary factors and can detect smaller amounts of blood in the feces. These tests have similar specificity to gFOBT but detect slightly more clinically relevant cancer lesions. Detection of polyps or adenomas is poor. The sensitivity of FIT methods for CRC is around 74% with a specificity of around 95%. The sensitivity of FIT is around 23% for advanced precancerous colorectal adenomas or polyps and around 8% for non-advanced adenomas. EXACT Sciences have developed a fecal CRC test that analyses fecal DNA in addition to fecal hemoglobin. This test (Cologuard) is reported to have a sensitivity for CRC of 92% at a specificity of 87% and detects 17% of non-advanced and 42% of Advanced colorectal adenomas or polyps (Imperiale et al. (2014)). However, this test is expensive and not widely used.

Whole population CRC screening programmes for people of screening age are established in many countries using FIT tests and have resulted in a reduction in the mortality caused by CRC. A major problem faced by CRC screening programmes is compliance. In the USA compliance is around 70% (/^'.e. around 70% of people of screening age are up to date with their colonoscopy screening). In Europe, the best performing FIT CRC screening

programmes have compliance rates around 60-70% but compliance in many is below 50%. Thus at least 30% of people of screening age are not screened. Another problem faced by CRC screening programmes is that, once the programmes have been running for a few years, their success leads to a fall in the prevalence of CRC in the screening population. Clearly, this is a success metric, but it does mean that identification of people with colorectal polyps to be removed before they develop into cancer becomes a high priority. Thus, tests that can identify people with adenomas, particularly High-Risk adenomas are becoming increasingly necessary.

It is thought that the reasons for non-compliance with FIT and colonoscopy include the inconvenience, requirement for time off work, high cost, discomfort and invasiveness of colonoscopy procedures and the unpleasantness and cultural taboos surrounding fecal collection. To address the medical needs of unscreened people, workers have tried for decades to develop blood tests for CRC which would overcome some of these limitations. However, no such tests have been widely adopted for clinical CRC screening procedures.

Many classical blood biomarkers, including carcinoembryonic antigen (CEA), have been investigated as possible blood based biomarkers for CRC but so far, their clinical accuracy is too low for routine use and, if they have any application at all, they are better used for patient monitoring than for CRC detection or diagnosis. Typically, such markers detect late stage IV CRC, but not early stage I or II CRC and not pre-cancerous colorectal polyps. In some cases, workers have combined multiple blood based biomarkers to increase the combined sensitivity for CRC detection. A recently published example involved the investigation of the levels of 93 plasma protein biomarkers in 226 CRC patients and 118 colonoscopy screened negative controls. From the results, a 5-biomarker panel model including GDF-15, AREG, FasL, Flt3L and P53 autoantibody was selected as optimal and validated in a separate patient cohort including 41 CRC cases, 106 Advanced adenoma cases and 107 colonoscopy screened negative controls. The biomarker panel was found to have a sensitivity of 56% for CRC and 22% for Advanced adenoma at 90% specificity (Chen et al. (2017)).

Recently, the Epi proColon test developed by Epigenomics AG became the first CRC blood test to be approved by the FDA in USA. This test measures hypermethylation of a Septin-9 gene DNA sequence as diagnostic biomarker for CRC in blood. The FDA study reported that the test has a sensitivity for CRC of 68% at 78% specificity. The sensitivity for colorectal polyps or adenomas was very low and barely above the false positive rate of the test.

In summary, around 8% of people of CRC screening age have one or more High-Risk colorectal polyps/adenomas that may develop into CRC and removal of these is an effective CRC prevention treatment. Colonoscopy identifies most or all cases of colorectal polyps, but has low patient compliance and is not widely used as a front-line CRC screening method outside of USA. Most countries perform population CRC screening using the FIT test but these methods also suffer from poor patient compliance and detect only around 23% of High-Risk colorectal polyps. There is therefore a need for blood tests that can identify persons with colorectal polyps/adenomas, but neither classical CRC blood biomarkers, nor the only blood test approved for CRC detection, are able to detect colorectal polyps.

We now report a simple low-cost ELISA blood test that detects >50% of Advanced or High- Risk colorectal adenomas with a specificity of >90%.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided the use of carbonic anhydrase, Tissue Inhibitor of Metalloproteinases-1 (TIMP-1 ) and/or cell-free nucleosomes in a blood, serum or plasma sample as a biomarker for diagnosing the presence of a colorectal adenoma.

According to a further aspect of the invention, there is provided a method for diagnosing or detecting a colorectal adenoma, in an animal or a human subject which comprises the steps of:

(i) detecting or measuring the level of the biomarker(s) as defined herein in a blood, serum or plasma sample obtained from the subject; and

(ii) using the measured level of biomarker(s) as indicative of the presence of said colorectal adenoma in the subject.

According to a further aspect of the invention, there is provided a method for determining the prognosis of an animal or a human subject with a colorectal adenoma, which comprises the steps of:

(ii) using the measured level of biomarker(s) as indicative of the prognosis of said colorectal adenoma.

According to a further aspect of the invention, there is provided a method for monitoring the efficacy of a therapy in an animal or a human subject having, suspected of having, or of being predisposed to colorectal adenoma, which comprises the steps of:

(i) detecting or measuring the level of the biomarker(s) as defined herein in a blood, serum or plasma sample obtained from the subject; and (ii) using the measured level of biomarker(s) compared with an earlier biological sample taken from said subject as indicative of the efficacy of said therapy.

According to a further aspect of the invention, there is provided a method of treating a colorectal adenoma in an animal or a human subject, which comprises the following steps:

(i) detecting or measuring the level of the biomarker(s) as defined herein in a blood, serum or plasma sample obtained from the subject;

(ii) using the measured level of biomarker(s) as indicative of the presence of said colorectal adenoma in the subject; and

(iii) treating surgically or administering a therapeutic agent to a subject diagnosed in step (ii) as a patient having said colorectal adenoma.

