use of a dna base associated with a cell-free nucleosome as a biomarker, methods for detecting a dna

专利摘要:
METHOD FOR DETECTING NUCLEOSOMES CONTAINING NUCLEOTIDES The invention relates to a method for detecting and measuring the presence of mononucleosomes and oligonucleosomes and nucleosomes containing certain nucleotides and the use of such measurements for the detection and diagnosis of diseases. The invention also relates to a method of identifying nucleotide-associated nucleotide biomarkers for the detection and diagnosis of diseases and to the biomarkers identified by said method
公开号:BR112014005087B1
申请号:R112014005087-2
申请日:2012-08-31
公开日:2020-12-22
发明作者:Jacob Vicent Micallef
申请人:Singapore Volition Pte Limited；
IPC主号:

专利说明:

Field of the Invention
[001] The invention relates to a method for detecting and measuring the presence of mononucleosomes and oligonucleosomes and nucleosomes containing certain nucleotides and the use of such measurements for the detection and diagnosis of diseases. The invention also relates to a method of identifying nucleotide biomarkers associated with nucleosomes for the detection and diagnosis of diseases and the biomarkers identified by said method. Background of the Invention
[002] The human body is composed of several hundred types of cells. All of these cell types contain the same genome, but have different phenotypes and different functions in the body. This phenotypic diversity is due to the differential expression of the genome in different types of cells. The control of differential expression of genes is not fully understood, but the basic mechanisms include regulation of genes by a series of interconnected epigenetic signals associated with the gene, including control of chromatin packaging, such as euchromatin or heterochromatin, control of the positioning of nucleosomes and sites accessible to nucleases, methylation, hydroxymethylation and other DNA modifications and variation in the structure of the nucleosomes around which DNA is involved.
[003] The nucleosome is the basic unit of the chromatin structure and consists of a protein complex of eight highly conserved nuclear histones (which comprise a pair of each of the histones H2A, H2B, H3 and H4). Around this complex, approximately 146 base pairs of DNA are involved. Another histone, H1 or H5, acts as a binding agent and is involved in the chromatin compaction. DNA is wrapped around successive nucleosomes in a structure, often referred to as "beads in a necklace" and this constitutes the basic open structure or eukromatin. In the compacted or heterochromatin structure, this rope is coiled and super coiled in a closed and complex structure (Herranz and Esteller, 2007).
[004] The structure of nucleosomes can vary through Post-Transcriptional Modification (MPT) of histone proteins and by the inclusion of histone variant proteins. MPT of histone proteins normally occurs on the tails of nuclear 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. It is known that changes in histones are involved in the epigenetic regulation of gene expression (Her- ranz and Esteller, 2007). The structure of the nucleosome can also vary according to the inclusion of isoforms or alternative histone variants, which are different gene or splicing products, which have different amino acid sequences. Histone variants can be classified into a number of families that are subdivided into individual types. The nucleotide sequences of a large number of histone variants are known and accessible to the public, for example, through the National Human Genome Research Institute NHGRI Histone DataBase (Marino-Ramírez, L., Levine, KM, Morales, M., Zhang, S., Moreland, RT, Baxevanis, AD, and Landsman, D. The Histone Database: an integrated re- source for histones and histone fold-containing proteins. Database Vol. 2011. (Submitted) and http: / /genome.nhgri.nih.gov/histones/complete.shtml), GenBank Data Bank (NIH genetic sequence), the EMBL Nucleotide Sequence Database and the DNA Data Bank of Japan (DDBJ).
[005] The renewal of normal cells in adult humans involves the creation, through cell division, of about 1011 cells per day and the death of a similar number, mainly by apoptosis. During the apoptosis process, chromatin is divided into mononucleosomes and oligonucleosomes that are released from cells. Under normal conditions, the levels of circulating nucleosomes found in healthy individuals are considered low. Elevated levels are found in individuals with a variety of conditions, including many types of cancer, autoimmune diseases, inflammatory diseases, stroke and myocardial infarction (Holdenreider & Stieber, 2009).
[006] Mononucleosomes and oligonucleosomes can be detected by ELISA (Enzyme-Linked ImmunoSorbant Assay) and by several methods already reported (Salgame et al .., 1997; Holdenrieder et al .., 2001; van Nieuwenhuijze et al. , 2003). These assays typically use an antihistone antibody (for example, anti-H2B, anti-H3 or anti-H1, H2A, H2B, H3 and H4), as the capture antibody and an anti-DNA antibody or antibody to the H2A complex -H2B-DNA (anti-H2A-H2B-DNA), as detection antibody. Using these assays, researchers / technicians in the field report that the level of nucleosomes in the serum is higher (up to an order of magnitude) than in plasma samples taken from the same patients. This is also true for serum and plasma DNA measurements performed by PCR (Holdenrieder et al., 2005). The reason for this is not known, but the authors speculate that this may be due to the additional release of DNA during the clotting process. However, it has been found that the results of ELISA assays for nucleosomes of the current state of the art are not congruent with each other. Furthermore, although it is reported that most of the circulating DNA in serum or plasma is in the form of mononucleosomes and oligonucleosomes (Holdenrieder et al., 2001), there is no agreement between the levels of nucleosomes and DNA measured in the serum or plasma. It was found that the correlation coefficient between ELISA results for circulating levels of free nucleosomes and circulating levels of DNA, as measured by real-time PCR (Polymerase Chain Reaction), was r = 0.531 in serum and r = 0.350 in plasma (Holdenrieder et al., 2005).
[007] Current ELISA methods for nucleosomes are used in cell culture, mainly as a method to detect apoptosis (Salgame et al., 1997; Holdenrieder et al., 2001; van Nieuwenhuijze et al., 2003) , and are also used for the measurement of free circulating nucleosomes in serum and plasma (Holdenrieder et al., 2001). The levels of free nucleosomes in serum and plasma released into the circulation by cells in the process of death were measured by ELISA methods in studies with a number of different types of cancer in order to evaluate its use as a potential biomarker (Holdenrieder et al., 2001). It was found that the average circulating levels of nucleosomes was high in most, but not all, of the studied cancers. The highest circulating levels of nucleosomes were observed in individuals with lung cancer. The lowest levels were seen in prostate cancer, which were within the normal range for healthy individuals. However, it was observed that patients with malignant tumors have serum nucleosome concentrations that varied considerably, and it was found that some patients with advanced cancer had low levels of circulating nucleosomes, being within the measured range. in healthy individuals (Holdenrieder et al., 2001). Due to the high levels of nucleosomes found above, as well as in a variety of non-cancerous causes, circulating levels of nucleosomes are not used clinically as a cancer biomarker (Holdenrieder and Stieber, 2009). Surprisingly, it has been shown that many individuals with cancer, whose circulating nucleosome levels (measured by current state ELISA methods for nucleosomes) are low or undetectable, in fact, have high levels of free circulating nucleosomes. It has been designed and demonstrated that the new ELISA methods for nucleosomes detect nucleosomes that were not detected by current state of the art ELISA methods.
[008] ELISA methods for the detection of MPTs in histones are also known in the art. ELISA methods for detecting MPT in free histone proteins (not bound to other histones and to DNA in a nucleosomal complex) are used to detect MPTs in histones extracted from cell layers, usually through extraction with acid. An immunoassay for the detection of MPTs in free circulating nucleosomes has been proposed (Bawden et al., 2005). A method for ELISA detection of MPTs in histones in purified nucleosomes adhered directly to microtiter wells has recently been reported (Dai et al., 2011). In this method, the nucleosomes, obtained by digesting the chromatin extracts from cultured cells, are applied directly to the microtiter wells, and reacted with anti-MPT antibodies. It will be clear to those skilled in the art that this method requires relatively pure nucleosome samples and that direct measurement of histone MPTs in complex biological media, such as blood, plasma or serum, is not adequate.
[009] A modified method of chromatin immunoprecipitation (ChIP) for the detection of an MPT in histone (H3K9Me, histone H3 monomethylated in the K9 lysine residue), in free nucleosomes associated with a particular DNA sequence, was plasma. It was observed that the methylation level of specific histone sequences was independent of the concentration of circulating nucleosomes (Deligezer et al., 2008).
[0010] Histone variants (also known as histone isoforms) are known to be epigenetic regulators of gene expression (Herranz and Esteller, 2007). Histone variants have been studied in vivo and in vitro using a variety of techniques, including studies to reduce the expression (knock-down) of the gene encoding a particular variant (for example, knock-down using RNAi), chromatin immunoprecipitation, stable amino acid isotope labeling and quantitative, proteomic, immunohistochemistry and Western Blotting mass spectrometry (Whittle et al., 2008; Boulard et al., 2010; Sporn et al., 2009; Kapoor et al., 2010 ; Zee et al., 2010; Hua et al., 2008).
[0011] Immunohistochemistry studies of the expression of the histone variant in tissue samples removed by surgery or by biopsy of individuals diagnosed with lung cancer, breast cancer and melanoma have been described. These immunohistochemistry studies report that the marking of histone variants macroH2A (mH2A) and H2AZ in samples of resected cancer tissue may have a prognostic application in these cancers (Sporn et al., 2009, Hua et al., 2008, Kapoor et al. , 2010). A disadvantage of immunohistochemistry methods for clinical use is that the tissue sample collection is invasive, involving surgery or biopsy. Another disadvantage of immunohistochemistry methods is that they are unsuitable for early diagnosis or diagnostic screening since the reasonable expectation of the disease must generally already exist before a biopsy or tissue resection is performed. Minimally invasive blood ELISA tests are suitable for a wide range of applications and would overcome these disadvantages, being preferable for the patient, as well as being faster, less costly and of higher yield for the healthcare provider.
However, variants of free histones in free nucleosomes have not been measured in the blood or by other means. No study on the presence or absence of histone variants in free nucleosomes in the blood has been described. Currently, there are no methods for the detection or measurement of histone variant in free intact nucleosomes, nor has any measure been suggested or contemplated.
[0013] In addition to the epigenetic signaling mediated by the position of the nucleosome and the structure of the nucleosome (both in terms of the variant of the constituent histone protein and MPT structures), the control of gene expression in cells is also mediated through changes in DNA nucleotides, including the cytosine methylation status of DNA. The fact that DNA can be methylated at position 5 of the cytosine nucleotide to form 5-methylcytosine has been known in the state of the art for some time. It has been observed that methylated DNA in the form of 5-methylcytosine occurs at positions in the DNA sequence where a cytosine nucleotide occurs next to a guanine nucleotide. These positions are referred to by the abbreviation “CpG”. More than 70% of CpG positions are reported to be methylated in vertebrates (Pennings et al., 2005). The regions of the genome that contain a high proportion of CpG sites are often referred to as “CpG islands”, and approximately 60% of the sequences of human gene promoters are associated with such CpG islands (Rodriguez-Paredes and Estel - read, 2011) The active genes of these CpG islands are generally hypomethylated. Methylation of the promoter sequences of the genes is associated with inactivation of the stable gene. DNA methylation also commonly occurs in repetitive elements, including repetitive elements Alu and long elements of intertwined nucleotides (Herranz and Estellar, 2007; Allen et al., 2004).
[0014] The involvement of DNA methylation in cancer was reported in early 1983 (Feinberg and Vogelstein, 1983). The patterns of DNA methylation observed in cancer cells differ from healthy cells. It was found that repetitive elements, especially around the pericentromeric areas, are hypomethylated in cancer in relation to healthy cells, but it has been observed that the promoters of specific genes are hypermethylated in cancer. The balance of these two effects results in global DNA hypomethylation in cancer cells (Rodiguez-Paredes; Esteller, 2007).
