TW201930881A - Method for the detection and treatment of colorectal adenomas - Google Patents

Abstract

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

Description

Detection and treatment of colorectal adenoma

The invention relates to a method for detecting and treating colorectal adenoma or colorectal polyps by blood test.

Colorectal cancer (CRC) is a common disease with high mortality. The biology of the disease is thought to be related to the progression of precancerous adenomas (polyps), with epithelial variability from low-risk adenomas to moderate-risk adenomas to high-risk adenomas, which may lead to I, II, Phase III and the final Phase IV CRC. Mortality varies widely, depending on whether the disease is detected in the pre-cancerous adenoma stage, early CRC in situ in the colon or rectum (eg, stage I CRC) or spread to a greater extent Treatment of advanced CRC (eg, stage IV CRC) is more difficult.

The detection and removal of adenomas is thought to prevent the development of cancer, and the prognosis of these patients is very good. If the disease progresses to CRC, the 5-year survival rate for patients with CRC disease detected in stage I is > 90%, but only 10% survival for patients with stage IV CRC metastatic disease. For this reason, many countries have CRC screening procedures to identify individuals with CRC or precancerous adenomas or polyps. The incidence of CRC is age- and gender-dependent; men are more common than women, and older people are more common. CRC is rare in people under the age of 50, so screening older people is more useful. In screening programs in different countries, the age range for actual screening varies, but is usually 50-74 years old.

In the screening population, the prevalence of CRC is approximately 0.6%. However, the prevalence of adenomas is higher. Approximately 36% of the screening age population has one or more colorectal adenomas. Of these, approximately 8% are high-risk or advanced adenomas, and 28% are low or medium-risk or non-advanced adenomas (Imperiale et al. (2014)). Although all or most of the CRC is thought to have evolved from high-risk adenomas, it is clear that not all patients with high-risk adenomas develop CRC.

It is important to remove or treat colorectal adenomas, especially for high-risk adenomas, because some patients with high-risk adenomas develop CRC if left untreated. Currently, adenomas are usually examined by colonoscopy. When detected, neutralizing high-risk adenomas are usually surgically removed at the time of colonoscopy. Effective medical treatment for adenomas is also feasible, including non-steroidal anti-inflammatory drugs (NSAIDs). For example, enzyme epoxidase (COX-2) is overexpressed in colorectal polyps, and COX-2 inhibitors such as aspirin, celebrex, and Vioxx are effective in reducing disease. Adenoma burden in people with familial adenomatous polyposis. COX-2 inhibitors reduce the amount and size of polyps. COX-2 inhibitors also reduce the recurrence of adenomas (especially high-risk adenomas) after surgical resection (Arber (2008)). A problem associated with these drugs is the identification of patients with adenomas, especially high-risk adenomas. Approximately 8% of the screening age population has one or more high-risk adenomas and is a potential candidate for drug treatment. However, high-risk adenomas can only be diagnosed by colonoscopy, and if determined in this way, they can be surgically removed without medication. Thus, blood tests that can identify high-risk adenomas have great potential for companion products as COX-2 inhibitors such as polyp therapeutics.

The CRC screening program is able to perform CRC testing earlier than in other situations, so it can treat earlier and extend life for more years. Early CRC testing saves healthcare providers money and resources by reducing the need for expensive advanced cancer medications and hospitalizations. Ideally, the CRC screening procedure can also detect precancerous colorectal polyps or adenomas that may develop into CRC if left untreated, but may be removed prior to cancer development if detected. The prognosis of these patients is good, and the removal of precancerous polyps on a population basis can reduce the incidence and prevalence of CRC.

Methods commonly used for CRC detection and/or screening have a number of disadvantages. The primary CRC screening method used in the United States is colonoscopy. Colonoscopy essentially involves visual inspection of the large intestine, which uses a lens that passes through the descending colon, transverse colon, and ascending colon to find out if there are any cancerous or potential precancerous lesions. The main advantage of colonoscopy is its detection accuracy, its clinical sensitivity to CRC is 95%, and the detection rate for precancerous adenomas is very high, with very high clinical specificity. This accuracy makes colonoscopy a golden criterion for CRC testing, especially in the detection of low-grade lesions and adenomas. However, colonoscopy as a first line CRC detection or screening method is subject to many limitations. Colonoscopy is a highly invasive procedure that requires surgical consent. This procedure is usually performed under anesthesia, and the patient needs to prepare the intestine first, in some cases (eg, intestinal tear) causing the patient to be injured and the procedure is expensive (> $1000). CRC was measured in about 0.5% of screening colonoscopy, and high-risk adenomas were measured in 8% of screening colonoscopy, so most people received surgery for a small benefit. Because of these shortcomings, patients have low compliance with colonoscopy, and many screening ages are reluctant to undergo the procedure. For these reasons, colonoscopy is not used as a first-line CRC test or screening method in most countries of the world.

Some health care providers use a so-called sigmoidoscopy related method in which a shorter lens is only used to examine the descending colon. Although this method misses two-thirds of the colon, it does check the most common areas of cancer. The shortcomings of sigmoidoscopy are similar to colonoscopy and are generally not used as first line tests for similar reasons. A virtual colonoscopy or computed tomography (CT) colon imaging can also be used. This process combines X-ray and computer technology to produce rectal and colon images to detect colorectal tumors and polyps.

The most commonly used CRC detection and screening methods involve a two-stage procedure that first screens the screening age population using a non-invasive first line stool test to identify subgroups of screening populations with a higher risk of CRC. People who tested positive in the stool test were referred for a subsequent colonoscopy. Usually about 5% of these people will be issued with a CRC. Most people have negative stool screening results, so the two-stage approach avoids unnecessary colonoscopy for most people without lesions.

The basic principle of CRC stool testing is to detect bleeding from the colon or rectum. In short, when the colon or rectum is partially blocked by the invasive cancerous or precancerous adenoma, the passage of the feces through the obstruction may cause damage and bleeding. This bleeding is detected by testing for the presence of hemoglobin in the stool sample. Because the degree of bleeding per day can vary widely, multiple tests may be performed on different days.