According to a further aspect of the invention, there is provided a method of treating a colorectal adenoma in an individual in need thereof, which comprises the step of treating surgically or administering a therapeutic agent to a patient identified as having differing levels of the biomarker(s) as defined herein in a blood, serum or plasma sample obtained from said patient, when compared to the levels of said biomarker(s) in a blood, serum or plasma sample obtained from a control subject.

According to a further aspect of the invention, there is provided a biomarker panel comprising two or more biomarkers selected from the group consisting of: carbonic anhydrase, TIMP-1 and cell-free nucleosomes.

According to a further aspect of the invention, there is provided the use of a kit comprising one or more binding agents capable of detecting and/or quantifying the biomarker(s) as defined herein, for the diagnosis of a colorectal adenoma.

DETAILED DESCRIPTION OF THE INVENTION

The terms“adenoma”,“polyp”,“colorectal adenoma” and“colorectal polyp” are used interchangeably herein. They refer to a growth on the lining of the colon or rectum.

Adenomas/polyps are considered pre-malignant, i.e. they may into develop into colon cancer, and therefore it is recommended that they are removed. The terms“Advanced adenoma” and“High-Risk adenoma” are used interchangeably herein. The definitions of these terms would be known to a person skilled in the art. They vary slightly in different countries and screening programmes, but broadly they refer to a colorectal adenoma measuring ^1 cm in the greatest dimension, including sessile or serrated adenomas, or an adenoma with villous histology or with high grade dysplasia (Imperiale et al. (2014)). The terms“non-advanced adenoma” and“Low and Medium-Risk adenomas” are used interchangeably herein. They refer to an adenoma that is not advanced or High-Risk.

In one embodiment, the colorectal adenoma is selected from the group consisting of: Low- Risk adenoma, Medium-Risk adenoma, and High-Risk adenoma. In a further embodiment, the colorectal adenoma is a High-Risk adenoma.

According to one aspect of the invention, there is provided the use of carbonic anhydrase as a biomarker for diagnosing the presence of a colorectal adenoma.

In one embodiment, the carbonic anhydrase is selected from: carbonic anhydrase 1 (CA-1 ) and carbonic anhydrase 9 (CA-9). In a further embodiment, the carbonic anhydrase is carbonic anhydrase 1 (CA-1 ). In an alternative embodiment, the carbonic anhydrase is carbonic anhydrase 9 (CA-9).

Carbonic anhydrase enzymes are a group of enzymes which control pH balance. They have been shown to be elevated in cancer, both in terms of tissue expression and in terms of circulating level, as they help maintain pH balance in the tumour environment when disturbed by poor angiogenesis and hypoxia. Circulating carbonic anhydrase levels are reported to be elevated in CRC. However, there is no evidence that circulating carbonic anhydrase levels are altered in colorectal adenomas and any association with

adenomas/polyps has to our knowledge, not been investigated. Surprisingly, the present inventors have found that circulating carbonic anhydrase (in particular, CA-9) levels are decreased in patients with High-Risk colorectal adenomas.

According to one aspect of the invention, there is provided the use of Tissue Inhibitor of MetalloProteinases-1 (TIMP-1 ) as a biomarker for diagnosing the presence of a colorectal adenoma. Circulating TIMP-1 levels are reported to be elevated in CRC. However, circulating TIMP-1 levels are reported not to be altered in subjects with polyps and TIMP-1 has been reported not to be a useful biomarker for polyp detection (Nielsen et al. (2004)). Surprisingly, the present inventors have found that circulating TIMP-1 levels are raised in patients with High- Risk colorectal adenomas.

In one embodiment, the use comprises carbonic anhydrase and TIMP-1. In an alternative embodiment, the use comprises carbonic anhydrase and cell-free nucleosomes. In a yet further alternative embodiment, the use comprises TIMP-1 and cell-free nucleosomes.

In one embodiment, carbonic anhydrase, Tissue Inhibitor of Metalloproteinases-1 (TIMP-1 ) and cell-free nucleosomes are used as a biomarker panel for diagnosing the presence of a colorectal adenoma.

When the levels of circulating levels of carbonic anhydrase, TIMP-1 and nucleosome levels are combined using a regression analysis method, a combined accuracy of 37.9% sensitivity for High-Risk colorectal adenomas was achieved at a specificity of 90%.

Therefore, according to a further aspect of the invention there is provided the use of a panel of circulating biomarkers including any combination, or all, of circulating levels of carbonic anhydrase, TIMP-1 , cell-free nucleosomes and cell-free nucleosomes containing an epigenetic signal structure as a biomarker panel for diagnosing the presence of a colorectal adenoma.

In one embodiment, the level of cell-free nucleosomes ( i.e . cell-free nucleosomes per se) are used as the biomarker. In an alternative embodiment, the cell-free nucleosomes contain an epigenetic signal structure which is used as the biomarker.

In one embodiment, the epigenetic signal structure is selected from the group consisting of: a post-translational histone modification, a histone variant, a particular nucleotide and a protein adduct. It will be understood that the terms“epigenetic signal structure” and “epigenetic feature” are used interchangeably herein.