[0015] Hypermethylation of certain specific genes can be used as a diagnostic biomarker for cancer. For example, a method described for the detection of the hypermethylation of the Septin 9 gene by the amplification of DNA by PCR, extracted from plasma, detected 72% of colon cancers with a false positive rate of 10% (Grutzmann et al ., 2008). The DNA methylation status of specific genes or loci is normally detected by selective cytosine bisulfite deamination, except for 5-methylcytosine, uracil, which leads to a change in the primary DNA sequence, which can be detected by sequencing or by other means (Allen et al., 2004).
[0016] Global DNA hypomethylation is a hallmark of cancer cells (Estellar 2007 and Hervouet et al., 2010). Global DNA methylation can be studied in cells using immunohistochemistry (IHC) techniques. Alternatively, DNA is extracted from the cells for analysis. A number of methods have been described for detecting global methylation in DNA extracted from cells or other media, such as restriction enzyme digestion and nearest-neighbor analysis, fluorescent assays using chloroacetaldehyde, reverse determination by methylation of all CpG sites using DNA methyltransferase, together with tritium-labeled S-adenosyl-methionine to calculate the amount of unmethylated CpG and the digestion of DNA into single nucleotides by analyzing high performance liquid chromatography, thin layer chromatography, or liquid chromatography, followed by mass spectroscopy. The disadvantages of these methods are that the work is intense and / or requires large amounts of DNA extracted from good quality (Allen et al. 2004). PCR-based methods that involve bisulfite deamination, surpass the need for large amounts of DNA, however specific regions of the genome should be amplified, usually repetitive sequences, as an indication of the total 5-methylcytosine content of the genome (Allen et al. 2004). These methods for measuring global DNA methylation have been used to study DNA extracted from a variety of cells and tissues. Some experts have studied the DNA extracted from white cells in whole blood, since it is easier to obtain it in a minimally invasive way (Moore et al., 2008; Ting Hsiung et al., 2007; Mansour et al. , 2010). Liquid chromatography with mass spectrometry is considered the gold standard for measuring global DNA methylation, but it is expensive, and DNA must be digested at the level of a single nucleotide before analysis (Vasser et al., 2009) .
[0017] Recent techniques for estimating global DNA methylation include ultra high-pressure liquid chromatography with mass spectrometry of hydrolyzed DNA extracted from tissue (Zhang et al., 2011) and a specific automatic sequencing method for methylation (MSDS) (Ogoshi et al. 2011). A classic competitive immunoassay for global DNA methylation (as well as a similar assay for global 5-hydroxymethylcytosine methylation) has been described. In this method the DNA extracted from cells or tissues is added to a microtiter well coated with a 5-methylated cytidine conjugate, an anti-5-methylcytidine antibody is added and the binding antibody distribution between the conjugate 5 - methylcitidine adhered and the methylated DNA in the extracted sample is compared with that of known standards in order to estimate the overall DNA methylation level, present in the cell sample (Biolabs, 2011). In another immunoassay method, DNA extracted from tissues or from plasma or serum samples is adhered to a microtiter well and methylated DNA is detected using an anti-5-methylcytosine antibody (Vasser et al., 2009; Epigentek, 2009). A disadvantage of these methods is that they require DNA extraction, which involves denaturation and removal of the entire nucleosome and chromatin structure from DNA. These, therefore, cannot measure nucleotides linked to nucleosomes and are not suitable, for example, for direct measurement of global DNA methylation in biological fluids, such as tissue, blood, plasma or serum lysate, without a passode. DNA extraction.
[0018] The 5-hydroxymethyl modification of cytosine bases in DNA was also observed. The role of 5-hydroxymethylation is not yet well understood, but it seems to be involved in the regulation of genes (Stroud et al., 2011).
[0019] Current methods for detecting global DNA methylation involve DNA extraction or purification, but they are not suitable methods for rapid, high-yield, low-cost and minimally invasive diagnostics. Likewise, DNA analysis for other modified or unusual bases (eg, uracil, inosine, xanthine, hypoxanthine) can only be performed by analyzing substantially pure or extracted DNA. Such an analysis cannot be performed directly on complex biological media, such as tissue lysate, blood, plasma or serum.
[0020] Free nucleosomes containing 5-methylcytosine, or any other specific nucleotides or modified nucleotides, have not been measured in the blood or by any other means. No study has been described regarding the presence or absence of free nucleosomes containing specific nucleotides in the blood. Assays for free nucleosomes containing specific nucleotides have not been suggested or contemplated. Currently, there are no methods for the detection or measurement of nucleotides associated with free nucleosomes.
[0021] Now simple immunoassay methods will be described for direct estimation of nucleotides associated with nucleosomes including, for example, 5-methylcytosine and 5-hydroxymethylcytosine, in biological samples without extraction. Surprisingly, it has been shown that nucleotides associated with nucleosomes can be detected in blood samples, in which nucleosomes are not detected by state-of-the-art ELISA methods. Summary of the Invention
[0022] According to the first aspect of the invention, a free nucleosome comprising a base of DNA, nucleotides or nucleosides is provided, for use as a biomarker for the diagnosis of cancer, cardiomyopathy, systemic lupus erythematosus, colitis, chronic obstructive pulmonary disease, Crohn's disease and rheumatoid arthritis.
[0023] According to the second aspect of the invention, a method is provided for detecting the presence of a nucleosome, containing a base of DNA, nucleotides or nucleosides in a sample, comprising the steps of: (i) placing the sample in contact with a binding agent that binds to the DNA base, nucleotides or nucleosides; (ii) detecting or quantifying the binding of said DNA, nucleotide or nucleoside-based binding agent in the sample; and (iii) using the presence or degree of such a bond as a measure of the presence of nucleosomes containing the base of DNA, nucleotides or nucleosides in the sample.
[0024] In accordance with the third aspect of the invention, a method is provided for detecting the presence of a nucleosome, containing a base of DNA, nucleotides or nucleosides in a sample, comprising the steps of: (i) bringing the sample into contact with a first binding agent that binds to nucleosomes; (ii) placing the nucleosomes or the sample in contact with a second binding agent that binds to the base of DNA, nucleotides or nucleosides; (iii) detecting or quantifying the binding of said second DNA, nucleotide or nucleoside-based binding agent in the sample; and (iv) using the presence or degree of such a bond as a measure of the presence of nucleosomes containing the base of DNA, nucleotides or nucleosides in the sample.
[0025] In accordance with the fourth aspect of the invention, a method is provided for detecting the presence of a nucleosome, containing a base of DNA, nucleotides or nucleosides in a sample, comprising the steps of: (i) bringing the sample into contact with a first binding agent that binds to the base of DNA, nucleotides or nucleosides; (ii) bringing the nucleosomes or the sample into contact with a second binding agent that binds to the nucleosomes; (iii) detecting or quantifying the binding of said second nucleosome binding agent in the sample; and (iv) using the presence or degree of such a bond as a measure of the presence of nucleosomes containing the base of DNA, nucleotides or nucleosides in the sample.
[0026] According to another aspect of the invention, a method is provided for detecting the presence of a nucleosome, containing a base of DNA, nucleotides or nucleoside in a cell, comprising the steps of: (i) isolating the chromatin from a cell; (ii) digest, sonicate, or otherwise break chromatin to form mononucleosomes and / or oligonucleosomes; and (iii) detecting or measuring the presence of the base of DNA, nucleotides or nucleosides in said nucleosomes, according to the method of the invention.
[0027] According to another aspect of the invention, a method is provided for detecting or diagnosing the state of the disease in an animal or a human being, comprising the steps of: (i) detecting or measuring nucleosomes, containing a base of DNA, nucleotides or nucleosides in an individual's body fluid, and (ii) using the detected level of DNA base, nucleotide or nucleoside associated with the nucleosomes to identify the individual's disease state.
[0028] In accordance with another aspect of the invention, a method is provided to assess an animal or human being for adaptation to medical treatment, comprising the steps of: (i) detecting or measuring nucleosomes, containing a base of DNA, nucleotides or nucleosides in an individual's body fluid, and (ii) using the base level of DNA, nucleotide or nucleoside associated with the nucleosomes as a parameter for selecting an appropriate treatment for the individual.
[0029] In accordance with another aspect of the invention, a method is provided for monitoring the treatment of an animal or a human being, comprising the steps of: (i) detecting or measuring nucleosomes containing a DNA base , nucleotides or nucleosides in an individual's body fluid; (ii) repeat the detection or measurement of nucleosomes containing a base of DNA, nucleotides or nucleosides in an individual's body fluid, one or more times, and (iii) use any changes in the detected level of DNA base, nucleotide or nucleoside associated with nucleosomes as a parameter for any change in the individual's condition.
[0030] According to another aspect of the invention, a method is provided to identify a base biomarker of DNA, nucleotides or nucleosides for detecting or diagnosing the disease state of an animal or a human, comprising the steps of: (i) detecting or measuring nucleosomes containing the base of DNA, nucleotides or nucleosides in an individual's body fluid; (ii) detecting or measuring nucleosomes containing the base of DNA, nucleotides or nucleosides in a body fluid of a healthy individual or a control subject; and (iii) use the difference between the levels detected in sick and control individuals to identify whether a DNA, nucleotide or nucleoside base is useful as a biomarker for disease status.
[0031] According to another aspect of the invention, a biomarker identified by said method of the invention is provided.
[0032] In accordance with another aspect of the invention, a kit is provided for the detection of a base of DNA, nucleotides or nucleosides associated with the nucleosome, which comprises a ligand ("ligand") or ligand ("binder") specific for the DNA base, nucleotides or nucleosides or component part of them, or a structural mimic / shape of the DNA base, nucleotides or nucleosides or component part thereof, together with the instructions for use of the kit. Brief Description of the Figures
[0033] Figure 1. Dose-response curve by ELISA for the detection of DNA methylated by 5-methylcytosine in free nucleosomes in digested cross-linked chromatin, extracted from MCF-7 cells diluted in bovine serum.
[0034] Figure 2. Dose-response curve by ELISA for the detection of DNA methylated by 5-hydroxymethylcytosine in free nucleosomes in digested reticulated chromatin, extracted from A375 cells diluted in bovine serum.
[0035] Figure 3. Levels of nucleosomes detected in serum and plasma samples with EDTA collected from 20 healthy volunteers, using the current state of the art ELISA methods for nucleosomes.
[0036] Figure 4. Levels of histone variant mH2A1.1 associated with free nucleosomes detected in serum and plasma samples with EDTA collected from 20 healthy volunteers.
[0037] Figure 5. Levels of the mH2A2 histone variant associated with the free nucleosomes detected in serum and plasma samples with EDTA collected from 20 healthy volunteers.
[0038] Figure 6. Levels of histone variant H2AZ associated with free nucleosomes detected in serum and plasma samples with EDTA collected from 20 healthy volunteers.
[0039] Figure 7. Levels of histone modification associated with free P-H2AX (Ser139) nucleosomes detected in serum and plasma samples with EDTA collected from 20 healthy volunteers.
[0040] Figure 8. Levels of DNA methylated by 5-methylcytosine associated with free nucleosomes detected in serum and plasma samples with EDTA collected from 20 healthy volunteers, using the ELISA test of the invention.
[0041] Figure 9. Levels of DNA methylated by 5-hydroxymethylcytosine associated with free nucleosomes detected in serum and plasma samples with EDTA collected from 20 healthy volunteers, using the ELISA test of the invention.
[0042] Figure 10. Levels of nucleotides and types of histones associated with free nucleosomes detected in EDTA plasma samples collected from 3 individuals with colon cancer.
[0043] Figure 11. Nucleotide levels and types of histones associated with free nucleosomes detected in EDTA plasma samples collected from 13 individuals with lung cancer.
[0044] Figure 12. Levels of nucleotides and types of histones associated with free nucleosomes detected in plasma samples with EDTA collected from 2 individuals with pancreatic cancer.