All fecal CRC tests are currently designed to detect fecal hemoglobin. The guaiac fecal occult blood test (FOBT or gFOBT) is a chemical test for hemoglobin, and patients or operators typically apply a small amount of feces to test paper or other substrates coated with alpha-guaiac acid. If blood is present in the stool, the addition of hydrogen peroxide to the test strip produces a rapid color change that oxidizes α-guaiac acid to blue in a reaction catalyzed by the blood matrix (the component of hemoglobin). . The intake of meat (and blood matrix) and some vegetables, containing other catalyst molecules, behaves like a blood matrix in the test, thus causing false positive results. Similarly, some substances containing vitamin C can cause a false negative reaction, so it is generally recommended to limit the diet before testing. The Guaiac FOBT can be highly clinically specific and has a sensitivity of about 60% for the detection of CRC, depending on the threshold used. The detection of precancerous polyps or adenomas is very poor. The chemical FOBT method has been one of the selected methods in the past, and although it is still widely used, it has been replaced by a fecal immunochemical test method.

The Fecal Immunochemical Test (FIT) method for CRC detection (also known as iFOBT or FOBTi) is essentially an immunoassay for human hemoglobin in stool samples. The FIT method is less affected by false positive and negative results due to dietary factors, and a smaller amount of blood in the feces can be detected. These tests have similar specificity to gFOBT but detect more clinically relevant cancer lesions. The detection of precancerous polyps or adenomas is poor. The FIT method has a sensitivity to CRC of approximately 74% and a specificity of approximately 95%. The sensitivity of the FIT method to advanced precancerous colorectal adenomas or polyps is approximately 23%, and sensitivity to non-advance adenomas is approximately 8%. EXAT Sciences has developed a fecal CRC test that analyzes fecal DNA in addition to fecal hemoglobin. The trial (Cologuard) reported a sensitivity of 92% for CRC, a specificity of 87%, and 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.

In many countries, a full CRC screening program using FIT testing was established for screening age groups, and mortality due to CRC was reduced. The main problem facing the CRC screening program is compliance. In the United States, compliance is about 70% (ie, about 70% of screening ages are colonoscopy). In Europe, the best performing FIT CRC screening program has a compliance rate of approximately 60-70%, but compliance rates in many countries are below 50%. Therefore, at least 30% of screening ages are not screened. Another problem facing the CRC screening program is that once the program has been in operation for a few years, their success will lead to a decline in the prevalence of CRC in the screening population. Obviously, this is a sign of success, but it means that identifying people with rectal polyps that need to be removed is a high priority before developing into cancer. Therefore, testing for people with adenomas, especially high-risk adenomas, is becoming more and more necessary.

It is reported that reasons for non-compliance with FIT and colonoscopy include: inconvenience, need for vacation, high cost, discomfort and invasiveness during colonoscopy, and unpleasant and cultural taboos on fecal collection. In order to address the medical needs of people who have not been screened, in recent decades, relevant workers have attempted to develop blood tests for CRC that may overcome some of these limitations. However, such tests have not been widely adopted in clinical CRC screening procedures.

Many typical blood biomarkers, including carcinoembryonic antigen (CEA), have been studied as possible blood-based biomarkers, but so far their clinical accuracy is too low for routine use if they have any application , is more suitable for monitoring patients rather than CRC detection or diagnosis. Typically, such markers measure late stage IV CRC rather than early stage I or II CRC, nor are precancerous rectal polyps. In some cases, related workers have combined multiple blood-based biomarkers to increase the combined sensitivity of CRC detection. A recently published example involved a survey of 93 plasma protein biomarkers in 226 CRC patients and 118 colonoscopy negative controls. From the results, the 5-biomarker kit model containing GDF-15, AREG, FasL, Flt3L and P53 autoantibodies was selected as the optimal combination and validated in separate patient groups, including 41 CRC cases, 106 cases of advanced adenoma and 107 negative controls screened for colonoscopy. The biomarker kit has a sensitivity to CRC of 56%, a sensitivity of 22% for advanced adenomas, and a specificity of 90% (Chen et al. (2017)).

The Epi proColon test developed by Epigenomics AG has recently become the first CRC blood test approved by the US FDA. This assay measures hypermethylation of the Septin-9 gene DNA sequence as a diagnostic marker for CRC in the blood. According to the FDA study, the test has a sensitivity to CRC of 68% and a specificity of 78%. The sensitivity of colorectal polyps or adenomas is very low, almost no higher than the false positive rate of the test.

In conclusion, approximately 8% of CRC screening age sufferers with one or more high-risk colorectal polyps/adenomas that may develop CRC, and removal is an effective CRC prophylaxis. Colonoscopy can identify most or all cases of colorectal polyps, but patient compliance is low and is not widely used as a first-line CRC screening method outside the United States. Most countries use the FIT test for population CRC screening, but these methods also have poor patient compliance and only detect 23% of high-risk colorectal polyps. Therefore, there is a need for a blood test that can identify a person with a colorectal polyp/adenomas, but neither the classic CRC blood biomarker nor the only blood test that detects CRC can detect colorectal polyps.

We now disclose a simple, low-cost ELISA blood test that detects >50% of advanced or high-risk colorectal adenomas with >90% specificity.

According to a first aspect of the present invention, a carbonic anhydrase, a tissue inhibitor of metalloproteinase-1 (TIMP-1) and/or a free nucleosome is provided as a biomarker in a blood, serum or plasma sample. For the diagnosis of the presence of colorectal adenoma.

According to another aspect of the present invention, a method of diagnosing or detecting a colorectal adenoma in an animal or a human subject is provided, comprising the steps of:
(i) detecting or measuring the content of a biomarker as defined herein in a blood, serum or plasma sample obtained from the subject;
(ii) using the measured biomarker content as an indicator of the presence of colorectal adenoma in the subject.

According to another aspect of the present invention, there is provided a method of determining the prognosis of an animal or a human subject having a colorectal adenoma comprising the steps of:
(i) detecting or measuring the content of a biomarker as defined herein in a blood, serum or plasma sample obtained from the subject;
(ii) using the measured biomarker content as an indicator of the presence of the colorectal adenoma in the subject.

According to another aspect of the present invention, there is provided a method of monitoring the efficacy of treatment in an animal, or a human subject, having, suspected of having or prone to a colorectal adenoma, comprising the steps of:
(i) detecting or measuring the concentration of a biomarker as defined herein in a blood, serum or plasma sample obtained from the subject;
(ii) using the measured biomarker concentration as compared to a biological sample previously obtained from the subject as an indicator of efficacy of the treatment.

According to another aspect of the present invention, a method of treating a colorectal adenoma in an animal or a human subject is provided, comprising the steps of:
(i) detecting or measuring the concentration of a biomarker as defined herein in a blood, serum or plasma sample obtained from the subject;
(ii) using the measured biomarker concentration as an indicator of the presence of a colorectal adenoma in the subject;
(iii) Surgical treatment or administration of a therapeutic agent to a subject diagnosed as having a colorectal adenoma in step (ii).