Circulating nucleosome levels are reported to be elevated in CRC as well as other cancer diseases (Holdenrieder ef a/. (2001 )). The present inventors have found that circulating nucleosome levels are increased in patients with High-Risk colorectal adenomas. Circulating nucleosomes are not a homogeneous group of protein-nucleic acid complexes. Rather, they are a heterogeneous group of chromatin fragments originating from the digestion of chromatin on cell death and include an immense variety of epigenetic structures including particular histone isoforms or variants, post-translational histone modifications, nucleotides or modified nucleotides and nucleosome adducts. It will be clear to those skilled in the art that an elevation in nucleosome levels will be associated with elevations in some circulating nucleosome subsets containing particular epigenetic signals including nucleosomes comprising particular histone isoforms or variants, comprising particular post- translational histone modifications, comprising particular nucleotides or modified nucleotides and comprising particular nucleosome adducts. Similarly, decreases will occur in patients with adenoma in the absolute levels and/or proportions of circulating nucleosomes containing other epigenetic signals including nucleosomes comprising particular histone isoforms or variants, comprising particular post-translational histone modifications, comprising particular nucleotides or modified nucleotides and comprising particular nucleosome adducts. Assays for these types of chromatin fragments are known in the art (for example, see W02005/019826, WO2013/030579, WO2013/030578, W02013/084002 which are herein incorporated by reference). These assays have previously been used to show that epigenetically altered circulating cell free nucleosomes can be detected in the blood of diseased patients. Therefore, in one embodiment, the detection or measurement of cell-free nucleosomes comprises:

(i) contacting the sample with a first binding agent which binds to cell-free nucleosomes or a component thereof;

(ii) contacting the sample comprising the first binding agent bound to cell-free nucleosomes or a component thereof, with a second binding agent which binds to an epigenetic feature within said cell-free nucleosomes; and

(iii) detecting or quantifying the binding of said second binding agent to the epigenetic feature within said cell-free nucleosomes in the sample.

In an alternative embodiment, the detection or measurement in step (b) comprises:

(i) contacting the sample with a first binding agent which binds to an epigenetic feature within said cell-free nucleosomes;

(ii) contacting the sample comprising the first binding agent bound to the epigenetic feature, with a second binding agent which binds to cell-free nucleosomes or a component thereof; and

(iii) detecting or quantifying the binding of said second binding agent to cell-free nucleosomes or a component thereof in the sample.

The nucleosome is the basic unit of chromatin structure and consists of a protein complex of eight highly conserved core histones (comprising of a pair of each of the histones H2A, H2B, H3, and H4). Around this complex is wrapped approximately 146 base pairs of DNA. Another histone, H1 or H5, acts as a linker and is involved in chromatin compaction. The DNA is wound around consecutive nucleosomes in a structure often said to resemble“beads on a string” and this forms the basic structure of open or euchromatin. In compacted or heterochromatin this string is coiled and super coiled into a closed and complex structure (Herranz and Esteller (2007)).

It will be appreciated that the term cell-free nucleosome throughout this document is intended to include any cell-free chromatin fragment that includes one or more nucleosomes. Epigenetic signal structures/features of a cell-free nucleosome as referred herein may comprise, without limitation, one or more histone post-translational modifications, histone isoforms, modified nucleotides and/or proteins bound to a nucleosome in a nucleosome- protein adduct.

Normal cell turnover in adult humans involves the creation by cell division of some 10¹¹ cells daily and the death of a similar number, mainly by apoptosis. During the process of apoptosis chromatin is broken down into mononucleosomes and oligonucleosomes which are released from the cells. Under normal conditions the levels of circulating nucleosomes found in healthy subjects is reported to be low. Elevated levels are found in subjects with a variety of conditions including many cancers, auto-immune diseases, inflammatory conditions, stroke and myocardial infarction (Holdenreider & Stieber (2009)).

Mononucleosomes and oligonucleosomes can be detected by Enzyme-Linked

ImmunoSorbant Assay (ELISA) and several methods have been reported (Salgame et al. (1997); Holdenrieder et al. (2001 ); van Nieuwenhuijze et al. (2003)). These assays typically employ an anti-histone antibody (for example anti-H2B, anti-H3 or anti-H 1 , H2A, H2B, H3 and H4) as capture antibody and an anti-DNA or anti-H2A-H2B-DNA complex antibody as detection antibody. In one embodiment, the anti-histone antibody comprises an anti-H3 antibody.

Current nucleosome ELISA methods are used primarily in cell culture, usually as a method to detect apoptosis (Salgame et al. (1997); Holdenrieder et al. (2001 ); van Nieuwenhuijze et a/. (2003)), but are also used for the measurement of circulating cell free nucleosomes in serum and plasma (Holdenrieder et al. (2001 )). Cell free serum and plasma nucleosome levels released into the circulation by dying cells have been measured by ELISA methods in studies of a number of different cancers to evaluate their use as a potential biomarker (Holdenrieder et al. (2001 )). Mean circulating nucleosome levels are reported to be high in most, but not all, cancers studied. However, patients with malignant tumours are reported to have serum nucleosome concentrations that varied considerably and some patients with advanced tumour disease were found to have low circulating nucleosome levels, within the range measured for healthy subjects (Holdenrieder et al. (2001 )). Because of this and the variety of non-cancer causes of raised nucleosome levels, circulating nucleosome levels have not been used clinically as a biomarker of cancer (Holdenrieder and Stieber, (2009)).

In one embodiment, the epigenetic signal structure of the cell-free nucleosome comprises one or more histone post-translational modifications. The structure of nucleosomes can vary by Post Translational Modification (PTM) of histone proteins. PTM of histone proteins typically occurs on the tails of the core histones and common modifications include acetylation, methylation or ubiquitination of lysine residues as well as methylation of arginine residues and phosphorylation of serine residues and many others. Histone modifications are known to be involved in epigenetic regulation of gene expression (Herranz and Esteller (2007)). For example, in one embodiment, the post-translational histone modification is H3K9Me3.

In one embodiment of the invention a group or class of related histone modifications (rather than a single modification) is detected. A typical example of this embodiment, without limitation, would involve a 2-site immunoassay employing one antibody or other selective binder directed to bind to nucleosomes and one antibody or other selective binder directed to bind the group of histone modifications in question. Examples of such antibodies directed to bind to a group of histone modifications would include, for illustrative purposes without limitation, anti-pan-acetylation antibodies (e.g. a Pan-acetyl H4 antibody), anti-citrullination antibodies or anti-ubiquitination antibodies.