[0045] Figure 13. Levels of nucleotides and types of histones associated with free nucleosomes detected in a plasma sample with EDTA collected from 1 individual with oral cancer.
[0046] Figure 14. Levels of nucleotides and types of histones associated with free nucleosomes detected in plasma samples with EDTA collected from 4 different types of cancer normalized as a proportion of the levels of DNA methylated by 5-methylcytosine associated with the nucleosomes detected by the ELISA data of the invention. The normalized levels for a sample containing nucleosomes from healthy volunteers produced by the method of * Holdenrieder et al. 2001 is shown for comparison purposes (mH2A2 and 5-hydroxymethylcytosine were not measured in this example).
[0047] Figure 15. Levels of 5-methylcytosine (5mC), mH2A1.1, H2AZ and P-H2AX (Ser139) associated with free nucleosomes detected in plasma samples with EDTA, plasma with citrate, plasma with heparin collected from volunteers healthy animals using the ELISA method of the invention.
[0048] Figure 16. Levels of 5-methylcytosine associated with free nucleosomes detected in serum samples collected from 3 healthy volunteers and 10 patients with colon cancer detected using the ELISA method of the invention.
[0049] Figure 17. Levels of 5-methylcytosine associated with free nucleosomes detected in EDTA plasma samples collected from 13 healthy volunteers and 55 cancer patients. The cutoff points shown were defined as the mean value of healthy samples plus one or two standard deviations from the mean.
[0050] Figure 18. Levels of 5-methylcytosine associated with free nucleosomes detected in EDTA plasma samples collected from 10 healthy volunteers and 61 cancer patients. The cutoff point shown was defined as the mean value of healthy samples plus two standard deviations from the mean.
[0051] Figure 19. Levels of 5-methylcytosine associated with free nucleosomes detected in EDTA plasma samples collected from patients with colon and lung cancer who have increased tumor size, stadium and nodal disease development .
[0052] Figure 20. Average detected levels of nucleotides and types of histones associated with free nucleosomes, using the ELISA methods of the invention, in EDTA plasma samples collected from 10 different types of cancer normalized by the proportion of DNA levels methylated by 5-methylcytosine (5mC) associated with nucleosomes and expressed in relation to the average proportions found in 11 healthy individuals.
[0053] Figure 21. Average detected levels of nucleotides and types of histones associated with free nucleosomes, using the ELISA methods of the invention, in EDTA plasma samples collected from 2 patients with cardiopathy, 10 patients with systemic lupus erythematosus ( lupus), 12 patients with ulcerative colitis, 10 patients with chronic obstructive pulmonary disease (COPD), 8 patients with Crohn's disease and 10 patients with rheumatoid arthritis (RA), were normalized by the proportion of DNA levels methylated by 5-methylcytosine ( 5mC) associated with nucleosomes and expressed in relation to the average proportions found in 11 healthy individuals. Detailed Description of the Invention
[0054] According to the first aspect of the invention, a free nucleosome is provided comprising a base of DNA, nucleotides or nucleosides, for use as a biomarker for the diagnosis of cancer, cardiomyopathy, systemic lupus erythematosus, colitis, chronic obstructive pulmonary disease, Crohn's disease and rheumatoid arthritis.
[0055] In one embodiment, the nucleosome is a mononucleosome or oligonucleosome.
[0056] According to a particular aspect of the invention, which can be mentioned, the use of a DNA, nucleotide or nucleoside base is provided as a biomarker for the diagnosis of cancer.
[0057] In one embodiment, cancer is a cancer of the bladder, breast, colon, cervix, esophagus, kidney, large intestine, lung, oral, ovaries, pancreas, prostate, rectum, skin or stomach. In a particular embodiment, which can be mentioned, cancer is a cancer of the colon, lung, oral cavity or pancreas.
[0058] ELISA tests were developed for the detection and measurement of nucleosomes, containing the DNA bases 5-methylcytosine and 5-hydroxymethylcytosine. An antihistone antibody was used as the capture antibody for these assays, in combination with an appropriate specific anti-nucleotide antibody. The tests were carried out in order to demonstrate that nucleosomes containing specific nucleotides can be measured in blood samples collected from cancer patients and are discriminant in their use as non-invasive or minimally invasive biomarkers. The levels of DNA with 5-methylcytosine associated with nucleosomes, in relation to the levels of other nucleosome epitopes, detected in serum and plasma samples, collected from sick individuals, differed from those detected in samples from healthy individuals. In addition, the pattern of nucleotide levels detected in the nucleosomes in samples collected from individuals with different diseases differed in such a way that a differential diagnosis of the disease was possible, particularly when the nucleotide patterns associated with the nucleosomes were examined in combination with the standards determined for nucleosomes containing different histone variants and histone modifications. It is clear to technicians on the subject that the inclusion of tests for nucleosomes containing different or additional nucleotides could improve discrimination in the differential diagnosis using these standards.
[0059] To investigate the levels of nucleosomes found in healthy individuals, using the methods of the current state of the art, the nucleosomes were measured in serum and plasma samples, collected from 20 healthy individuals. Both methods of the current state of the art produced higher signals in serum samples collected from healthy individuals than in plasma samples. The results are shown in Figure 3. This is consistent with published data that nucleosome levels are higher in serum than in plasma (* Holdenrieder et al., 2001).
[0060] To investigate the levels of nucleosomes found in healthy individuals, using the methods of the invention, nucleosomes containing the modified 5-methylcytosine nucleotide were measured in the serum of 20 healthy individuals and in healthy bovine serum. Serum results were low or undetectable for all 20 healthy individuals. The nucleosomes containing the modified 5-methylcytosine nucleotide were measured in EDTA plasma samples collected from 20 healthy individuals, and, surprisingly, higher signals were observed. Elevated levels of free nucleosomes containing the modified 5-methylcytosine nucleotide sequences were detected by the methods of the present invention in healthy human plasma with EDTA, but lower levels were detected in healthy human serum, as shown in Figure 8. Figures 4 to 9 show that similar results were obtained for other nucleosome structures. This discovery is unexpected and different for both published results (* Holdenrieder et al., 2001), and the results found for the ELISA methods for nucleosomes of the current state of the art. Thus, surprisingly, the methods of the invention produce results opposite to the methods of the current state of the art in relation to the relative levels of nucleosomes that occur in serum and plasma samples with EDTA.
[0061] It has been investigated whether nucleosome structures are detectable in all common types of plasma that can be collected. Elevated levels of 5-methylcytosine associated with free nucleosomes were found to be detectable by the method of the invention in plasma with EDTA and, to a lesser extent, in plasma with citrate collected from healthy individuals, but levels of 5-methylcytosine associated with nucleosomes were low or undetectable in relation to the background signals of the buffer and horse serum in most heparin plasma samples (3 of 5) collected from healthy individuals. The results are shown in Figure 15. To summarize, free nucleosomes are found in relatively high concentrations in all or most of the EDTA and citrate plasma samples collected from healthy individuals using the method of the invention, but are low or absent in most serum or plasma samples with heparin collected from healthy individuals. Therefore, it is clear that the precise choice of the sample type will be fundamental for the different applications.
[0062] It has been shown that sample selection for the detection of free nucleosomes containing specific nucleotide structures involves several parameters. These include: the low levels of free nucleosomes generally present in serum and plasma samples with heparin collected from healthy individuals, and the higher levels, usually present in EDTA and citrate plasma samples collected from healthy individuals; the recommendation that serum samples containing free nucleosomes should be stabilized by adding EDTA after separating the serum from the clot (* Holdenreider et al., 2001); and the serum sampling protocol. Other stabilizing agents (for example, protease inhibitors), can also be used. Whenever possible, centrifuged serum samples were used within 1 hour after the venipuncture, after which 10 mM EDTA was added and the sample was frozen.
[0063] The choice of the type of blood sample for clinical samples should be made based on the optimal clinical discrimination for the particular test. Following the finding that low levels of nucleosomes in the serum of healthy individuals using the method of the invention are consistent, nucleosomes containing the nucleotide 5-methylcytosine were measured in serum samples collected from individuals with cancer . The clinical sensitivity of up to 100% was observed as shown in Figure 16 for colon cancer samples.
[0064] The relative levels of free nucleosomes containing the 5-methylcytosine and 5-hydroxymethylcytosine nucleotides and other nucleosome structures were also measured in EDTA plasma samples collected from individuals with a variety of diseases. Free nucleosome levels are high in EDTA plasma samples collected from both healthy individuals and sick individuals, so it appears unlikely that EDTA plasma samples are the best choice for a sensitive discriminator of sensitive sick individuals and healthy. However, it was found that the levels and composition of free circulating nucleosomes, in terms of the relative levels of nucleosomes containing different nucleotides (as well as other nucleosome structures), vary between healthy and sick individuals and also between different diseases. tions. It is therefore being first reported that (i) elevated levels of circulating nucleosomes are present in all or most of the EDTA plasma samples collected from both healthy sick individuals, not being true for all types of blood samples ; and also that (ii) surprisingly, disease detection and disease type discrimination can, however, be done by analyzing these nucleosomes in EDTA plasma based on the levels and structural profile of one or more of the relative types of nucleosome structures present in the plasma of sick and healthy individuals.
[0065] Free nucleosomes were measured in plasma with EDTA taken from healthy individuals and in 117 individuals with a variety of types of cancer in two experiments consisting of 55 and 62 individuals with cancer, respectively. In total, 78% (91 of 117) of cancer samples were correctly identified as positive for cancer using the inventive method for 5-methylcytosine associated with nucleosomes using a cut-off level from the resulting mean for healthy subjects + 2 standard deviations from the mean.
[0066] In the first of these two experiments, EDTA-free plasma nucleosomes collected from 13 healthy individuals and 55 individuals with stomach cancer, large intestine, rectum, lung (small cell carcinoma and various non-small cell carcinomas ), breast, ovary, pancreas, prostate, kidney and various oral cancers (oral cavity, palate, pharynx and larynx). All 13 samples from healthy individuals and cancer patients were positive for nucleosomes. However, the levels detected in samples collected from individuals with cancer were higher than those found in samples from healthy individuals and the results showed that healthy individuals with cancer could be discriminated against. For example, the normal range calculated in terms of OD as the mean ± 2 standard deviations from the mean, for 5-methylcytosine associated with nucleosomes was 0 - 1.41. Using this cutoff value all 13 healthy samples were negative and 30 of the 55 cancer samples were positive (the overall clinical sensitivity of 55%), including 38% (3 of 8) of stomach, 60% (3 5) large intestine, 33% (1 of 3) rectum, 33% (2 of 6) small lung cells, 64% (9 of 14) non-small lung cells, 33% (2 of 6) breast, 100% (1 of 1) ovary, 100% (1 of 1) pancreas, 33% (2 of 6) prostate, 100% (1 of 1) kidney and 60% (3 of 5) of oral cancer samples. The results are shown in Figure 17.
[0067] Likewise, the normal range for the H2AZ assay associated with nucleosomes was 0 - 0.95. Using this cut-off level of 0.95; all 13 healthy individuals were negative for elevated levels of H2AZ in nucleosomes. In contrast, a positive result was found for elevated levels of H2AZ in nucleosomes in 84% (46 of 55) of cancer samples (a total clinical sensitivity of 84%), including 100% (8 of 8) of stomach, 100 % (5 of 5) of large intestine, 67% (2 of 3) of rectum, 83% (5 of 6) of small lung cells, 79% (11 of 14) of non-small lung cells, 50% (3 of 6) breast, 100% (1 of 1) ovary, 100% (1 of 1) pancreas, 80% (4 of 5) prostate, 100% (1 of 1) kidney, and 100% (5 out of 5) of oral cancer samples.