According to another aspect of the present invention, there is provided a method of treating a colorectal adenoma in a subject in need thereof, comprising the steps of: surgically treating or administering a therapeutic agent to a patient, the patient being identified as having blood, serum or Plasma samples have different concentrations of biomarkers as defined herein, as compared to biomarker concentrations in blood, serum or plasma samples obtained from a control subject.

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

According to another aspect of the invention, there is provided a kit comprising a kit for detecting and/or quantifying one or more binding agents of a biomarker as defined herein for use in diagnosing a colorectal adenoma.

According to a first aspect of the present invention, a carbonic anhydrase, a tissue inhibitor of metalloproteinase-1 (TIMP-1) and/or a free nucleosome is provided as a biomarker in a blood, serum or plasma sample. For the diagnosis of the presence of colorectal adenomas.

The terms "adenomas", "polyps", "colorectal adenomas" and "large intestine rectal polyps" are used interchangeably herein. They refer to the growth of the colon or the inner layer of the rectum. Adenomas/polyps are considered precancerous, ie they may develop into colon cancer, so it is recommended to remove them. The terms "advanced adenoma" and "high risk adenoma" are used interchangeably herein. The definition of these terms is well known to those of ordinary skill in the art. They vary slightly in different countries and screening programs, but broadly they refer to colorectal adenomas with a maximum size of ≥1 cm, including sessile or serrated adenomas, or with villus tissue type or highly epithelial variation. Adenoma (Imperiale et al. (2014)). The terms "non-advanced adenoma" and "low and medium risk adenoma" are used interchangeably herein. They refer to non-advanced or high-risk adenomas.

In one embodiment, the colorectal adenoma is selected from the group consisting of: low risk adenoma, moderate risk adenoma, and high risk adenoma. In another 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 the group consisting of carbonic anhydrase 1 (CA-1) and carbonic anhydrase 9 (CA-9). In another embodiment, the carbonic anhydrase is carbonic anhydrase 1 (CA-1). In yet another embodiment, the carbonic anhydrase is carbonic anhydrase 9 (CA-9).

Carbonic anhydrase is a group of enzymes that control pH balance. They have been shown to rise in cancer (in terms of tissue performance and circulating concentrations) because they help maintain pH balance in the tumor environment when exposed to undesirable angiogenesis and hypoxia. Cyclic carbonic anhydrase is reported to rise in the CRC. However, there is no evidence that circulating carbonic anhydrase concentrations change in colorectal adenomas, and as far as we know, any association with adenoma/polyps has not been studied. Surprisingly, the inventors have found that the concentration of circulating carbonic anhydrase (especially CA-9) is reduced in patients with high risk of colorectal adenoma.

According to one aspect of the invention, the use of matrix metalloproteinase tissue inhibitor-1 (TIMP-1) as a biomarker for the diagnosis of colorectal adenomas is provided.

In the CRC, circulating TIMP-1 concentrations were reported to be rising. However, it has been reported that circulating TIMP-1 concentrations have not changed in subjects with polyps, and TIMP-1 has been reported to be not a useful biomarker for polyp detection (Nielsen et al. (2004)). Surprisingly, the inventors have found an increase in circulating TIMP-1 concentrations in patients with high-risk colorectal adenomas.

In one embodiment, the use comprises carbonic anhydrase and TIMP-1. In another embodiment, the use comprises carbonic anhydrase and free nucleosomes. In yet another embodiment, the use comprises TIMP-1 and free nucleosomes.

In one embodiment, carbonic anhydrase, matrix metalloproteinase tissue inhibitor-1 (TIMP-1), and free nuclear mini-system are used as biomarker kits to diagnose the presence of colorectal adenomas.

When a regression analysis method was used to combine concentrations of circulating concentrations of carbonic anhydrase, TIMP-1, and nucleosome concentrations, a combination accuracy of 37.9% sensitivity was achieved for high-risk colorectal adenomas with a specificity of 90%.

Thus, in accordance with another aspect of the present invention, there is provided a use of a cyclic biomarker kit comprising carbonic anhydrase, TIMP-1, free nucleosomes, and any combination of free nucleosomes comprising an epigenetic signal structure Or all, as a biomarker kit for diagnosing the presence of colorectal adenomas.

In one embodiment, the concentration of free nucleosomes (i.e., free nucleosomes themselves) is used as a biomarker. In another embodiment, free nucleosomes containing epigenetic signal structures are used as biomarkers.

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

Circulating nucleosome concentrations are reported to rise in CRC and other cancer diseases (Holdenrieder et al. (2001)). The inventors have found that circulating nucleosome concentrations increase in patients with high risk of colorectal adenoma.

The circulating nucleosome is not a homogenous group of protein-nucleic acid complexes. Conversely, they are heterogeneous populations of chromatin fragments derived from the breakdown of chromatin at the time of cell death and contain a variety of epigenetic structures, including specific tissue protein isoforms or variants, post-translational tissue protein modifications. , nucleotide or modified nucleotides as well as nucleotide adducts. It is well known to those of ordinary skill in the art that elevated nucleosome concentrations are associated with a subset of circulating nucleosomes containing specific epigenetic signals, including inclusion of specific tissue protein isoforms or variants, including specific translations. Post-tissue protein modification, inclusion of specific nucleotides or modified nucleotides, and nucleosomes comprising specific nucleosome adducts. Similarly, a reduction in the absolute concentration and/or proportion of circulating nucleosomes in patients with adenomas, including other epigenetic signals, including specific tissue protein isoforms or changes, may occur. Body, comprising a specific post-translational tissue protein modification, comprising a specific nucleotide or modified nucleotide, and a nucleosome comprising a specific nucleosome adduct. The determination of these types of chromatin fragments is known in the art (for example, see WO2005/019826, WO2013/030579, WO2013/030578, WO2013/084002, which is incorporated herein by reference). These assays have previously been used to demonstrate circulating free nucleosomes that can detect epigenetic changes in the blood of diseased patients. Thus, in one embodiment, the detection or measurement of free nucleosomes comprises:
(i) contacting the sample with a first binding agent that binds to the free nucleosome or a component thereof;
(ii) contacting the sample comprising the first binding agent bound to the free nucleosome or a component thereof with a second binding agent, the second binding agent and an apparent in the free nucleosome Combination of genetic features;
(iii) detecting or quantifying the binding of the second binding agent in the free nucleosome to the epigenetic feature in the sample.