In one embodiment, the epigenetic signal structure of the cell-free nucleosome comprises one or more histone variants or isoforms. The structure of the nucleosome can also vary by the inclusion of alternative histone isoforms or variants which are different gene or splice products and have different amino acid sequences. Histone variants can be classed into a number of families which are subdivided into individual types. The nucleotide sequences of a large number of histone variants are known and publicly available for example in the

National Human Genome Research Institute NHGRI Histone Database (Marino-Ramirez, L, Levine, K.M., Morales, M., Zhang, S., Moreland, R.T., Baxevanis, A.D., and Landsman, D. The Histone Database: an integrated resource for histones and histone fold-containing proteins. Database Vol.2011. and http://genome.nhgri.nih.gov/histones/complete.shtml), the GenBank (NIH genetic sequence) Database, the EMBL Nucleotide Sequence Database and the DNA Data Bank of Japan (DDBJ). For example, variants of histone H2 include H2A1 , H2A2, mH2A1 , mH2A2, H2AX and H2AZ.

In one embodiment, the epigenetic signal structure of the cell-free nucleosome comprises one or more DNA modifications. In addition to the epigenetic signalling mediated by nucleosome histone isoform and PTM composition, nucleosomes also differ in their nucleotide and modified nucleotide composition. Global DNA hypomethylation is a hallmark of cancer cells and some nucleosomes may comprise more 5-methylcytosine residues (or 5- hydroxymethylcytosine residues or other nucleotides or modified nucleotides) than other nucleosomes. In one embodiment, the DNA modification is selected from 5-methylcytosine or 5-hydroxymethylcytosine.

In one embodiment, the epigenetic signal structure of the cell-free nucleosome comprises one or more protein-nucleosome adducts or complexes. A further type of circulating nucleosome subset is nucleosome protein adducts. It has been known for many years that chromatin comprises a large number of non-histone proteins bound to its constituent DNA and/or histones. These chromatin associated proteins are of a wide variety of types and have a variety of functions including transcription factors, transcription enhancement factors, transcription repression factors, histone modifying enzymes, DNA damage repair proteins and many more. These chromatin fragments including nucleosomes and other non-histone chromatin proteins or DNA and other non-histone chromatin proteins are described in the art.

In one embodiment, the protein adducted to the nucleosome (and which therefore may be used as a biomarker) is selected from: a transcription factor, a High Mobility Group Protein or chromatin modifying enzyme. References to“transcription factor” refer to proteins that bind to DNA and regulate gene expression by promoting ( i.e . activators) or suppressing ( i.e . repressors) transcription. Transcription factors contain one or more DNA-binding domains (DBDs), which attach to specific sequences of DNA adjacent to the genes that they regulate. All of the circulating nucleosomes and nucleosome moieties, types or subgroups described herein may be useful in the present invention.

In one embodiment, the method and uses of the present invention comprise two or more measurements of cell-free nucleosomes per se and/or cell-free nucleosome epigenetic features are performed as a panel of nucleosome features.

It will be understood that methods and uses of the present invention find particular use in blood, serum or plasma samples obtained from a subject. In one embodiment, the sample is a blood or serum sample. In a further embodiment, the sample is a serum sample.

(ii) using the measured level of biomarker(s) compared with an earlier biological sample taken from said subject as indicative of the efficacy of said therapy. References to“subject” or“patient” are used interchangeable herein. In one embodiment, the subject is a human subject.

In one embodiment, said detection or measurement comprises an immunoassay, immunochemical, mass spectroscopy, chromatographic, chromatin immunoprecipitation or biosensor method.

In one embodiment, the detection or measurement comprises an immunoassay. In a preferred embodiment of the invention there is provided a 2-site immunoassay method. In particular, such a method is preferred for the measurement of nucleosome incorporated epigenetic features in situ employing an immobilized anti-nucleosome binding agent in combination with a labelled anti-histone modification or anti-histone variant or anti-DNA modification or anti-adducted protein detection binding agent. In another embodiment of the invention, there is provided a 2-site immunoassay employing a labelled anti-nucleosome detection binding agent in combination with an immobilized anti-histone modification or anti- histone variant or anti-DNA modification or anti-adducted protein binding agent.

Detecting or measuring the level of the biomarker(s) may be performed using a suitable binding agent. In one embodiment, the one or more binding agents comprises a ligand or binder specific for the desired biomarker, e.g. the cell-free nucleosome or component part thereof, or a structural/shape mimic of the nucleosome or component part thereof.

It will be clear to those skilled in the art that the terms antibody, binder or ligand in regard to any aspect of the invention is not limiting but intended to include any binder capable of binding to particular molecules or entities and that any suitable binder can be used in the method of the invention. It will also be clear that the term“nucleosomes” is intended to include mononucleosomes and oligonucleosomes and any such chromatin fragments that can be analysed in fluid media.

In one embodiment, the ligands or binders of the invention include naturally occurring or chemically synthesised compounds, capable of specific binding to the desired target. A ligand or binder may comprise a peptide, an antibody or a fragment thereof, or a synthetic ligand such as a plastic antibody, or an aptamer or oligonucleotide, capable of specific binding to the desired target. The antibody can be a monoclonal antibody or a fragment thereof. A ligand may be labelled with a detectable marker, such as a luminescent, fluorescent, enzyme or radioactive marker; alternatively or additionally a ligand according to the invention may be labelled with an affinity tag, e.g. a biotin, avidin, streptavidin or His (e.g. hexa-His) tag. Alternatively, ligand binding may be determined using a label-free technology for example that of ForteBio Inc.