[0068] In an embodiment of the invention, a control sample is provided and the cut-off level to distinguish between positive or negative results is defined in relation to the result for the control sample. This can be any proportion equal to or above or below the level of the control sample result. The results of the patient below this level are considered negative and the results of the patients above this level are considered positive. There may also be a "gray zone" band when patient results are very close to the cutoff level, in which the decision is considered indeterminate and / or the test must be repeated.
[0069] Likewise, for the mH2A1.1 assay associated with nucleosomes, the normal range was 0 - 0.91. Using this cutoff value, all 13 healthy samples were negative and 64% (35 of 55) of the cancer samples were positive. For the P-H2AX assay (Ser139) associated with nucleosomes, the normal range was 0 - 1.08. Using this cutoff value, all 13 healthy samples were negative and 60% (33 of 55) of the cancer samples were positive. Thus, some nucleosome assays have better clinical sensitivity than others.
[0070] In addition, it is possible to use the pattern of nucleosome structures to improve the clinical utility of the invention. This can be done, for example, by reducing the cut-off point of the 5-methylcytosine assay associated with nucleosomes to mean + 1 standard deviation, which gives a range of up to 1.01. In this case, the number of false negatives is reduced to 4, giving a better clinical sensitivity of 93% (51 out of 55), at the expense of an increase in false positive results for samples collected from healthy individuals, from 0% to 23% ( 3 of 13). The results are shown in Figure 17.
[0071] Samples with positive results for nucleosomes associated with 5-methylcytosine, or any nucleosomes, can be questioned regarding the profile of the structure of the nucleosomes. The nucleosome profile can be used to distinguish healthy and sick patients, as shown in Figures 20 and 21, in which the relative proportions of the various nucleosome structures in sick patients are expressed in relation to those found in healthy patients and patients with other diseases, excluding cancer. This shows that investigating multiple nucleosome structures on a test panel can facilitate better clinical discrimination.
[0072] Likewise the specificity and / or sensitivity of the diagnosis of the invention can be increased by combining data from more than one assay in the form of reasons. For example, the use of the P-H2AX: 5-nucleosome-associated methylcytosine ratio increases the detection of true positive cancer cases from 55% (30 of 55) to 67% (37 of 55) for 5-nucleosome-associated 5-methylcytosine alone, at the cut-off level of 2 standard deviations, maintaining 100% (13 of 13) of negative results for samples collected from healthy individuals.
[0073] Levels of free circulating nucleosomes containing two different nucleotides in EDTA plasma samples collected from 3 colon cancer patients, 13 lung cancer patients, 2 pancreatic cancer patients and 1 oral cancer patient and compared them with the levels present in the blood samples of 20 healthy individuals, as well as with an artificially produced preparation of serum nucleosomes from healthy individuals, prepared as described in the literature (* Holdenreider et al., 2001). The observed levels were also expressed in a normalized way, such as the ratio of the level of nucleosomes containing a specific nucleotide, and shown so that such reasons or patterns of reasons are useful for the diagnosis of both cancer in general and for the differential diagnosis of specific types of cancer. It was also investigated whether the level of 5-methylcytosine associated with nucleosomes varies according to the progression of the disease. It was observed that the mean level of free nucleosomes containing 5-methylcytosine increases with the severity of the disease and increases with the advance of the spread of the disease to the lymph nodes. This provides evidence that the detected nucleosomes are associated with the tumor.
[0074] The nucleosomes present in these 19 cancer samples were also measured using two current state of the art ELISA methods for nucleosomes. Of the 19 individuals with cancer studied, most had low levels of EDTA plasma nucleosomes, as determined by ELISA 1 and 2 for nucleosomes of the current state of the art. This result illustrates a reason why current state of the art assays are not used for routine clinical purposes.
[0075] ELISA methods of the present invention were used to measure nucleosomes containing the nucleotides 5-methylcytosine and 5-hydroxymethylcytosine in the same 19 samples. Surprisingly, elevated levels of nucleosomes containing 5-methylcytosine were detected in all 19 samples. Thus, in one embodiment, the invention provides a new ELISA method for nucleosomes capable of detecting nucleosomes not detected by assays for nucleosomes of the current state of the art.
[0076] The levels of nucleosomes containing three different variants of histones and one MPT in histone were also measured in the same 19 samples collected from individuals with cancer, as well as a sample of nucleosomes generated from healthy individuals by a method described in the literature (* Holdenrieder et al., 2001). These measurements were also used in conjunction with the nucleotide measurements associated with the nucleosomes described here, as a panel of the diversity of free nucleosomes present in biological fluids collected from individuals with 4 different types of cancer, and with the nucleosomes generated from healthy individuals. Surprisingly, the pattern of nucleosomes found in the 4 types of cancer investigated (lung, colon, pancreas and oral) was completely different from that found in the sample of nucleosomes generated from healthy individuals. In addition, the different types of cancer were also distinguished from each other based on the pattern of free nucleosomes detectable in the blood of individuals. In this way, in the form of carrying out the invention, a method is provided for the detection or diagnosis of the presence, type, recurrence or severity of a disease or the evaluation of the ideal drug or other treatment options, through the testing a sample for a panel of different nucleosome epitopes consisting of two or more measurements of nucleosomes containing different bases of DNA or a combination of one or more bases of DNA and one or more variants of histone and / or one or more modi - nucleosome and / or nucleosome measurements, per se, or in any combination or ratio of any of these, as an indicator of an individual's state of health or disease.
[0077] Similarly, ELISA methods of the invention were used to detect variability in the nucleotide and histone structures of free circulating nucleosomes in a variety of cancers and other diseases (except cancer) and compare these with the structures of the nucleosomes found in healthy individuals. Nucleosomes were present in all cancers and other diseases (except cancer) investigated and it was found that the profiles differed from healthy individuals.
[0078] EDTA plasma samples collected from 2 patients with cardiomyopathy, 10 patients with systemic lupus erythematosus (lupus), 12 patients with ulcerative colitis, 10 patients with chronic obstructive pulmonary disease (COPD), 8 patients with Crohn's disease and 10 with rheumatoid arthritis (RA) and the levels of several nucleosome structures detected were normalized by the proportion of the average levels of 5-methylcytosine associated with the nucleosomes and the results were expressed in relation to those found in the 11 healthy individuals. It was found that the diseases were associated with nucleosome structure profiles that differed from those found in healthy individuals or those with cancer. Thus, nucleosome structure profiles can be used as a diagnostic tool for the detection, prognosis prediction, monitoring and prediction of therapeutic efficacy in a wide variety of non-cancerous diseases. The results are shown in Figure 21.
[0079] The variability in the structure of free nucleosomes was also studied in terms of nucleotides and types of histones detected using ELISA methods of the invention for EDTA plasma samples collected from 55 patients with 10 different cancers. The levels of several nucleosome structures detected were normalized by the proportion of the levels of DNA methylated by 5-methylcytosine (5mC) associated with the nucleosomes and expressed in relation to the average proportions found in 11 healthy individuals. The nucleosomes are present in all individuals and the structure profiles of the nucleosomes that varied between cancer, diseases (except cancer) and healthy individuals. Thus, the structure profiles of the nucleosomes can be used as a diagnostic tool for the detection, prediction of prognosis, monitoring and prediction of therapeutic efficacy in cancer and other diseases. The results are shown in Figures 20 and 21.
[0080] As most of the circulating DNA in serum or plasma is in the form of mononucleosomes and oligonucleosomes (Holdenrieder et al., 2001), it is clear to those skilled in the art that the methods of the present invention can also be used in order to detect or measure free methylated DNA per se (as DNA associated with the nucleosome containing, for example, 5-methylcytosine or 5-hydroxymethylcytosine) directly in biological fluids, including blood, serum and plasma. The methods of the invention employed here have advantages in terms of simplicity and speed over methods for measuring methylated DNA of the current state of the art, especially when DNA extraction is not involved or unnecessary.
[0081] It will also be evident that the method of the present invention can be used to detect or measure any nucleic acid or a base of DNA or a nucleic acid or nucleosome derivative analogue. Such bases include, without limitation, adenine, thymine, guanine, cytosine, uracil, inosine, xanthine, hypoxanthine, 7,8-dihydro-8-oxo-guanine and any derivatives or analogues thereof. It will be clear to those skilled in the art that a common nucleotide (for example, without limitation, guanine, cytosine, thymine or adenine), will occur in all or most nucleosomes and that the method of the invention, using an antibody to a nucleotide common, will provide a method for binding and detecting virtually all nucleosomes in a sample. Thus, in one embodiment, the invention provides a new method for detecting nucleosomes, per se, in which nucleosomes, containing a common nucleotide are measured as a way of ensuring that all or most of the nucleosomes are detected.
[0082] In another embodiment, the invention provides a new method for the detection of all DNA associated with nucleosomes, in which nucleosomes containing a common nucleotide are measured as a way of ensuring that all or most of the DNA bound to the nucleosomes is detected. In addition, measuring two or more bases of DNA will provide the basis for measuring the ratio of the relative DNA content of said DNA bases. Such ratios of the relative levels of 5-methylcytosine and 5-hydroxymethylcytosine in the samples are illustrated in Figures 10 to 14. The data shows that the detectable relative levels of 5-methylcytosine and 5-hydroxymethylcytosine differ in different types of cancer and can be used to distinguish these types of cancer. Other similar reasons would also be useful in the art. For example, when using the present invention to measure an appropriate DNA base (or bases), as a metric for DNA bound to the total nucleosome, and when determining the relative level of another base (for example, 5-methylcytosine ), it will be clear that the method of the invention can be used to detect the proportion of DNA that comprises any particular base (for example, the percentage of DNA that is methylated in the sample). Thus, the methods of the present invention provide a simple and quick method for measuring the percentage of the DNA content of any base in a sample. The method can be used quickly and effectively on several samples, for example, blood. The methods of the invention can be used to detect and measure nucleosome DNA bases in any sample where such nucleosomes occur, including, for example, samples obtained from the digestion of chromatin extracted from cells. It will be clear to those skilled in the art that the term nucleotide is intended here to include, without limitation, purines, pyrimidines or any other nucleic acid bases and similar molecules, with or without associated sugars, and with or without phosphorylation and including any analogues, derivatives or imitations thereof.
[0083] It is concluded that the method of the present invention is an effective method for the detection and measurement of DNA associated with nucleosomes containing certain nucleotides, that this method can also be used effectively, as a method for the detection of nucleosomes, per se, and that it is a superior method for detecting nucleosomes, per se, than the methods of the current state of the art, and that this method can also be used effectively, as a method for the direct detection of free DNA , per se, and for the composition of free DNA nucleotides per se, and which is a superior method for the detection of DNA associated with nucleosomes and their nucleotide composition than the methods of the current state of the art. The method is fast, inexpensive and suitable for use in complex and fluid biological media. It has been shown that the method of the present invention can be used to detect nucleosomes and nucleosomes containing DNA methylated in the blood, and that it can be used as a biomarker for cancer. It will be clear to those skilled in the art that a biomarker present in blood samples collected from cancer patients has value for a wide range of purposes for the screening / screening of diseases and diagnosis of cancer and other diseases that are associated with circulating nucleosomes. high levels (Holdenrieder et al., 2001).
[0084] In order to confirm that elevated levels of nucleosomes are not found in healthy subjects when using the methods of the invention, nucleosomes containing the nucleotides 5-methylcytosine and 5-hydroxymethylcytosine were measured in the serum of 20 healthy subjects and in serum healthy beef. The results of the nucleosomes circulating in the serum for both ELISA tests of the invention were low or undetectable in all 20 healthy subjects. A similar test was also performed on the plasma samples collected from the same 20 healthy individuals, and surprisingly, higher signs were observed. This discovery is unexpected and quite different from the results found for the current state of the art ELISA methods for nucleosomes.