In another embodiment, the detecting or measuring in step (b) comprises:
(i) contacting the sample with a first binding agent that binds to an epigenetic feature in the free nucleosome;
(ii) contacting the sample comprising the first binding agent bound to the epigenetic feature with a second binding agent that binds to the free nucleosome or a component thereof;
(iii) detecting or quantifying the binding of the second binding agent to the free nucleosome or a component thereof in the sample.

The nucleosome is the basic unit of the chromatin structure and consists of a protein complex of eight highly retained core tissue proteins (containing each pair of tissue proteins H2A, H2B, H3 and H4). Approximately 146 base pairs of DNA are coated around this complex. Another tissue protein, H1 or H5, acts as a linker and is involved in the compaction of chromatin. DNA is entangled in a so-called "beaded" structure by successive nucleosomes and forms the basic structure of open or true chromatin. In compacted or true chromatin, the string is in a helical and supercoiled structure, forming a closed and complex structure (Herranz and Esteller (2007)).

It will be understood that the term free nucleosome in the present invention is intended to incorporate any free chromatin fragment comprising one or more nucleosomes. The epigenetic signal structure/characteristic of a free nucleosome referred to herein may include, but is not limited to, one or more post-translationally modified tissue proteins, tissue protein isoforms, modified nucleotides, and/or A protein that binds to a nucleosome in a protein adduct.

Normal cell turnover in adults involves cell division of approximately 10 ¹¹ cells per day and a similar number of deaths (primarily apoptosis). During apoptosis, chromatin is broken down into mononuclear bodies and oligonucleosomes, which are released from the cells. The report states that under normal conditions, the concentration of circulating nucleosomes in healthy subjects is small. An increase in concentration is found in subjects with different conditions, including many cancers, autoimmune diseases, inflammatory conditions, strokes, and myocardial infarction (Holdenreider & Stieber (2009)).

It has been reported that mononuclear and oligonucleosomes can be assayed by enzyme-linked immunosorbent assay (ELISA) and several methods (Salgame et al. (1997); Holdenrieder et al. (2001); van Nieuwenhuijze et al. (2003)). . These assays typically use anti-tissue protein antibodies (eg, anti-H2B, anti-H3 or anti-H1, H2A, H2B, H3, and H4) as capture antibodies, as well as anti-DNA or anti-H2A-H2A-H2B-DNA. The complex antibody acts as a detection antibody. In one embodiment, the anti-tissue protein antibody comprises an anti-H3 antibody.

Current nucleosome ELISA methods are mainly used for cell culture and are commonly used as a method for detecting apoptosis (Salgame et al. (1997); Holdenrieder et al. (2001); van Nieuwenhuijze et al. (2003)), but also Circulating free nucleosomes in serum and plasma were measured (Holdenrieder et al. (2001)). In many studies of different cancers, free serum and plasma nucleosome concentrations released into the circulation by dead cells were measured by ELISA to assess their potential as biomarkers (Holdenrieder et al. (2001)). It has been reported that the mean circulating nucleosome concentration is high in most but not all cancer studies. However, it has been reported that serum nucleosome concentrations in patients with malignant tumors vary widely, and some patients with advanced tumor disease are found to have low concentrations of circulating nucleosomes, which are measured in healthy subjects. Internal (Holdenrieder et al. (2001)). Because of this and various non-cancer causes of elevated nucleosome concentrations, circulating nucleosome concentrations have not been clinically used as biomarkers for cancer (Holdenrieder and Stieber (2009)).

In one embodiment, the epigenetic signal structure of the free nucleosome comprises one or more post-translational tissue protein modifications. The structure of nucleosomes can be altered by Post Translational Modification (PTM). The PTM of histoproteins usually occurs at the end of the core tissue proteins. Common modifications include acetamylation, methylation or ubiquitination of amino acid residues, and methylation of arginine residues and serine residues. Phosphorylation of the radical as well as many other modifications. Tissue protein modification is known to be involved in epigenetic regulation of gene expression (Herranz and Esteller (2007)). For example, in one embodiment, the post-translational tissue protein modification is H3K9Me3.

In one embodiment of the invention, one or a class of related tissue protein modifications (rather than a single modification) are detected. A typical example of this embodiment, but not limited to, relates to a 2-site immunoassay that uses one of the antibodies or other selective binding agents that bind to the nucleosome, and one of the groups that are modified with the tissue protein in question. An antibody or other selective binding agent. Examples of such antibodies that bind to tissue protein-modified groups include, but are not limited to, anti-pan-ethylated antibodies (eg, panthenyl H4 antibody), anti-citrullylated antibodies, or anti-ubiquitinated antibodies.

In one embodiment, the epigenetic signal structure of the free nucleosome comprises one or more tissue protein variants or isomeric isomers. The structure of the nucleosome can also be altered by the inclusion of shear tissue protein isoforms or variants that are different genes or splicing products and have different amines. Base acid sequence. Tissue protein variants can be divided into a number of families that are subdivided into individual types. Nucleotide sequences of a large number of tissue protein variants are well known and publicly available, for example, in the NHGRI Tissue Protein Library of the National Institute of Human Genetics (Mariño-Ramírez, L., Levine, KM, Morales, M., Zhang, S., Moreland, RT, Baxevanis, AD, 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), GenBank (NIH Gene Sequence) database, EMBL nucleotide sequence database, EMBL nucleotide sequence database and Japanese DNA database (DDBJ). For example, variants of tissue protein H2 include H2A1, H2A2, mH2A1, mH2A2, H2AX, and H2AZ.

In one embodiment, the epigenetic signal structure of the free nucleosome comprises one or more DNA modifications. In addition to epigenetic signal transmission mediated by nucleosome tissue isoforms and PTM, the composition of nucleotides and modified nucleotides of nucleosomes is also different. Comprehensive low-level methylation of DNA is a hallmark of cancer cells, and some nucleosomes may contain more 5-methylcytosine residues (or 5-hydroxymethylcytosine residues than other nucleosomes or Other nucleotides or modified nucleotides). In one embodiment, the DNA modification is selected from the group consisting of 5-methylcytosine or 5-hydroxymethylcytosine.

In one embodiment, the epigenetic signal structure of the free nucleosome comprises one or more protein-nucleosome adducts or complexes. Another type of circulating nucleosome subset is a nucleosome protein adduct. It has been known for many years that chromatin contains a large amount of non-tissue protein proteins bound to its constituent DNA and/or tissue proteins. These chromatin-related proteins are of many types and have a variety of functions, including transcription factors, transcription enhancers, transcriptional repressors, tissue protein modifying enzymes, DNA damage repair proteins, and others. These chromatin fragments include nucleosomes and other non-tissue protein chromatin proteins or DNA, and have been described in the art for chromatin proteins of other non-tissue proteins.