The term“detecting” or“diagnosing” as used herein encompasses identification,

confirmation, and/or characterisation of a disease state. Methods of detecting, monitoring and of diagnosis according to the invention are useful to confirm the existence of a disease, to monitor development of the disease by assessing onset and progression, or to assess amelioration or regression of the disease. Methods of detecting, monitoring and of diagnosis are also useful in methods for assessment of clinical screening, prognosis, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development.

The immunoassays of the invention include any method employing one or more antibodies or other specific binders directed to bind to the biomarkers defined herein. Immunoassays include 2-site immunoassays or immunometric assays employing enzyme detection methods (for example ELISA), fluorescence labelled immunometric assays, time-resolved

fluorescence labelled immunometric assays, chemiluminescent immunometric assays, immunoturbidimetric assays, particulate labelled immunometric assays and

immunoradiometric assays as well as single-site immunoassays, reagent limited

immunoassays, competitive immunoassay methods including labelled antigen and labelled antibody single antibody immunoassay methods with a variety of label types including radioactive, enzyme, fluorescent, time-resolved fluorescent and particulate labels. All of said immunoassay methods are well known in the art, see for example Salgame et al. (1997) and van Nieuwenhuijze et al. (2003).

Identifying and/or quantifying can be performed by any method suitable to identify the presence and/or amount of a specific protein in a biological sample from a subject or a purification or extract of a biological sample or a dilution thereof. In methods of the invention, quantifying may be performed by measuring the concentration of the target in the sample or samples. Biological samples that may be tested in a method of the invention include those as defined hereinbefore. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner.

Identification and/or quantification of biomarkers may be performed by detection of the biomarker or of a fragment thereof, e.g. a fragment with C-terminal truncation, or with N- terminal truncation. Fragments are suitably greater than 4 amino acids in length, for example 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. It is noted in particular that peptides of the same or related sequence to that of histone tails are particularly useful fragments of histone proteins.

For example, detecting and/or quantifying can be performed by one or more method(s) selected from the group consisting of: SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spec (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC and other LC or LC MS- based techniques. Appropriate LC MS techniques include ICAT® (Applied Biosystems, CA, USA), or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography (e.g. high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin-layer chromatography, NMR (nuclear magnetic resonance) spectroscopy could also be used.

In one embodiment, the method comprises comparing the amount of the biomarker(s) present in said blood, serum or plasma sample with one or more controls, such as comparing the amount of biomarker(s) present in a blood, serum or plasma sample obtained from the subject with the amount of biomarker(s) present in a blood, serum or plasma sample obtained from a normal subject. It will be understood that a“normal” subject refers to a healthy/non-diseased subject.

The invention described herein has particular use in further screening patients which have undergone fecal occult blood testing (e.g. FIT testing). Therefore, in one embodiment, the subject has been identified as positive for fecal occult blood. In an alternative embodiment, the subject has been identified as negative for fecal occult blood. In one embodiment, the method additionally comprises testing for fecal occult blood in a fecal sample obtained from the subject.

In one embodiment, the fecal occult blood test is selected from: a Fecal Immunochemical Test (FIT, also known as immunochemical fecal occult blood test or iFOBT), stool guaiac test for fecal occult blood (gFOBT) or Fecal porphyrin quantification (such as HemoQuant).

In a further embodiment, the fecal occult blood test is a Fecal Immunochemical Test. FIT products utilize specific antibodies to detect globin and is currently one of the most commonly used colon cancer screening tests. It will be understood that a patient is deemed to have been tested“positive” when fecal occult blood is determined to be present in the fecal sample at a level above the (single) threshold cut-off level used for the test. For example, in one embodiment, the cut-off level for the numerical fecal occult blood result is at least 10 pg Hb/g, such as at least 20, 30, 40 or 50 pg Hb/g, in particular at least about 20pg Hb/g.

In one embodiment, the method additionally comprises using a clinical parameter to diagnose the subject with a colorectal adenoma. This parameter can be used in the interpretation of results. Clinical parameters may include any relevant clinical information for example, without limitation, gender, weight, Body Mass Index (BMI), smoking status and dietary habits. Therefore, in one embodiment, the clinical parameter is selected from the group consisting of: age, sex and body mass index (BMI).

Patient parameters including age and sex are known to be associated with the development of High-Risk adenomas. We have shown that addition of age as a patient parameter increases the accuracy of these tests for High-Risk adenomas.

Age is well known risk-factor for development of both CRC and adenomas. Addition of age as patient parameter to a logistic regression model including age, circulating carbonic anhydrase, TIMP-1 and nucleosome levels gave a combined accuracy of >50% sensitivity for High-Risk colorectal adenomas at a specificity of 90%. These are the highest accuracy figures for any blood test and compare highly favourably, both to the FIT test which has 23% sensitivity for High-Risk adenomas at 95% specificity, and to the more expensive Cologuard fecal haemoglobin and DNA test which has a sensitivity for High-Risk adenomas of 42% (which is the highest sensitivity of any IVD test for High-Risk adenomas), at a specificity of 87% (Imperiale et al. (2014)). Thus, the accuracy of the method of the invention for High- Risk adenoma detection surpasses that of the most accurate in vitro diagnostic test known in both sensitivity and specificity.

In one embodiment, the method additionally comprises examining the subject by

colonoscopy, capsule camera, sigmoidoscopy or an MRI method to identify the number and location of colorectal adenoma(s).

In one embodiment, the method additionally comprises, treating by colonoscopy and/or surgically and/or administering a therapeutic agent to a subject diagnosed with a colorectal adenoma. Treatment by surgery may comprise laproscopic surgery. Effective drug treatments for adenomas include Non-Steroidal-Anti-Inflammatory Drugs (NSAIDs), such as aspirin and COX-2 inhibitor drugs, e.g. Celebrex and Vioxx. Treatment may also comprise radiation therapy, such as external beam radiation therapy or intraoperative radiation therapy ( i.e . given during surgery).