[0085] The invention has been tested on several cancers and non-cancerous diseases, and this has been shown to be effective in detecting all the diseases tested. This includes the detection of cases of prostate cancer, which is not detectable by current state of the art ELISA assays for nucleosomes (Holdenrieder, 2001). It is clear that the invention is effective for the detection of all or most cancers. It will be clear to those skilled in the art that the clinical performance of the invention can be further improved by including other nucleosome structure tests and by examining the reasons for the different nucleosome structures present.
[0086] In accordance with one aspect of the invention, a double antibody, immunometric or sandwich immunoassay method is provided for the detection and measurement of free nucleosomes containing nucleotides in a sample. An embodiment of this aspect is an immunoassay that comprises the steps of: (i) putting the sample that may contain nucleosomes in contact with a first antibody or another ligand that binds to the nucleosomes; (ii) placing the nucleosomes or sample in contact with a second antibody or other ligand that binds to a nucleotide; (iii) detecting and / or quantifying the binding of said second antibody or other linker to a nucleotide in the sample; and (iv), using the presence or degree of such a bond as a measure of the presence of a nucleotide associated with the nucleosome in the sample.
[0087] According to a second embodiment, a method is provided to detect and measure the free nucleosomes containing nucleotides in a sample by means of an immunometric immunoassay comprising the steps of: (i) placing the sample that may contain nucleosomes in contact with a first antibody or other linker that binds to a nucleotide; (ii) placing the nucleosomes or sample in contact with a second antibody or other ligand that binds to the nucleosomes; (iii) detecting and / or quantifying the binding of said second antibody or other ligand to the nucleosomes in the sample; and (iv), using the presence or degree of such a bond as a measure of the presence of a nucleotide associated with the nucleosome in the sample.
[0088] A variety of antibodies or other ligands can be used in the invention as a ligand that binds to nucleosomes. These include ligands targeted to bind to epitopes that occur in intact nucleosomes and not free histones (for example, an epitope found at the junction between two histones of a nucleosome) and also ligands targeted to any component of the nucleosome , including common epitopes of nucleosome protein, histone or nucleic acids.
[0089] It will be clear to those skilled in the art that the invention methods described include a variety of embodiments, including biosensor tests and label-free tests of the type marketed, for example, by ForteBio Incorporated of USA. Immunometric immunoassays employ an antibody (or other ligand) to bind to the analyte. The analyte, then bound, is detected as a direct measure of the level or concentration in the original test sample. In contrast, “competitive” immunoassays often use a much smaller amount of antibody (or other ligand) that binds to part of the analyte, and a preparation of the labeled analyte (or analyte analog) is used to distribute between the fractions free and linked analytes (with sample analyte). The amount of labeled and bound analyte is measured as an indirect measure of the analyte concentration in the original sample. In a variation of the “competitive” immunoassay, a labeled antibody is used, along with a solid phase analyte (or analyte analog) preparation. The binding of the marked antibody is distributed between the sample analyte and the solid phase analyte (or analyte analogue). The amount of antibody bound to the solid phase analyte (or analyte analog) preparation is used as an indirect measure of the analyte concentration in the sample.
[0090] In accordance with a third embodiment of the invention, a method is provided for the detection and measurement of a nucleotide, including a nucleotide associated with the nucleosome, in a sample by means of a label-free immunometric immunoassay comprising the steps of: (i) bringing the sample into contact with an antibody or other ligand that binds to a nucleotide; (ii) detecting and / or quantifying the binding of said antibody or other linker to a nucleotide in the sample; and (iii) using the presence or degree of binding as a measure of the presence of a nucleotide in the sample.
[0091] According to a fourth embodiment of the invention, a method is provided for the detection and measurement of a nucleotide, including a nucleotide associated with the nucleosome in a sample through a competitive immunoassay, which comprises the steps of: (i) placing the sample in contact with an antibody or other ligand that binds to a nucleotide; (ii) detecting and / or quantifying the binding of said antibody or other linker to a nucleotide in the sample; and (iii) using the presence or degree of binding as a measure of the presence of a nucleotide in the sample.
[0092] It will be clear to those skilled in the art that these invention immunoassay methods measure nucleotides and nucleotides associated with nucleosome directly without any need for DNA extraction. In contrast, the current state of the art nucleotide immunoassay methods detect nucleotides (not associated with the nucleosome) after DNA extraction from a sample. The methods of the invention have advantages in terms of speed, simplicity and adaptation for direct measurements in complex biological samples, including blood or blood derivatives.
[0093] According to a fifth embodiment of the invention, a method is provided to detect the proportion of free DNA that comprises a particular nucleotide in a sample comprising the steps of: (i) detecting or measuring the level of free DNA in a sample; (ii) detecting or measuring the level of a nucleotide associated with the nucleosome according to a method of the invention; and (iii) use both measures to determine the proportion of DNA that comprises the nucleotide.
[0094] According to an embodiment of this aspect of the invention, both the level of free DNA in the sample and the nucleotide of interest are measured using the method of the invention. In another embodiment, the nucleotide of interest is a methylated cytosine nucleotide and the proportion of DNA that comprises the nucleotide provides a measure of overall DNA methylation.
[0095] It has been observed that the detection and measurement of nucleosomes containing nucleotides in the blood collected from individuals can be used as a diagnostic method to identify individuals with cancer and differentiate them from healthy individuals. In addition, it has been shown that patterns of nucleosomes containing a panel of different nucleotides, histone variants and MPTs in histones can be used to distinguish the various types of cancer. It will be clear to those skilled in the art that this provides a blood test for cancer that will detect this disease in individuals and can be used to distinguish the types of cancer in cancer-positive individuals. According to a further aspect of the invention, a method for detecting or diagnosing the presence of a disease is provided by measuring or detecting the presence and / or level or concentration of free nucleosomes containing a nucleotide in a body fluid, and using the level detected as a biomarker of an individual's disease state, including, without limitation, a clinical diagnosis of a disease, a differential diagnosis of the type or subtype of the disease, or a prognosis of the disease, or a recurrence disease, or a diagnosis of the individual's susceptibility to treatment regimens. It will be clear to those skilled in the art that the body fluids used for diagnostic tests include, without limitation, blood, serum, plasma, urine, cerebrospinal fluid and other fluids. In a preferred embodiment, the body fluid selected as a sample is blood, serum or plasma. The assay response, the level, concentration or quantity of a nucleoside associated with the nucleosome in a body fluid can be expressed in absolute or relative terms, for example, without limitation, as a proportion of the total nucleosome level present or as a ratio of the level of nucleosomes containing another nucleotide or variant of histone or MPT to histone or total DNA level.
[0096] In one embodiment of the invention, measurement of nucleosomes associated with nucleosomes is used as a member of a diagnostic testing and measurement panel for the detection or diagnosis of an individual's disease state, including, without limitation, a clinical diagnosis of a disease, a differential diagnosis of the type or subtype of the disease, or a prognosis of the disease, or a recurrence of the disease, or a diagnosis of the individual's susceptibility to treatment regimes.
[0097] As there are reports that all or most of the free circulating DNA is in the condition of DNA associated with the nucleosome, it will be clear to those skilled in the art that diagnosis or detection of the disease state can be achieved through the detection or measurement of nucleotides per se using a direct nucleotide immunoassay of the invention, without the step of extracting DNA in a biological fluid, instead of, or in addition to, an immunoassay for nucleosomes associated with nucleosomes. According to a further aspect of the invention, a method of immunoassay of nucleotides, without extraction, is provided to detect or diagnose the presence of a disease by measuring or detecting the presence and / or level or concentration of a nucleotide in body fluid, and the use of the level detected as a biomarker (either alone, as a member of a test panel) of an individual's disease status, including, without limitation, a clinical diagnosis of a disease, a differential diagnosis of the type or subtype of the disease, or a prognosis of the disease, or a recurrence of the disease, or a diagnosis of the susceptibility of individuals to treatment regimens. It will be clear to those skilled in the art that the body fluids used for diagnostic tests include, without limitation, blood, serum, plasma, urine, cerebrospinal fluid and other fluids. In a preferred embodiment, the body fluid selected as a sample is blood, serum or plasma. The assay response, level, concentration or quantity of a nucleotide in a body fluid can be expressed in absolute or relative terms, for example, without limitation, as a proportion of the total nucleosome level present or as a ratio of the level of another nucleotide or variant of histone or MPT to histone or level of total DNA.
[0098] According to another aspect of the invention, a method is provided to detect or measure the presence and / or the level of nucleosomes containing a nucleotide in a cell comprising the steps of: (i) isolating the chromatin from a cell; (ii) breaking down chromatin to form mononucleosomes and / or oligonucleosomes; and (iii) detecting or measuring the presence of a nucleotide in mononucleosomes and / or oligonucleosomes, by means of an immunoassay method of the present invention.
[0099] Methods for producing mononucleosomes and / or oligonucleosomes from chromatin are well known in the art and include enzymatic digestion and sonication (Dai et al., 2011). In one embodiment, the nucleotide selected for detection by the method is a common nucleotide that occurs in all or most of the intact nucleosomes, providing a method for detecting or measuring nucleosomes, per se. In another embodiment, the nucleotide selected for detection by the method is a common nucleotide that occurs in all or most of the intact nucleosomes, providing a method for detecting or measuring the DNA bound to the nucleosome.
[00100] It will be clear to those skilled in the art that the described method of detecting nucleotides associated with nucleosomes in cells or tissues has advantages over currently used methods, including IHC, or detecting nucleotides in DNA extracted from cells by digestion by restriction enzymes and analysis of the nearest neighbor, or by fluorescent assays using chloroacetaldehyde, by inverse determination by methylation of all CpG sites using DNA methyltransferase, together with tritium-labeled S-adenosyl-methionine to calculate the amount of Unmethylated CpG, or by digesting DNA into single nucleotides by analyzing high performance liquid chromatography, thin layer chromatography, or liquid chromatography, followed by mass spectroscopy. The level, concentration or quantity of a particular nucleotide associated with the nucleosome can be expressed in absolute or relative terms, for example, as a proportion of the total nucleosomes present or as a ratio of the total level of nucleosomes or the level of nucleosomes containing other nucleotides or histone or MPT variant in histone, or the total DNA level.
[00101] It will be clear to those skilled in the art that the terms antibody, ligand or ligand in relation to any aspect of the invention, are not limiting, but are intended to include any ligands capable of binding molecules or entities that any suitable binders can be used in the method of the invention. It will also be apparent that the term nucleosomes is intended to include mononucleosomes and oligonucleosomes and any chromatin fragments that can be analyzed in fluid media.
[00102] In accordance with another aspect of the invention, a kit for detecting or measuring nucleosomes is provided, which comprises a ligand or ligand specific for the nucleotide or component part thereof, or a structural mimic / shape of the nucleosome or component part thereof, together with the instructions for use of the kit according to any of the methods defined here.
[00103] In accordance with another aspect of the invention, a kit for detecting or measuring nucleosomes containing a nucleotide comprising a specific ligand or ligand for the nucleotide sequences or a part of the same component, or a structural mimic / form of the nucleotide or part of the same component, together with instructions for using the kit according to any of the methods defined herein.
[00104] According to another aspect of the invention, a method is provided for the identification of a nucleotide-associated nucleotide biomarker or a nucleotide biomarker for the detection or diagnosis of the disease state in animals or humans, which comprises the steps of: (i) detecting or measuring the level of free nucleosomes containing a nucleotide in a body fluid of sick individuals; (ii) detecting or measuring the level of free nucleosomes containing a nucleotide in a body fluid from control individuals, and (iii) using the difference between the levels detected in sick and control individuals to identify whether a nucleotide is useful as a biomarker for that disease.