In one embodiment, the protein added to the nucleosome (and thus useful as a biomarker) is selected from the group consisting of: a transcription factor, a high mobility group protein, or a chromatin modified enzyme. "Transcription factor" refers to a protein that binds to DNA and regulates gene expression by promoting (ie, activator) or inhibiting (ie, inhibiting) transcription. A transcription factor contains one or more DNA binding domains (DBDs) attached to a specific DNA sequence adjacent to a gene it regulates.

All of the cyclic nucleosomes and nucleosome moieties, types, or subgroups described herein can be used in the present invention.

In one embodiment, the methods and uses of the invention comprise a method of measuring two or more free nucleosomes themselves and/or epigenetic features of free nucleosomes as a set of nucleosome features.

It will be appreciated that the methods and uses of the invention are particularly applicable to blood, serum, or plasma samples obtained from a subject. In an embodiment, the sample is a blood or serum sample. In another embodiment, the sample is a serum sample.

According to another aspect of the present invention, a method of diagnosing or detecting a colorectal adenoma in an animal or a human subject is provided, comprising the steps of:
(i) detecting or measuring the concentration of a biomarker as defined herein in a blood, serum or plasma sample obtained from the subject;
(ii) Using the measured biomarker concentration as an indicator of the presence of colorectal adenoma in the subject.

According to another aspect of the present invention, there is provided a method of determining the prognosis of an animal or a human subject having a colorectal adenoma comprising the steps of:
(i) detecting or measuring the concentration of a biomarker as defined herein in a blood, serum or plasma sample obtained from the subject;
(ii) using the measured biomarker concentration as an indicator of the prognosis of the colorectal adenoma in the subject.

According to another aspect of the present invention, there is provided a method of monitoring the efficacy of treatment in an animal, or a human subject, having, suspected of having or prone to a colorectal adenoma, comprising the steps of:
(i) detecting or measuring the concentration of a biomarker as defined herein in a blood, serum or plasma sample obtained from the subject;
(ii) using the measured biomarker concentration as compared to a biological sample previously obtained from the subject as an indicator of efficacy of the treatment.

References to "subject" or "patient" are used interchangeably herein. In an embodiment, the subject is a human subject.

In one embodiment, the detecting or measuring comprises immunoassay, immunochemistry, mass spectrometry, chromatography, chromatin immunoprecipitation or biosensor methods.

In an embodiment, the detecting or measuring comprises an immunoassay. In a preferred embodiment of the invention, a 2-site immunoassay method is provided. In particular, this method is preferably used for in situ measurement of nucleosomes containing apparent transfer characteristics using immobilized anti-nucleosome binding agents with labeled anti-tissue protein modifications or anti-tissue Protein variant or anti-DNA modified or anti-additive protein detection binding agent. In another embodiment of the invention, a 2-site immunoassay is provided which uses a labeled anti-nucleosome detection binding agent and an immobilized anti-tissue protein modification or anti-tissue protein variant or anti- DNA modified or anti-additive protein binding agent.

The concentration of the biomarker can be detected or measured using a suitable binding agent. In one embodiment, the one or more binding agents comprise a ligand or conjugate for the biomarker to be measured, such as a free nucleosome or a component thereof, or a structure/shape of a free nucleosome or a component thereof Simulated object.

It will be apparent to those skilled in the art that the term "antibody, binding agent or ligand" is not limiting with respect to any aspect of the invention, but is intended to include any binding agent capable of binding to a particular molecule or substance, and the method of the invention Any suitable binder can be used. It is also clear that the term "nucleosome" is intended to include both mononucleosomes and oligonucleosomes as well as any such chromatin fragments that can be analyzed in a fluid medium.

In one embodiment, a ligand or binding agent of the invention comprises a naturally occurring or chemically synthesized compound that is capable of specifically binding to a desired target. The ligand or binding agent may comprise a peptide, an antibody or fragment thereof, or a synthetic ligand, such as a plastic antibody, or an aptamer or oligonucleotide, capable of specifically binding to a desired target. The antibody may be a monoclonal antibody or a fragment thereof. The ligand may be labeled with a detectable label, such as a luminescent, fluorescent, enzymatic or radioactive label; alternatively or additionally, the ligand according to the invention may be labeled with an affinity label, eg, biotin, avidin Protein, streptavidin, or His (eg, hexa-His) marker. Alternatively, ligand binding can be determined using, for example, the label-free technique of ForteBio Inc.

The term "detection" or "diagnosis" as used herein includes the identification, confirmation, and/or characterization of a disease state. The detection, monitoring and diagnostic methods according to the present invention can be used to confirm the presence of a disease, monitor the progression of the disease by assessing the onset and progression, or assess the improvement or regression of the disease. Methods of detection, monitoring, and diagnosis can also be used to assess clinical screening, prognosis, treatment options, and assessment of therapeutic benefit, ie, drug screening and drug development methods.

Immunoassays of the invention include any method of using one or more antibodies or other specific binding agents that are intended to bind to a biomarker as defined herein. Immunoassays include 2-site immunoassay or immunoassay (eg ELISA) using enzyme detection methods, fluorescent-labeled immunoassays, jet fluoro-immunoassay, chemiluminescence immunoassay, immunoturbidimetry, particle-labeled immunoassay And immunoradiometric assays, as well as single-site immunoassays, reagent-restricted immunoassays, competitive immunoassay methods, including labeled antigens and labeled antibody single-antibody immunoassays, including radioactivity, enzymes, fluorescence, jet variability, and particles A variety of tag types for tagging. All such immunoassay methods are well known in the art, see, for example, Salgame et al. (1997) and van Nieuwenhuijze et al. (2003).

Identification and/or quantification can be performed by any suitable method to identify the presence and/or amount of a particular protein in a biological sample from a subject or in a purified or extract of the biological sample or a dilution thereof. In the method of the present invention, quantification can be performed by measuring a target concentration in one or more samples. Biological samples that can be detected in the methods of the invention comprise those as defined above. The sample can be prepared (e.g., suitably diluted or concentrated) and stored in a conventional manner.

Identification and/or quantification of the biomarker can be performed by detecting the biomarker or a fragment thereof, for example, a fragment having a C-terminal truncation or having an N-terminal truncation. Preferably, the length of the fragment is greater than 4 amino acids, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. . Of particular note is that a peptide that is identical or related to a tissue protein terminal peptide is a particularly useful fragment of tissue proteins.