(iii) treating surgically (e.g. by colonoscopy) or administering a therapeutic agent to a subject diagnosed in step (ii) as a patient having said colorectal adenoma.

According to a further aspect of the invention, there is provided a method of treating a colorectal adenoma in an individual in need thereof, which comprises the step of treating surgically (e.g. by colonoscopy) or administering a therapeutic agent to a patient identified as having differing levels of the biomarker(s) as defined herein in a blood, serum or plasma sample obtained from said patient, when compared to the levels of said biomarker(s) in a blood, serum or plasma sample obtained from a control subject.

According to a further aspect of the invention there is provided a method of treatment for a colorectal adenoma in a subject which comprises the steps of:

(i) obtaining a blood, serum or plasma sample from the subject;

(ii) measuring the level of carbonic anhydrase and/or TIMP-1 and/or cell-free nucleosomes in the sample;

(iii) using the level of carbonic anhydrase and/or TIMP-1 and/or cell-free nucleosomes measured in the sample as an indicator of the presence of a colorectal adenoma in the subject; and

(iv) treating the patient surgically (e.g. by colonoscopy) or with a therapy or drug aimed to slow or prevent progression of the colorectal adenoma to a colorectal cancer.

Optionally, the cell-free nucleosomes measured may include nucleosomes that contain an epigenetic signal structure which is used as the biomarker. According to a further aspect of the invention there is provided a method of treatment for a colorectal adenoma in a subject which comprises the steps of:

(i) obtaining a blood, serum or plasma sample from the subject;

(iii) using the level of carbonic anhydrase and/or TIMP-1 and/or cell-free nucleosomes measured in the sample as an indicator of the presence of a colorectal adenoma in the subject;

(iv) examining the patient by colonoscopy, capsule camera, sigmoidoscopy or MRI methods to identify the number and location of colorectal adenoma(s) in the subject; and

(v) removing or treating the adenoma(s).

Optionally, the cell-free nucleosomes measured may include nucleosomes that contain an epigenetic signal structure which is used as the biomarker.

Methods of the invention may be used as stand-alone methods for the detection of subjects with a colorectal adenoma, or may be used in conjunction with a test for CRC to select candidates with either or both of CRC and/or adenomas for further investigation by colonoscopy or other methods. Similarly, methods of the invention may be used to test for the presence of colorectal polyps/adenomas among symptomatic patients who display symptoms that may be consistent with a diagnosis of colorectal adenoma.

Diagnostic kits for the diagnosis and monitoring of the presence of colorectal adenoma are described herein.

According to a further aspect of the invention, there is provided a biomarker panel (or kit) comprising two or more biomarkers selected from the group consisting of: carbonic anhydrase, TIMP-1 and cell-free nucleosomes.

It will be understood that the reference to cell-free nucleosomes includes cell-free nucleosomes per se and/or cell-free nucleosomes containing an epigenetic signal structure. Therefore, in one embodiment, the cell-free nucleosomes contain an epigenetic signal structure. In another embodiment, the panel comprises measuring the level of cell-free nucleosomes per se and at least one cell-free nucleosome containing an epigenetic signal structure (as described hereinbefore). In one embodiment, the biomarker panel comprises: carbonic anhydrase, TIMP-1 and cell- free nucleosomes. In a further embodiment, the biomarker panel comprises: carbonic anhydrase, TIMP-1 and cell-free nucleosomes containing an epigenetic signal structure. In a yet further embodiment, the biomarker panel comprises: carbonic anhydrase (such as CA-9 or CA-1 ), TIMP-1 , cell-free nucleosomes (per se) and cell-free nucleosomes containing an epigenetic signal structure (e.g. a post-translational histone modification, such as H3K9Me).

In one embodiment, the biomarker panel consists of: carbonic anhydrase, TIMP-1 and cell- free nucleosomes. In a further embodiment, the biomarker panel consists of: carbonic anhydrase, TIMP-1 and cell-free nucleosomes containing an epigenetic signal structure. In a yet further embodiment, the biomarker panel consists of: carbonic anhydrase, TIMP-1 , cell- free nucleosomes (per se) and cell-free nucleosomes containing an epigenetic signal structure.

In one embodiment, the biomarker panel is for use in diagnosing the presence of a colorectal adenoma.

According to a further aspect of the invention, there is provided the use of a kit comprising one or more binding agents capable of detecting and/or quantifying the biomarker(s) as defined herein for the diagnosis of a colorectal adenoma.

Suitably a kit according to the invention may contain one or more components selected from the group: a ligand binder, or ligands, specific for the biomarkers defined herein, one or more controls, one or more reagents and one or more consumables; optionally together with instructions for use of the kit in accordance with any of the methods defined herein.

It will be understood that the embodiments described herein may be applied to all aspects of the invention. Furthermore, all publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.

The invention will now be illustrated with reference to the following non-limiting examples.

EXAMPLE 1 Serum samples were taken from 521 asymptomatic subjects aged 50-74 who were undergoing FIT testing for CRC screening purposes. The FIT test used was the EIKEN OC- SENSOR. Of the 521 subjects, 67 were tested FIT positive at the recommended 20 pg hemoglobin/g feces cut off and were subsequently diagnosed with one or more High-Risk adenomas on colonoscopy. The remaining 454 subjects tested negative for FIT and did not undergo colonoscopy and are assumed to be free of High-Risk adenomas. However, as FIT has a poor detection rate of 23% for High-Risk adenomas, the FIT negative population in fact contains the majority of High-Risk adenomas cases. Using published data (Imperiale et al. (2014)) we have estimated that the 454 FIT negative subjects will actually include 28 subjects (6%) with a High-Risk adenoma. This means that the accuracy estimates for the specificity of High-Risk adenoma detection quoted herein are likely to be underestimated by up to 6%.