[00105] It will be clear to those skilled in the art that control individuals can be selected from a variety of bases which may include, for example, individuals who are disease free or may be individuals with different diseases (for example for differential diagnosis investigation).
[00106] According to another aspect of the invention, a method is provided for the identification of a nucleotide-associated nucleotide biomarker or a nucleotide biomarker to assess the prognosis of a sick animal or human comprising the steps to: (i) detect or measure the level of free nucleosomes containing a nucleotide in a body fluid of sick individuals, and (ii) correlate the level of free nucleosomes containing a nucleotide detected in a body fluid of sick individuals with the evolution disease of individuals.
[00107] According to another aspect of the invention, a method is provided for the identification of a nucleotide biomarker to be used for the selection of a treatment regimen for a sick animal or human, in need of treatment comprising the steps of: (i) detecting or measuring the level of free nucleosomes containing a nucleotide in a body fluid of sick individuals, and (ii) correlating the level of free nucleosomes containing a nucleotide detected in a body fluid of sick individuals with the effectiveness of the treatment regimen observed in these individuals. [00108] According to another aspect of the invention, a method is provided for the identification of a nucleotide biomarker associated with the nucleosome or a nucleotide biomarker to be used to monitor the treatment of an animal or human being patient, which comprises the steps of: (i) detecting or measuring the level of free nucleosomes containing a nucleotide in a body fluid of a sick individual; (ii) repeat such detection or measurement one or more times during the individual's disease progression; and (iii) correlating the level of free nucleosomes containing a nucleotide detected in a sick person's body fluid with the progression of the disease in the individual.
[00109] According to another aspect of the invention, a biomarker identified by the method as defined herein is provided.
[00110] It is already known in the state of the art that it is possible to detect by immunoassay the presence of a portion that is part of a complex containing other portions. It will be clear to those skilled in the art that free nucleosomes containing a nucleotide can be detected in a biological fluid, including blood, plasma, serum and urine, by a procedure that involves direct immunoassay of the nucleotide itself in the fluid. In this procedure, a single antibody immunoassay, using an antibody specific for an epitope present in a nucleotide, or a two-site immunoassay (2-site immunoassay), using two antibodies directed to two epitopes present in a nucleotide, it is used to detect the presence of a nucleotide within a nucleosome. Thus, in another embodiment of the invention, a nucleotide contained within a nucleosome is detected directly in a biological fluid, including blood, plasma, serum and urine, using an immunoassay method for a nucleotide.
[00111] Thus, in an embodiment of the invention, the nucleotide associated with the nucleosome is detected directly, without prior extraction in a biological fluid, including blood, plasma, serum and urine using an immunoassay for the nucleotide.
[00112] Another aspect of the invention provides ligands or linkers, such as chemically synthesized or natural compounds, capable of binding to the specific biomarker. A ligand or linker according to the invention can comprise a peptide, an antibody or a fragment thereof, or a synthetic linker, such as a plastic antibody, or an aptamer or oligonucleotide, capable of binding to the specific biomarker. The antibody can be a monoclonal antibody or a fragment of it capable of binding to the specific biomarker. A ligand according to the invention can be labeled with a detectable marker, such as a luminescent, fluorescent, enzyme or radioactive marker, a ligand according to the invention can be labeled, alternatively or additionally, with a affinity tag, for example, biotin, avidin, strepta-vidin or His tag (for example, hexa-His). Alternatively, the ligand binding can be determined using unmarked technology, for example, from ForteBio Inc.
[00113] A biosensor, according to the invention, can comprise the biomarker or a structural mimic / shape capable of binding an antibody against the specific biological marker. Also provided is an arrangement comprising a ligand or mimic, as described herein.
[00114] Also provided by the invention is the use of one or more ligands, as described herein, which may be naturally occurring or chemically synthesized, which are, appropriately, a peptide, antibody or fragment thereof, apamer or oligonucleotide ; or the use of a biosensor of the invention, or an arrangement of the invention, or a kit of the present invention to detect and / or quantify the biomarker. In these uses, detection and / or quantification can be performed on a biological sample, as defined herein.
[00115] Diagnostic or monitoring kits are provided to carry out the methods of the invention. Such kits suitably comprise a ligand, according to the invention, for the detection and / or quantification of the biomarker, and / or a biosensor, and / or an arrangement, as optionally described here, together with the instructions for using the kit.
[00116] Another aspect of the invention is a kit for detecting the presence of a disease state, which comprises a biosensor capable of detecting and / or quantifying one or more of the biomarkers, as defined herein.
[00117] Biomarkers for detecting the presence of a disease are essential goals for discovering new targets and drug molecules that slow progression or stop the disorder. As the level of the marker is indicative of the disorder and response to the drug, the biomarker is useful for the identification of assays of new therapeutic compounds in vitro and / or in vivo. The biomarkers of the invention can be used in screening methods for compounds that modulate the activity of the biomarker.
Thus, in another aspect of the invention, there is provided the use of a ligand or ligand, as described, which can be a peptide, antibody or fragment thereof or aptamer or oligonucleotides according to the invention, or the use of a biosensor according to the invention, or an arrangement according to the invention, or a kit according to the invention, to identify a substance capable of promoting and / or suppressing the generation of the marker.
[00119] A method of identifying a substance capable of promoting or suppressing the generation of the marker in an individual is also provided, comprising administering a test substance to an animal and detecting and / or quantifying the level of the marker present in a test sample from the individual.
[00120] The term "biomarker" means a biological or biologically derived indicator distinct from a process, event or condition. Biomarkers can be used in diagnostic methods, for example, in clinical screening and in the evaluation of prognosis and monitoring of therapy results, identifying patients most likely to respond to a particular therapeutic treatment, selecting drugs and developing biomarkers and their use are important for identifying treatments with new drugs and for the discovery of new targets for drug treatment.
[00121] The terms "detect" and "diagnose", as used herein, include the identification, confirmation, and / or characterization of a disease state. Detection, monitoring and diagnosis methods, according to the invention, are useful to confirm the existence of a disease, to accompany the development of the disease, by assessing its onset and progression, or to evaluate the improvement or regression of the disease. Detection, monitoring and diagnosis methods are also useful in methods for evaluating clinical screening, prognosis, choosing therapy, evaluating therapeutic benefit, that is, for drug screening and drug development.
[00122] Efficient diagnosis and follow-up methods provide very powerful "solutions for the patient" with the potential to improve the prognosis which, when establishing the correct diagnosis, allows the rapid identification of the most appropriate treatment (thus decreasing , unnecessary exposure to the side effects of harmful drugs), and reduces recidivism rates.
[00123] In one embodiment, said biomarker is released from the cells of a tumor. Thus, according to another aspect of the invention, a method is provided for detecting the growth of a tumor, which comprises the steps of (i) measuring a biomarker in a biological sample that is associated with or released from the cells of a tumor and (ii) demonstrate that the level of said biomarker is associated with the size, phase, aggressiveness or spread of the tumor.
[00124] It is known that increased cell turnover, cell death and apoptosis lead to an increase in circulatory levels of free nucleosomes (Holdenrieder et al., 2001). The level of free circulating nucleosomes is a non-specific indicator and occurs in a variety of conditions, including inflammatory diseases, a wide variety of benign and malignant conditions, autoimmune diseases, as well as after trauma or ischemia ( Holdenrieder et al. 2001). It will be clear to those skilled in the art that the invention will have application in a variety of therapeutic areas where circulating nucleosomes have been found in individuals. These include, without limitation, trauma (eg, serious bodily injury or surgery), extreme exercise (eg, running a marathon), stroke and heart attack, septicemia or other serious infections and endometriosis. The immunoassay method of the present invention was used to measure nucleosome levels and to investigate their variability in the structure of nucleotides and histones in a variety of diseases, including cardiomyopathy, systemic lupus erythematosus, ulcerative colitis, chronic obstructive pulmonary disease , Crohn's disease and rheumatoid arthritis, and compare these with the results of healthy individuals. It was possible to detect nucleosomes and determine their structures in relation (in terms of histone and nucleotide composition) to all these diseases. Since the methods of the present invention are capable of detecting a wider range of nucleosomes than ELISA methods for current nucleosomes, the methods of the invention have applications in a wide variety of areas in cancer and non-cancerous diseases .
[00125] Immunoassays of the invention include immunometric assays employing enzymatic detection methods (eg, ELISA), immunometric assays with fluorescent labeling, time-resolved fluorescence labeled immunometric assays, immunometric assays, chemiluminescent, immunoturbidimetric assays, immunometric assays and immunoradiometric assays with labeled particles and competitive immunoassay methods including labeled antigen and competitive immunoassay methods with antibodies labeled with a variety of labeling types, including enzyme, fluorescent, fluorescent, fluorescent staining - in time and particles. All such immunoassay methods are well known in the art, see, for example, Salgame et al., 1997 and van Nieuwenhuijze et al., 2003.
[00126] In one embodiment, said biological sample comprises a body fluid. For example, biological samples that can be tested in a method of the invention include cerebrospinal fluid (CSF), whole blood, serum, plasma, menstrual blood, endometrial fluid, urine, saliva or other body fluid (feces, fluid lacrimal, synovial fluid, sputum), breathing, for example, condensed breathing, or an extract or purification of them, or dilution of them. Biological samples also include samples from a living individual, or collected post-mortem. Samples can be prepared, for example, diluted or concentrated, where appropriate, and stored in the usual way.
[00127] In one embodiment, the method of the invention is repeated on several occasions. This embodiment provides the advantage of allowing the detection results to be tracked over a period of time. Such an organization will provide the benefit of monitoring or assessing the effectiveness of treating a disease state. Such methods of tracking the invention can be used to track onset, progression, stabilization, improvement, recurrence and / or remission.
[00128] Thus, the invention also provides a method of monitoring the effectiveness of a therapy for a disease state in an individual, suspected of having such a disease, which comprises the detection and / or quantification of the present biomarker in a biological sample of that individual. When monitoring the methods, test samples can be collected on two or more occasions. The method may also include comparing the level of the marker (s) present in the test sample with one or more control (s) and / or with one or more previous test sample (s) collected (s) before the same test subject, for example, before the start of treatment, and / or from the same test subject in a previous phase of therapy. The method may include detecting a change in the nature or quantity of the biomarker (s) in test samples collected on different occasions.
[00129] Thus, according to another aspect of the invention, a method is provided to monitor the effectiveness of therapy for a disease state in a human or animal, comprising: (i) quantifying the amount of the biomarker, as defined herein , and (ii) comparing the amount of said biomarker in a test sample with the amount present in one or more control (s) and / or one or more previous test sample (s) collected at a time previous test from the same test subject.
[00130] A variation in the level of the biomarker in the test sample in relation to the level of a previous test sample collected at a previous moment from the same test individual can be indicative of a beneficial effect, for example, stabilization or improvement, of said therapy on the disorder or suspected disorder. In addition, after the treatment has been completed, the method of the invention can be repeated periodically in order to monitor the occurrence of a disease.
[00131] Methods for monitoring the effectiveness of a therapy can be used to control the therapeutic effectiveness of existing therapies and new therapies in humans and non-human animals (for example, in animal models). These monitoring methods can be incorporated into the screening of new drug substances and combinations of substances.
[00132] In another embodiment, the monitoring of faster changes due to quick-acting therapies can be performed in shorter intervals of hours or days.
[00133] According to another aspect of the invention, a method is provided for the identification of a biomarker for detecting the presence of a disease state. The term “identify / identification”, as used here, means that it confirms the presence of the marker present in the biological sample. Quantifying the amount of the marker present in the sample may include determining the concentration of the marker present in the sample. The identification and / or quantification can be performed directly on the sample, or indirectly, on an extract of the sample, or its dilution.