For example, detection and/or quantification can be performed by one or more methods selected from: SELDI (-TOF), MALDI (-TOF), 1-D gel-based analysis, two-dimensional 2-D gel-based analysis, mass spectrometry (MS), reverse phase (RP) LC, size infiltration (gel filtration), ion exchange, affinity, HPLC, UPLC and other LC or LC MS based technologies The group consisting of. Suitable LC MS technologies include ICAT® (Applied Biosystems, CA, USA) or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography (for example, high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin layer chromatography, NMR (nuclear magnetic resonance) spectroscopy can also be used.

In one embodiment, the method comprises comparing the amount of biomarker present in the blood, serum or plasma sample to one or more controls, such as blood, serum or plasma samples obtained from the subject. The amount of biomarker present is compared to the amount of biomarker present in a blood, serum or plasma sample obtained from a normal subject. It should be understood that a "normal" subject refers to a healthy/non-affected subject.

The invention described herein is particularly useful for further screening patients who have experienced fecal occult blood tests (eg, the FIT test). Thus, in one embodiment, the subject has been identified as positive for fecal occult blood. In another embodiment, the subject has been identified as fecal occult blood negative. In one embodiment, the method additionally comprises detecting fecal occult blood in the stool sample from the subject.

In one embodiment, the fecal occult blood test is selected from the group consisting of fecal immunochemistry (FIT, also known as immunochemical fecal occult blood test or iFOBT), fecal guaiac test (gFOBT) or fecal fistula for fecal occult blood test. Porphyrin quantification (eg HemoQuant). In another embodiment, the fecal occult blood test is a fecal immunochemical test. FIT products use specific antibodies to detect globulin, which is currently one of the most commonly used screening tests for colorectal cancer.

It should be understood that the patient is considered to be "positive" when it is determined that there is fecal occult blood in the stool sample that exceeds the (single) threshold critical concentration used in the test. For example, in one embodiment, the fecal occult blood result value has a critical concentration of at least 10 μg Hb/g, such as at least 20, 30, 40 or 50 μg Hb/g, in particular at least about 20 μg Hb/g.

In one embodiment, the method additionally comprises using a clinical parameter to diagnose the subject having a colorectal adenoma. This parameter can be used to interpret the results. Clinical parameters may include any relevant clinical information such as, but not limited to, gender, weight, body mass index (BMI), smoking status, and eating habits. Thus, in one embodiment, the clinical parameters are selected from the group consisting of: age, gender, and body mass index (BMI).

Patient parameters including age and gender are known to be involved in the development of high-risk adenomas. This specification discloses that adding age as a patient parameter can improve the accuracy of these high-risk adenoma tests.

Age is a well-known risk factor for CRC and adenoma development. Adding age as a patient parameter to a logistic regression model (including age, circulating carbonic anhydrase, TIMP-1, and nucleosome concentration) yields a combination of >50% high-risk colorectal adenoma with a specificity of >50%. 90%. This is the highest accuracy data for any blood test and is very advantageous compared to the FIT test and the more expensive Cologuard fecal hemoglobin and DNA assays. The FIT assay is 23% sensitive for high-risk adenomas, and its specificity is 95%, Cologuard fecal hemoglobin and DNA detection have a sensitivity of 42% (which is the highest sensitivity in any high-risk adenoma IVD test) with a specificity of 87% (Imperiale et al. (2014)). Thus, the accuracy of the method of the present invention for the detection of high-risk adenomas exceeds the accuracy of the most accurate in vitro diagnostic tests known to be sensitive and specific.

In one embodiment, the method further comprises examining the subject by colonoscopy, capsule camera, sigmoidoscopy, or MRI to identify the number and location of colorectal adenomas.

In one embodiment, the method further comprises treating the subject diagnosed with the colorectal adenoma by colonoscopy and/or surgery and/or administering a therapeutic agent. Surgical treatment may include laparoscopic surgery. Effective drug treatments for adenomas include non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and COX-2 inhibitor drugs, for example, celebrex and Vioxx. Treatment may also include radiation therapy, such as extracorporeal radiation therapy or intraoperative radiation therapy (ie, administered during surgery).

According to another aspect of the present invention, a method of treating a colorectal adenoma in an animal or a human subject is provided, comprising the steps of:
(i) detecting or measuring the concentration of a biomarker as defined herein in a blood, serum or plasma sample obtained from the subject;
(ii) using the measured biomarker concentration as an indicator of the presence of a colorectal adenoma in the subject;
(iii) surgical treatment (eg, by colonoscopy) or administration of a therapeutic agent to a subject diagnosed as having a colorectal adenoma in step (ii).

According to another embodiment of the present invention, there is provided a method of treating a colorectal adenoma in an individual in need thereof, comprising the steps of: surgical treatment (e.g., by colonoscopy) or administration of a therapeutic agent to a patient, the patient being A biomarker as defined herein having a different concentration in its blood, serum or plasma sample is identified as compared to the biomarker concentration in a blood, serum or plasma sample obtained from a control subject.

According to another aspect of the present invention, a method of treating a colorectal adenoma in a subject is provided, comprising the steps of:
(i) obtaining blood, serum or plasma samples from the subject;
(ii) measuring the concentration of carbonic anhydrase and/or TIMP-1 and/or free nucleosomes in the sample;
(iii) using the concentration of carbonic anhydrase and/or TIMP-1 and/or free nucleosomes measured in the sample as an indicator of the presence of colorectal adenoma in the subject;
(iv) treating the subject by surgery (eg, through a colonoscope) or a therapy or drug to slow or prevent the progression of the colorectal adenoma to colorectal cancer.

Alternatively, the measured free nucleosomes may comprise nucleosomes containing an epigenetic signal structure for use as biomarkers.

According to another aspect of the present invention, a method of treating a colorectal adenoma in a subject is provided, comprising the steps of:
(i) obtaining blood, serum or plasma samples from the subject;
(ii) measuring the concentration of carbonic anhydrase and/or TIMP-1 and/or free nucleosomes in the sample;
(iii) using the concentration of carbonic anhydrase and/or TIMP-1 and/or free nucleosomes measured in the sample as an indicator of the presence of colorectal adenoma in the subject;
(iv) examining the subject by colonoscopy, capsule camera, sigmoidoscopy or MRI to identify the number and location of colorectal adenomas;
(v) removing or treating adenomas.

Alternatively, the measured free nucleosomes may comprise nucleosomes containing an epigenetic signal structure for use as biomarkers.