The samples were analysed for TIMP-1 using the Human TIMP-1 Quantikine ELISA Kit available commercially from R&D Systems Inc. according to the manufacturer’s instructions. The results showed that circulating TIMP-1 levels are raised in patients with High-Risk adenomas over subjects tested FIT negative. Receiver Operating Characteristic (ROC) analysis gave an Area Under the Curve (AUC) of 58.5% and a sensitivity of 22.7% for High- Risk adenoma detection at a specificity of 90% (as stated above this is likely to be an underestimate of specificity).

EXAMPLE 2

The same serum samples described in EXAMPLE 1 above were analysed for Carbonic Anhydrase 9 using the CA-9 Quantikine ELISA Kit available commercially from R&D

Systems Inc. according to the manufacturer’s instructions. The results showed that circulating CA-9 levels are lowered in patients with High-Risk adenomas compared to subjects tested FIT negative. In contrast, circulating CA-9 levels measured in patients with late stage CRC were elevated compared to subjects tested FIT negative. Receiver

Operating Characteristic (ROC) analysis gave an Area Under the Curve (AUC) of 69.7% and a sensitivity of 28.8% for High-Risk adenoma detection at a specificity of 90% (as stated above this is likely to be an underestimate of specificity).

EXAMPLE 3

The same serum samples described in EXAMPLE 1 above were analysed for Nucleosomes using an ELISA developed in house. The ELISA used an anti-histone H3 monoclonal antibody immobilised on plastic microtitre wells together with a biotinylated monoclonal antibody that binds to a conformational nucleosome epitope not available on free histones.

In brief, 10mI of serum sample and 90mI of buffer were added to anti-histone H3 coated microtitre wells and incubated for 2.5 hours at room temperature with gentle shaking. The diluted sample was then removed and the wells were washed with a wash buffer. 10OmI biotinylated monoclonal antibody diluted in buffer was added and incubated for 1.5 hours. The diluted biotinylated antibody was removed and the wells were again washed with a wash buffer. 10OmI streptavidin-horse radish peroxidase conjugate diluted in buffer was added and incubated for 0.5 hours. The diluted streptavidin-horse radish peroxidase conjugate was removed and the wells were again washed with a wash buffer. 200mI of an enzyme substrate solution (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acidj- diammonium salt) was added and incubated 20 minutes. The optical density (OD) of the wells was measured using a standard microtitre plate reader and the relative nucleosome concentration interpolated from a calibration curve. The results showed that circulating nucleosome levels are raised in patients with High-Risk adenomas over subjects tested FIT negative. Receiver Operating Characteristic (ROC) analysis gave an Area Under the Curve (AUC) of 67.5% and a sensitivity of 16.7% for High-Risk adenoma detection at a specificity of 90% (as stated above this is likely to be an underestimate of specificity).

EXAMPLE 4

The same serum samples described in EXAMPLE 1 above were analysed for Nucleosomes containing Histone H3 post-translationally modified by tri-methylation of the lysine residue at position 9 (H3K9Me3) using an ELISA developed in house. The ELISA used an anti- H3K9Me3 monoclonal antibody immobilised on plastic microtitre wells together with a biotinylated monoclonal antibody that binds a conformational nucleosome epitope not available on free histones. In brief, 20mI of serum sample and 80mI of buffer were added to anti-histone H3K9Me3 coated microtitre wells and incubated for 2 hours at room temperature with gentle shaking. The diluted sample was then removed and the wells were washed with a wash buffer. 10OmI biotinylated monoclonal antibody diluted in buffer was added and incubated for 1 hour. The diluted biotinylated antibody was removed and the wells were again washed with a wash buffer. 10OmI streptavidin-horse radish peroxidase conjugate diluted in buffer was added and incubated for 0.5 hours. The diluted streptavidin-horse radish peroxidase conjugate was removed and the wells were again washed with a wash buffer. 100mI of an enzyme substrate solution (3,3',5,5'-Tetramethylbenzidine) was added and incubated 10 minutes. 50mI of a STOP solution (1 M HCI) was added and the optical density (OD) of the wells was measured using a standard microtitre plate reader. The results showed that circulating levels of nucleosomes containing the H3K9Me3 post translational modification are raised in patients with High-Risk adenomas over subjects tested FIT negative. Receiver Operating Characteristic (ROC) analysis gave an Area Under the Curve (AUC) of 62.9% and a sensitivity of 16.7% for High-Risk adenoma detection at a specificity of 90% (as stated above this is likely to be an underestimate of specificity).

EXAMPLE 5

A serum sample cohort including FIT negative subjects and FIT positive subjects diagnosed with a High-Risk adenoma were analysed for Carbonic Anhydrase 1 using a commercially available kit purchased from Nordic BioSite AB and performed according to the

manufacturer’s instructions. The results are expected to show that circulating CA-1 levels are altered in patients with High-Risk adenomas compared to subjects tested FIT negative.

EXAMPLE 6

The results obtained for the 4 assays described in EXAMPLES 1 , 2, 3 and 4 above were combined into a panel test for High-Risk adenomas using regression analysis. Receiver Operating Characteristic (ROC) analysis gave an Area Under the Curve (AUC) of 76.1% and a sensitivity of 43.3% for High-Risk adenoma detection at a specificity of 90% (as stated above this is likely to be an underestimate of specificity).

EXAMPLE 7

Other patient parameters may be added to the assay results to increase the accuracy of the test. We added patient age to the results obtained for the 4 assays described in EXAMPLES 1 , 2, 3 and 4 above and combined all five parameters into a panel test for High-Risk adenomas using regression analysis. Receiver Operating Characteristic (ROC) analysis gave an Area Under the Curve (AUC) of 76.5% and a sensitivity of 50.7% for High-Risk adenoma detection at a specificity of 90% (as stated above this is likely to be an

underestimate of specificity).