[00134] According to the alternative aspects of the present invention, the presence of the biomarker is assessed by detecting and / or quantifying antibodies, or fragments thereof, capable of specifically binding to the biomarker and which are generated by the patient's body in response to the biomarker and thus are present in a biological sample from an individual with a disease state.
[00135] The identification and / or quantification can be performed by any suitable method to identify the presence and / or quantity of a specific protein in a biological sample from a patient or from an extract or purification of a sample or a dilution thereof. In the methods of the invention, quantification can be performed by measuring the concentration of the biomarker in the sample or samples. Biological samples that can be tested in a method of the invention include those, as defined above. Samples can be prepared, for example, diluted or concentrated, where appropriate, and stored in the usual way.
[00136] The identification and / or quantification of biomarkers can be performed by detecting the marker or its fragment, for example, a fragment with truncation in the C-terminal region, or with truncation in the N-terminal region. The fragments are suitably more than 4 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of lenght. It is noted, in particular, that peptides of the same sequence or related to histone tails, are particularly useful fragments of histone proteins.
[00137] The biomarker can be detected directly, for example, by SELDI or MALDI-TOF. Alternatively, the biomarker can be detected directly or indirectly, by interacting with a ligand or ligands, such as an antibody or a biomarker binding fragment thereof, or another peptide, or ligating, for example, aptamer, or oligonucleotide, able to specifically bind to the biomarker. The ligand or ligand may have a detectable marker, such as a luminescent, fluorescent or radioactive marker, and / or an affinity tag.
[00138] For example, the detection and / or quantification can be performed by one or more method (s) selected from the group consisting of: SELDI (-TOF), MALDI (-TOF), an analysis 1-D gel based, 2-D gel based analysis, mass spectrography (MS), reverse phase CL (RP), techniques based on HPLC, UPLC and other size permeation CL or CL MS ( si-permeation) (gel filtration), ion exchange and affinity. Appropriate CL MS techniques include ICAT ® (Applied Biosystems, CA, USA), or iTRAQ ® (Applied Biosystems, CA, USA). Liquid phase chromatography (for example, high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin layer chromatography, NMR spectroscopy (nuclear magnetic resonance) could also be used.
[00139] Diagnostic or monitoring methods according to the invention can comprise the analysis of a sample by SELDI TOF MALDI TOF or detect the presence or level of the biomarker. These methods are also suitable for clinical screening, prognosis, monitoring of therapy results, identification of patients most likely to respond to a specific therapeutic treatment, selection and development of drugs and identification of new targets for the treatment. drug treatment.
[00140] The identification and / or quantification of the analyte biomarkers can be performed using an immunological method, involving an antibody, or a fragment thereof, capable of specifically binding to the biomarker. Suitable immunological methods include sandwich immunoassays, such as sandwich ELISA, where the detection of analyte biomarkers is performed using two antibodies that recognize different epitopes in the analyte biomarker; radioimmunoassays (RIA), direct, indirect or competitive enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), fluoride immunoassays (FIA), Western Blotting, immunoprecipitation and any particle based immunoassay (for example , using gold, silver, or latex particles, magnetic particles, or Q-dots). Immunological methods can be performed, for example, on microtiter plates or in strip format.
[00141] In one embodiment, one or more of the biomarkers can be replaced by a molecule, or a fragment of the measurable molecule, found upstream or downstream of the biomarker in a biological pathway.
[00142] The identification of key biomarkers specific to a disease is crucial for the integration of diagnostic procedures and therapeutic regimens. The use of appropriate diagnostic tools with predictive biomarkers, such as biosensors, can be developed; therefore, in the methods and uses of the present invention, identification and quantification can be performed using a biosensor, microanalysis system, microfabricated system, microseparation system, immunochromatography system or other suitable analytical devices. The biosensor can include an immunological method for detecting the biomarker (ES), electrical, thermal, magnetic, optical (for example hologram) or acoustic technologies. Using such biosensors, it is possible to detect the target biomarker (s) for the predicted concentrations found in biological samples.
[00143] As used here, the term "biosensor" means anything capable of detecting the presence of the biomarker. Examples of biosensors are described here.
[00144] The biosensors, according to the invention, can comprise a ligand or ligands, as described herein, capable of binding to the specific biomarker. Such biosensors are useful in the detection and / or quantification of a biomarker of the invention.
[00145] The biomarker (s) of the invention can be detected using technologies incorporating biosensors based on "smart" holograms, or high frequency acoustic systems, such systems are particularly feasible with ”barcode” or arrangement settings.
[00146] In smart hologram sensors (Smart Holograms Ltd, Cambridge, UK), a holographic image is stored in a thin polymer film that is sensitized in order to react specifically with the biomarker. At the exhibition, the biomarker reacts with the polymer leading to a change in the image presented by the hologram. The result of the reading test may be a change in the optical brightness, image, color and / or position of the image. For qualitative or semi-quantitative applications, a hologram of the sensor can be read with the naked eye, thus eliminating the need for detection equipment. A simple color sensor can be used to read the signal when quantitative measurements are required. The opacity or color of the sample does not interfere with the sensor's operation. The shape of the sensor allows multiplexing to simultaneously detect multiple substances. The reversible and irreversible sensors can be designed to meet different needs, making it possible to continuously monitor a biomarker of particular interest.
[00147] Suitably, the biosensors for detecting one or more biomarkers of the invention combine biomolecular recognition with appropriate means to convert the detection of the presence, or quantification, of the biomarker in the sample into a signal. Biosensors can be adapted for diagnostic tests in “alternative locations”, for example, in the infirmary, in outpatient clinics, in surgeries, at home, in the field and in the workplace.
[00148] To detect one or more biomarkers of the invention, biosensors include acoustic, plasmon resonance, holographic sensors, by Bio-Layer interferometry (BLI) and microfabricated. The printed recognition elements, thin film transistor technology, magnetic acoustic resonators and other new acusto-electric systems can be used in the biosensors to detect one or more biomarkers of the invention.
[00149] The methods involving the identification and / or quantification of one or more biomarkers of the invention can be performed on bench instruments, or they can be incorporated in disposable diagnostic or monitoring platforms that can be used in non-laboratory environments, for example example, in the doctor's office or in the patient's bed. Biosensors suitable for carrying out the methods of the invention include "credit" cards with optical or acoustic readers. Biosensors can be configured to allow the collected data to be transmitted electronically to the physician for interpretation and, therefore, can form the basis for e-medicine.
[00150] Diagnostic kits for diagnosing and monitoring the presence of a disease state are described here. In one embodiment, the kits additionally contain a biosensor capable of identifying and / or quantifying a biomarker. Suitably, a kit according to the invention may contain one or more components selected from the group: a ligand ligand, or ligands, specific for the biomarker or a structural mimic / shape of the bi-marker, one or more controls , one or more reagents and one or more consumables; optionally together with the instructions for use of the kit according to any of the methods defined here.
[00151] The identification of biomarkers for a disease state allows the integration of diagnostic procedures and therapeutic regimes. The detection of a biomarker of the invention can be used to screen individuals before participating in clinical trials. Biomarkers provide the means to indicate the therapeutic response, the inability to respond, the profile of unfavorable side effects, the degree of medication adherence and the achievement of adequate serum levels of drugs. Biomarkers can be used to provide an alert for an adverse drug response. Biomarkers are useful for the development of personalized therapies (for example, response assessment can be used to fine-tune the dosage), minimizing the number of prescribed drugs, reducing the delay in obtaining a therapy effective and avoid adverse drug reactions. Thus, through the monitoring of a biomarker of the invention, patient care can be precisely adjusted to match the needs determined by the disease and the pharmacogenic profile of the patient, the biomarker can thus be used to titrate the optimal dose , predict a positive therapeutic response and identify in these patients the high risk of serious side effects.
[00152] Tests based on biomarkers provide a first line of evaluation of the “new” patients and provide objective measures for the accurate and rapid diagnosis, which is not achievable with the current measures.
[00153] In addition, diagnostic biomarker tests are useful for identifying family members or patients with mild or asymptomatic disease or who may be at high risk of developing symptomatic disease. This allows for the initiation of appropriate treatment, or preventive measures, such as managing risk factors. These approaches are recognized in the improvement of the result and can prevent the onset of the manifestation of the disease.
[00154] The methods of monitoring biomarkers, biosensors and kits are also vital as tools for monitoring patients, as they allow the doctor to determine whether the recurrence is due to the worsening of the disease. If pharmacological treatment is assessed as inadequate, then therapy can be reintegrated or increased, a change in therapy may be offered, if applicable. Since biomarkers are sensitive to the state of the disease, they provide an indication of the impact of drug therapy.