The method of the invention can be used as an independent method for detecting a subject having a colorectal adenoma, or can be used in conjunction with the detection of CRC to select candidates having either or both of CRC and/or adenoma for Further research is done by colonoscopy or other methods. Similarly, the methods of the invention can be used to detect the presence of colorectal polyps/adenomas in symptomatic patients who exhibit symptoms that may be consistent with the diagnosis of colorectal adenomas.

Described herein are diagnostic kits for diagnosing and monitoring the presence of colorectal adenomas.

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

It should be understood that references to free nucleosomes include free nucleosomes themselves and/or free nucleosomes containing epigenetic signal structures. Thus, in one embodiment, the free nucleosomes comprise an epigenetic signal structure. In another embodiment, the kit includes measuring the concentration of the free nucleosome itself and at least one free nucleosome containing an epigenetic signal structure (as described above).

In one embodiment, the biomarker kit comprises: carbonic anhydrase, TIMP-1, and free nucleosomes. In another embodiment, the biomarker kit comprises: carbonic anhydrase, TIMP-1, and free nucleosomes having an epigenetic signal structure. In yet another embodiment, the biomarker kit comprises: carbonic anhydrase (eg, CA-9 or CA-1), TIMP-1, free nucleosome (self), and free nucleosomes containing epigenetic signal structures (eg post-translational tissue protein modifications such as H3K9Me).

In one embodiment, the biomarker kit consists of carbonic anhydrase, TIMP-1, and free nucleosomes. In another embodiment, the biomarker kit consists of carbonic anhydrase, TIMP-1, and free nucleosomes containing an epigenetic signal structure. In yet another embodiment, the biomarker kit consists of carbonic anhydrase, TIMP-1, free nucleosomes (self), and free nucleosomes containing epigenetic signal structures.

In one embodiment, the biomarker kit is used to diagnose the presence of a colorectal adenoma.

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

Suitably, the kit according to the invention may contain one or more components selected from the group consisting of a ligand binding agent or ligand specific for a biomarker as defined herein, one or more controls, one or more reagents and one or A multi-consumable product; alternatively, there is an indication that the indication is for the use of a kit according to any of the methods defined herein.

It should be understood that the embodiments described herein are applicable to all aspects of the invention. In addition, all publications cited in this specification, including but not limited to patents and patent applications, are hereby incorporated by reference.

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 years who underwent FIT testing for CRC screening. The FIT test used was EIKEN OC-SENSOR. Of the 521 subjects, 67 were tested for FIT-positive at the recommended critical concentration of 20 μg hemoglobin/g feces, and subsequently diagnosed with one or more high-risk adenomas in colonoscopy. The remaining 454 subjects were negative for FIT, did not undergo colonoscopy, and were considered to have no high-risk adenomas. However, since FIT has a lower detection rate (23%) for high-risk adenomas, the FIT-negative population actually contains most cases of high-risk adenomas. Using published data (Imperiale et al. (2014)), we estimated that 454 FIT-negative subjects actually included 28 subjects (6%) with high-risk adenomas. This means that the accuracy estimates for the specificity of high-risk adenomas cited in this paper may be underestimated by up to 6%.

TIMP-1 in the samples was analyzed using a Human TIMP-1 Quantikine ELISA Kit purchased from R&D Systems Inc. according to the manufacturer's instructions. The results showed an increase in circulating TIMP-1 concentrations in patients with high-risk adenomas compared to FIT-negative subjects. Received operating characteristic curve (ROC) analysis yielded 58.5% area under the curve (AUC), and the sensitivity of high-risk adenoma detection was 22.7%, and the specificity was 90% (as mentioned above, this may underestimate specificity) .

Example 2

Carbonic anhydrase 9 in the same serum sample as in the previous Example 1 was analyzed using a CA-9 Quantikine ELISA Kit purchased from R&D Systems Inc. according to the manufacturer's instructions. The results showed a decrease in circulating CA-9 concentration in patients with high-risk adenomas compared to FIT-negative subjects. Conversely, circulating CA-9 concentrations were measured in patients with advanced CRC compared to FIT negative subjects. Received operating characteristic curve (ROC) analysis yielded an area under the curve (AUC) of 69.7%, a high-risk adenoma detection sensitivity of 28.8%, and a specificity of 90% (as described above, this may underestimate specificity).

Example 3

Nucleosomes in serum samples identical to those of Example 1 above were analyzed using an internally developed ELISA. The ELISA used an anti-tissue protein H3 monoclonal antibody immobilized in a plastic microtiter well, and a biotinylated monoclonal antibody that binds to a non-configurable nucleosome epitope on the free tissue protein. Briefly, 10 μl of serum sample and 90 μl of buffer were added to anti-tissue protein H3 coated microtiter wells and incubated for 2.5 hours with gentle shaking at room temperature. The diluted sample is then removed and the microtiter well is rinsed with wash buffer. 100 μl of biotinylated monoclonal antibody diluted in buffer was added and incubated for 1.5 hours. The diluted biotinylated antibody was removed and the microtiter well was rinsed again with wash buffer. 100 μl of horseradish peroxidase-labeled streptavidin diluted in buffer was added and incubated for 0.5 hours. The diluted horseradish peroxidase-labeled streptavidin was removed and the microtiter well was rinsed again with wash buffer. 200 μl of an enzyme substrate solution (2,2'-diazobis[3-ethylbenzothiazoline-6-sulfonic acid] diammonium salt) was added and incubated for 20 minutes. The optical density (OD) of the wells was measured using a standard microtiter plate measuring instrument and the relative nucleosome concentration was interpolated from the calibration curve. The results showed that circulating nucleosome concentrations were elevated in patients with high-risk adenomas compared with FIT-negative subjects. Received operating characteristic curve (ROC) analysis yielded 67.5% area under the curve (AUC), and the sensitivity of high-risk adenoma detection was 16.7%, and the specificity was 90% (as mentioned above, this may underestimate specificity) .