REFERENCES

Arber. Cyclooxygenase-2 Inhibitors in Colorectal Cancer Prevention: Point. Cancer

Epidemiology Biomarkers & Prevention; 17(8): 1852-87, 2008

Chen et al. Development and validation of a panel of five proteins as blood biomarkers for early detection of colorectal cancer. Clinical Epidemiology; 9: 517-26, 2017

Herranz and Esteller, DNA methylation and histone modifications in subjects with cancer: potential prognostic and therapeutic targets. Methods Mol. Biol.; 361 : 25-62, 2007 Holdenrieder et al. Nucleosomes in serum of patients with benign and malignant diseases. Int. J. Cancer (Pred. Oncol.); 95: 114-120, 2001

Holdenrieder and Stieber, Clinical use of circulating nucleosomes. Critical Reviews in Clinical Laboratory Sciences; 46(1 ): 1-24, 2009

Holten-Anderson et al. Plasma TIMP-1 in patients with colorectal adenomas: a prospective study. European Journal of Cancer; 40: 2159-64, 2004

Imperiale et al. Multitarget Stool DNA Testing for Colorectal-Cancer Screening. New Engl. J. Med.; 370: 1287-97, 2014

Salgame et al. An ELISA for detection of apoptosis. Nucleic Acids Research; 25(3): 680-681 , 1997 van Nieuwenhuijze et al. Time between onset of apoptosis and release of nucleosomes from apoptotic cells: putative implications for systemic lupus erythematosus. Ann Rheum Dis; 62: 10-14, 2003

Claims

1. Use of carbonic anhydrase, Tissue Inhibitor of Metalloproteinases-1 (TIMP-1 ) and/or cell-free nucleosomes in a blood, serum or plasma sample as a biomarker for diagnosing the presence of a colorectal adenoma.

2. The use as defined in claim 1 , wherein the carbonic anhydrase is carbonic anhydrase 9 (CA-9).

3. The use as defined in claim 1 or claim 2, wherein the cell-free nucleosomes contain an epigenetic signal structure.

4. The use as defined in claim 3, wherein the epigenetic signal structure is selected from the group consisting of: a post-translational histone modification, a histone variant, a particular nucleotide and a protein adduct.

5. The use as defined in claim 4, wherein the post-translational histone modification is H3K9Me3.

6. The use as defined in any one of claims 1 to 5, wherein carbonic anhydrase, Tissue Inhibitor of Metalloproteinases-1 (TIMP-1 ) and cell-free nucleosomes are used as a biomarker panel for diagnosing the presence of a colorectal adenoma.

7. The use as defined in any one of claims 1 to 6, wherein the colorectal adenoma is a High-Risk adenoma.

8. A method for diagnosing or detecting a colorectal adenoma, in an animal or a human subject which comprises the steps of:

(i) detecting or measuring the level of the biomarker(s) as defined in any one of claims 1 to 7 in a blood, serum or plasma sample obtained from the subject; and

9. A method for determining the prognosis of an animal or a human subject with a colorectal adenoma, which comprises the steps of: (i) detecting or measuring the level of the biomarker(s) as defined in any one of claims 1 to 7 in a blood, serum or plasma sample obtained from the subject; and

10. A method for monitoring the efficacy of a therapy in an animal or a human subject having, suspected of having, or of being predisposed to colorectal adenoma, which comprises the steps of:

(ii) using the measured level of biomarker(s) compared with an earlier biological sample taken from said subject as indicative of the efficacy of said therapy.

1 1. The method as defined in any one of claims 8 to 10, wherein said detection or measurement comprises an immunoassay, immunochemical, mass spectroscopy, chromatographic, chromatin immunoprecipitation or biosensor method.

12. The method as defined in any one of claims 8 to 11 , comprising comparing the amount of the biomarker(s) present in said blood, serum or plasma sample with one or more controls, such as comprising the amount of biomarker(s) present in a blood, serum or plasma sample obtained from the subject with the amount of biomarker(s) present in a blood, serum or plasma sample obtained from a normal subject.

13. The method as defined in any one of claims 8 to 12, wherein the subject has been identified as positive for fecal occult blood.

14. The method as defined in any one of claims 8 to 13, which additionally comprises using a clinical parameter to diagnose the subject with a colorectal adenoma.

15. The method as defined in claim 14, wherein the clinical parameter is selected from the group consisting of: age, sex and body mass index (BMI).

16. A method of treating a colorectal adenoma in an animal or a human subject, which comprises the following steps:

(i) detecting or measuring the level of the biomarker(s) as defined in any one of claims 1 to 7 in a blood, serum or plasma sample obtained from the subject; (ii) using the measured level of biomarker(s) as indicative of the presence of said colorectal adenoma in the subject; and

17. A method of treating a colorectal adenoma in an individual in need thereof, which comprises the step of treating surgically or administering a therapeutic agent to a patient identified as having differing levels of the biomarker(s) as defined in any one of claims 1 to 7 in a blood, serum or plasma sample obtained from said patient, when compared to the levels of said biomarker(s) in a blood, serum or plasma sample obtained from a control subject.

18. A biomarker panel comprising two or more biomarkers selected from the group consisting of: carbonic anhydrase, TIMP-1 and cell-free nucleosomes.

19. The biomarker panel of claim 18, wherein the cell-free nucleosomes contain an epigenetic signal structure.

20. The biomarker panel of claim 18 or claim 19, for use in diagnosing the presence of a colorectal adenoma.

21. Use of a kit comprising one or more binding agents capable of detecting and/or quantifying the biomarker(s) as defined in any one of claims 1 to 7, for the diagnosis of a colorectal adenoma.