Example 4

The nucleosome containing the tissue protein H3 post-translational modification in the same serum sample as in the previous Example 1 was analyzed by an internally developed ELISA, and the tissue protein H3 post-translational modification was transmitted through the ninth position of the amino acid residue. Basic (H3K9Me3) modification. The ELISA used an anti-H3K9Me3 monoclonal antibody immobilized in a plastic microtiter well, and a biotinylated monoclonal antibody that binds to a non-configurable nucleosome epitope on the free tissue protein. Briefly, 20 μl of serum sample and 80 μl of buffer were added to anti-tissue protein H3K9Me3 coated microtiter wells and incubated for 2 hours at room temperature with gentle shaking. The diluted sample is then removed and the microtiter well is rinsed with wash buffer. 100 μl of biotinylated monoclonal antibody diluted in buffer was added and incubated for 1 hour. The diluted biotinylated antibody was removed and the microtiter well was rinsed again with wash buffer. 100 μl of horseradish peroxidase-labeled streptavidin diluted in buffer was added and incubated for 0.5 hours. The diluted horseradish peroxidase-labeled streptavidin was removed and the microtiter well was rinsed again with wash buffer. 100 μl of the enzyme substance solution (3,3',5,5'-tetramethylbenzidine) was added and incubated for 10 minutes. 50 μl of stop solution (1 M HCl) was added and the optical density (OD) of the wells was measured using a standard microtiter plate measuring instrument. The results showed that circulating concentrations of nucleosomes containing post-translationally modified histoprotein H3 were elevated in patients with high-risk adenomas compared with FIT-negative subjects. Received operating characteristic curve (ROC) analysis yielded 62.9% of the area under the curve (AUC), and the sensitivity of the high-risk adenoma test was 16.7%, and the specificity was 90% (as mentioned above, this may underestimate the specificity) .

Example 5

The serum sample group of carbonic anhydrase 1 was analyzed using a kit purchased from Nordic BioSite AB according to the manufacturer's instructions for use, including a FIT-negative subject and a FIT-positive test diagnosed as having a high-risk adenoma. By. The expected results show that circulating CA-1 levels are altered in patients with high-risk adenomas compared to FIT-negative subjects.

Example 6

Using the regression analysis, the results from the four trials described in Examples 1, 2, 3 and 4 above were combined into a kit trial for high-risk adenomas. Received operating characteristic curve (ROC) analysis yielded 76.1% of the area under the curve (AUC), and the sensitivity of the high-risk adenoma test was 43.3%, and the specificity was 90% (as mentioned above, this may underestimate the specificity) .

Example 7

Other patient parameters can be added to the test results to increase the accuracy of the test. We added the patient's age to the results of the four trials described in Examples 1, 2, 3, and 4 above, and used regression analysis to combine all 5 parameters into a kit trial for high-risk adenomas. Received operating characteristic curve (ROC) analysis yielded 76.5% area under the curve (AUC), and the sensitivity of high-risk adenoma detection was 50.7%, and the specificity was 90% (as mentioned above, this may underestimate 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 (21)

A use of carbonic anhydrase, tissue inhibitor of metalloproteinase-1 (TIMP-1) and/or free nucleosomes as a biomarker in the diagnosis of colorectal adenomas in a blood, serum or plasma sample. The use according to claim 1, wherein the carbonic anhydrase is carbonic anhydrase 9 (CA-9). The use of claim 1 or 2, wherein the free nucleosome comprises an epigenetic signal structure. The use according to claim 3, wherein the epigenetic signal structure is selected from the group consisting of: a post-translational tissue protein modification, a tissue protein variant, a specific nucleotide, and a protein adduct. Group of. The use according to claim 4, wherein the post-translational tissue protein modification system is H3K9Me3. The use according to any one of claims 1 to 5, wherein carbonic anhydrase, tissue inhibitor of metalloproteinase-1 (TIMP-1) and free nucleosome are used as a biomarker kit. Diagnosis of the presence of colorectal adenoma. The use according to any one of claims 1 to 6, wherein the colorectal adenoma is a high risk adenoma. A method of diagnosing or detecting a colorectal adenoma in an animal or a human subject, comprising the steps of: (i) detecting or measuring the concentration of the biomarker according to any one of claims 1 to 7 in the blood, serum or plasma sample obtained from the subject; (ii) Using the measured biomarker concentration as an indicator of the presence of colorectal adenoma in the subject. A method of determining the prognosis of an animal or a human subject having a colorectal adenoma, comprising the steps of: (i) detecting or measuring the concentration of the biomarker according to any one of claims 1 to 7 in the blood, serum or plasma sample obtained from the subject; (ii) using the measured biomarker concentration as an indicator of the prognosis of the colorectal adenoma in the subject. A method of monitoring the efficacy of treatment in an animal, or a human subject, having, suspected of having or prone to a colorectal adenoma, comprising the steps of: (i) detecting or measuring the concentration of the biomarker according to any one of claims 1 to 7 in the blood, serum or plasma sample obtained from the subject; (ii) using the measured biomarker concentration as compared to a biological sample previously obtained from the subject as an indicator of efficacy of the treatment. The method of any one of claims 8 to 10, wherein the detecting or measuring comprises immunoassay, immunochemistry, mass spectrometry, chromatography, chromatin immunoprecipitation or biosensor method. The method of any one of claims 8 to 11, comprising comparing the amount of the biomarker present in the blood, serum or plasma sample to one or more control groups, for example from the subject The amount of biomarker present in the obtained blood, serum or plasma sample is compared to the amount of biomarker present in the blood, serum or plasma sample obtained from a normal subject. The method of any one of claims 8 to 12, wherein the subject has been identified as positive for fecal occult blood. The method of any one of claims 8 to 13 further comprising diagnosing the subject having a colorectal adenoma using a clinical parameter. The method of claim 14, wherein the clinical parameter is selected from the group consisting of: age, gender, and body mass index (BMI). A method of treating a colorectal adenoma in an animal or a human subject, comprising the steps of: (i) detecting or measuring the concentration of the biomarker according to any one of claims 1 to 7 in the blood, serum or plasma sample obtained from the subject; (ii) using the measured biomarker concentration as an indicator of the presence of a colorectal adenoma in the subject; (iii) Surgical treatment or administration of a therapeutic agent to a subject diagnosed as having a colorectal adenoma in step (ii). A method for treating a colorectal adenoma in an individual in need thereof, comprising the steps of: surgically treating or administering a therapeutic agent to a patient, the patient being identified as having a different concentration in a blood, serum or plasma sample, such as a patent application The biomarkers of clauses 1 to 7 are compared to biomarker concentrations in blood, serum or plasma samples obtained from a control subject. A biomarker kit comprising two or more biomarkers selected from the group consisting of: carbonic anhydrase, TIMP-1, and free nucleosomes. The biomarker kit of claim 18, wherein the free nucleosome comprises an epigenetic signal structure. The biomarker kit of claim 18 or 19 is for use in diagnosing the presence of a colorectal adenoma. A kit comprising a kit capable of detecting and/or quantifying one or more binding agents of a biomarker according to any one of claims 1 to 7 for use in diagnosing a colorectal adenoma.