[00155] The invention will now be explained with reference to the non-limiting examples, below. EXAMPLE 1
[00156] A commercially available nucleosome preparation, produced by digesting the chromatin extracted from MCF-7 cells, in which the DNA and proteins in the nucleosome are cross-linked to remain stable (thus ensuring that all histones present in the preparation are incorporated into the intact nucleosomes) was tested for methylated DNA using an ELISA method for the nucleosome-associated 5-methylcytosine nucleotide, using a solid phase anti-histone capture antibody that binds intact nucleosomes and a biotinylated anti-5-methylcytosine detection monoclonal antibody. The nucleosome sample was serially diluted in fetal bovine serum and tested, in duplicate, in the ELISA. Pure fetal bovine serum was also evaluated in the ELISA as a control sample where there are no free nucleosomes. The test method was as follows: A solution with antihistone antibody in 0.1 M phosphate buffer at pH 7.4 was added to the microtiter wells (100 μL / well) and incubated overnight at 4 ° C to cover the wells with capture antibody. The excess of antihistone antibody was decanted. A solution of bovine serum albumin (20 g / L) was added to the wells (200 μL / well) and incubated for 30 minutes at room temperature to block excess protein binding sites in the wells. The excess bovine serum albumin solution was decanted and the wells were washed three times with washing buffer (200 μL / well, 0.05 M TRIS / HCl pH 7.5 containing 1% Tween 20). The sample (10 μL / well) and the test buffer (50 μL / well, 0.05 M TRIS / HCl - pH 7.5 containing 0.9% NaCl, 0.05% sodium deoxycholate and substitute Nonidet P40 1%) were added to the wells and incubated for 90 minutes at room temperature with light agitation. The sample mixture with assay buffer was decanted and the wells were washed three times with wash buffer (200 μL / well). The solution with biotinylated anti-5-methylcytosine detection antibody was added (50 μL / well) and incubated for 90 minutes at room temperature with light agitation. The excess detection antibody was decanted and the wells were again washed three times with wash buffer (200 μL / well). A solution containing the streptavidin-horseradish peroxidase conjugate was added (50 μL / well) and incubated for 30 minutes at room temperature with light agitation. The excess conjugate was decanted and the wells were washed again three times with washing buffer (200 μL / well). A colored substrate solution (100 μL / well, 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonic] diamonium salt) was incubated for 20 minutes at room temperature with light agitation. The optical density (OD) of the wells was measured at a wavelength of 405 nm using a standard microtiter plate reader. A dose-response curve of color increase with increased nucleosome-associated anti-5-methylcytosine concentration was observed with a low background signal observed in the absence of 5-methylcytosine (fetal bovine serum). The positive ELISA signal indicates that the 5-methylcytosine detected by the ELISA is embedded within an intact nucleosome comprising both the histone protein and DNA since (i) the capture antibody bound to the histones in the sample and (ii) the detection antibody bound to the 5-methylcytosine component of DNA. The results are shown in Figure 1. EXAMPLE 2
[00157] A commercially available nucleosome preparation, produced by digesting chromatin extracted from A375 cells, in which the DNA and proteins in the nucleosome are cross-linked to remain stable (thus ensuring that all histones present in the preparation are incorporated into intact nucleosomes) was tested for 5-hydroxymethylated DNA using an ELISA method for the nucleosome-associated 5-hydroxymethylcytosine nucleotide, using a solid phase anti-histone capture antibody that binds intact nucleosomes and an antibody monoclonal biotinylated anti-5-hydroxymethylcytosine detection. The nucleosome sample was serially diluted in fetal bovine serum and tested, in duplicate, in the ELISA. Pure fetal bovine serum was also evaluated in the ELISA as a control sample where there are no free nucleosomes. The test method was as follows: A solution with antihistone antibody in 0.1 M phosphate buffer at pH 7.4 was added to the microtiter wells (100 μL / well) and incubated overnight at 4 ° C to cover the wells with capture antibody. The excess of antihistone antibody was decanted. A solution of bovine serum albumin (20 g / L) was added to the wells (200 μL / well) and incubated for 30 minutes at room temperature to block excess protein binding sites in the wells. The excess bovine serum albumin solution was decanted and the wells were washed three times with washing buffer (200 μL / well, 0.05 M TRIS / HCl pH 7.5 containing 1% Tween 20). The sample (10 μL / well) and the test buffer (50 μL / well, 0.05 M TRIS / HCl - pH 7.5 containing 0.9% NaCl, 0.05% sodium deoxycholate and substitute Nonidet P40 1%) were added to the wells and incubated for 90 minutes at room temperature with light agitation. The sample mixture with assay buffer was decanted and the wells were washed three times with wash buffer (200 μL / well). The solution with biotinylated anti-5-hydroxymethylcytosine detection antibody was added (50 μL / well) and incubated for 90 minutes at room temperature with light agitation. The excess detection antibody was decanted and the wells were again washed three times with wash buffer (200 μL / well). A solution containing the streptavidin-horseradish peroxidase conjugate was added (50 μL / well) and incubated for 30 minutes at room temperature with gentle agitation. The excess of conjugate was decanted and the wells were again washed three times with washing buffer (200 μL / well). A colored substrate solution (100 μL / well, 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonic] diamonium salt) was incubated for 20 minutes at room temperature with light agitation. The optical density (OD) of the wells was measured at a wavelength of 405 nm using a standard microtiter plate reader. A dose-response curve of color increase with increased concentration of anti-5-hydroxymethyl cytosine associated with the nucleosome was observed with a low background signal observed in the absence of 5-hydroxymethyl cytosine (fetal bovine serum). The positive ELISA signal indicates that the 5-hydroxymethylcytosine detected by the ELISA is incorporated into an intact nucleosome comprising both the histone protein and DNA since (i) the capture antibody bound to the histones in the sample and (ii) the detection antibody bound to the 5-hydroxymethylcytosine component of DNA. The results are shown in Figure 2. EXAMPLE 3
[00158] Two current state of the art ELISA methods for nucleosomes were used to measure the content of free circulating nucleosomes in serum and plasma collected from blood samples from 20 healthy individuals. The first current ELISA method (ELISA 1) was that of the Roche Cell Death ELISA and the other (ELISA 2), an ELISA that employs an antihistone capture antibody and an antihistone complex detection antibody -DNA. The levels of nucleosomes detected by both ELISA methods for current nucleosomes were lower in normal plasma than in normal serum. The normal range (expressed in units of optical density) for the level of nucleosomes in the serum was calculated (mean ± 2 standard deviations from the mean of the 20 serum results of healthy subjects) at 0 - 4.3 DO units for ELISA 1 and 0 - 1.4 for ELISA 2. The respective plasma ranges were 0 - 0.95 and 0 - 0.96. The results are shown in Figure 3.
[00159] The levels of nucleosomes containing the two nucleotides associated with the nucleosomes were also measured, as well as the three histone variants associated with the nucleosomes and an MPT in histone in the same 20 samples collected from healthy individuals. The results show that healthy serum samples have uniformly low levels of nucleosomes containing histone or MPT variants or nucleotides. The normal ranges (expressed in optical density) for the level of nucleosomes in the serum containing histone, MPT or nucleotide variants were: (a) 0 - 0.36 for mH2A1.1, (b) 0.05 - 0, 78 for mH2A2, (c) 0.11 - 0.58 for H2AZ, (d), 0.06 - 0.61 for P- H2AX (Ser139), (e), 0.06 - 0.36 for 5- methylcytosine and (f), 0.03 - 0.36 for 5 - hydroxymethylcitrosine. The plasma results with EDTA measured were higher for all 20 healthy individuals. The results are shown in Figures 4, 5, 6, 7, 8 and 9. EXAMPLE 4
[00160] Free nucleosomes containing 5-methylcytosine in plasma were measured with EDTA collected from 13 healthy individuals and 55 subjects with stomach cancer, large intestine, rectum, lung (small cell carcinoma and various non-small cell carcinomas ), breast, ovary, pancreas, prostate, kidney and various oral cancers (oral cavity, palate, pharynx and larynx). All 13 samples from healthy individuals were positive for one or more types of free nucleosomes. All 55 samples from cancer patients were positive for all types of free nucleosomes evaluated. However, the levels detected in samples collected from individuals with cancer were higher than those found in samples from healthy individuals; the results showed that healthy individuals with cancer can be discriminated against. For example, the normal range calculated, in terms of OD, as the mean ± 2 standard deviations from the mean for 5-methylcytosine associated with nucleosomes was 0 - 1.41. Using this cutoff value, all 13 healthy samples were negative and 30 of the 55 cancer samples were positive (global clinical sensitivity of 55%), including 38% (3 of 8) of stomach, 60% (3 of 5 ) large intestine, 33% (1 of 3) rectum, 33% (2 of 6) small lung cells, 64% (9 of 14) non-small lung cells, 33% (2 of 6) breast, 100% (1 of 1) ovary, 100% (1 of 1) pancreas, 33% (2 of 6) prostate, 100% (1 of 1) kidney and 60% (3 of 5) of oral cancer samples. The results are shown in Figure 17.
[00161] The methods of the invention were also used to measure a variety of other structures associated with nucleosomes in the same samples. The results of these immunoassays have been compiled to provide a profile of nucleosome structures in samples collected from cancer patients, and normalized to the detected levels of nucleosomes containing 5-methylcytosine. The resulting profiles were compared regarding the structure of the nucleosomes of the samples collected from healthy individuals. The nucleosome structure profile of free nucleosomes was different from that of healthy individuals. The results are shown in Figure 20. Similarly, the nucleosome structure profiles of samples collected from a variety of non-cancerous diseases were compiled and compared to the nucleosome profile of samples collected from cancer patients and healthy individuals . The results are shown in Figure 21.
[00162] Then, another similar experiment was carried out including samples from 10 healthy individuals and another 62 patients with cancer of various types. The results were similar to the first experiment. For example, using the results of nucleosome-associated 5-methylcytosine and a cutoff point of mean + 2 standard deviations from the mean of the results for healthy individuals, negative results were obtained for all 10 healthy individuals and positive results were obtained for 95% (61 of 62) of cancer patients, including 9 out of 9 prostate cancer patients, 5 out of 5 skin cancer patients, 8 out of 8 esophageal cancer patients, 12 out of 13 cancer patients bladder, 2 out of 2 cervical cancer patients and 1 out of 1 colon cancer patients, 4 out of 4 breast cancer patients, 7 out of 7 ovarian cancer patients, 7 out of 7 laryngeal cancer patients, 3 of 3 lung cancer patients and 3 of 3 renal cancer patients. The results are shown in Figure 18. This result indicates that the levels of nucleotides and the levels of nucleotides associated with nucleosomes in serum, including particularly 5-methylcytosine, are clinically sensitive biomarkers for cancer.
EXAMPLE 5
[00163] Two current state of the art ELISA methods for nucleosomes were used to measure the free circulating nucleosome content of samples collected from 3 individuals with colon cancer, 13 individuals with lung cancer, 2 individuals with pancreatic cancer, 1 individual with oral cancer and a sample of nucleosomes produced from healthy individuals, according to the Holdenrieder method (* Holdenrieder et al., 2001). The first current ELISA method (ELISA 1) was that of Roche Cell Death ELISA and the other (ELISA 2), an ELISA that employs an anti-histone capture antibody and an anti-histone-DNA complex detection antibody .
[00164] The levels of nucleosomes containing the two nucleotides associated with the nucleosomes were also measured, as well as the three histone variants associated with the nucleosomes and an MPT in histone in the same 19 samples collected from cancer patients. The results show that while low nucleosome results have been detected for current state of the art ELISA methods, for most individuals, particularly for patients with pancreatic and oral cancer, most of these samples have higher detected levels nucleosomes that contain one or more nucleotides or variant histones associated with the nucleosomes. The results for the samples collected from 3 individuals with colon cancer, 13 individuals with lung cancer, 2 individuals with pancreatic cancer and 1 individual with oral cancer are shown in Figures 10, 11, 12 and 13, respectively . Levels of histone-associated variants and significant levels of MPT in histones were detected in 16 of the 19 cancer samples (all except 3 lung cancer samples). In addition, significant levels of nucleosome-associated 5-hydroxymethylcytosine were detected in 12 of the 19 cancer samples. In addition, significant levels of 5-methylcytosine associated with nucleosomes were detected in all 19 cancer samples.
[00165] Furthermore, the pattern of nucleosome levels containing different levels of nucleotides, histone variants and histone MPT, is not uniform for all individuals, but presents different patterns for the different types of cancer tested. To facilitate the comparison between the results of individuals with the same or different types of cancer, the results of nucleosome tests (for nucleosomes containing macroH2A1.1, macroH2A2, H2AZ, P-H2AX (Ser139), 5- methylcytosine, 5-hydroxymethylcytosine) were normalized by the proportion of the DO signal observed in nucleosomes containing 5-methylcytosine. The normalized results (with error bars showing the standard deviation in the results where samples from more than one individual were tested) are shown for each cancer in Figure 14, as well as the same results for the nucleosome sample produced from healthy individuals (mH2A2 and 5-hydroxymethylcytosine were not measured in this sample). Figure 14 shows that the distribution pattern of the nucleosomes, containing different nucleotides, histone variants or normalized MPT, in all four cancers investigated, differs greatly from the distribution of nucleosomes in the sample prepared from healthy individuals. For example, the relative level of nucleosomes containing macH2A1.1 in the healthy nucleosome sample differs from that detected in samples of any type of cancer. Thus, the present invention can be used as a method for detecting cancer in a simple blood screening test. It will be clear to those skilled in the art that the invention includes the testing of nucleosomes containing yet other nucleotides and / or histone variants and / or modifications in histones for new or better discrimination between the free nucleosomes circulating in the tumor or tumor cells. another disease origin.
[00166] In addition, the pattern of the types of nucleosomes observed differs from different types of cancer. For example, samples collected from individuals with colon, pancreas and oral cancer can be distinguished through the different normalized levels of 5-hydroxymethylcytosine and H2AZ associated with the nucleosome. Similarly, oral cancer has different normalized levels of both nucleosomes containing mH2A2 or P-H2AX (Ser139) than any of the other three types of cancer, and samples from patients with pancreatic cancer can be distinguished from samples from of colon cancer patients based on a different relative level of nucleosomes containing the macroH2A1.1 variant. Thus, the present invention can be used as a method to diagnose cancer in general and to distinguish a particular type of cancer. It will be clear to those skilled in the art that the invention includes testing nucleosomes containing yet other variants of histones and / or modifications and / or histone nucleotides for a new or better discrimination between free circulating nucleosomes of different or other specific tumor origin disease of origin. EXAMPLE 6
[00167] The method of the invention was tested on serum samples collected from three healthy individuals and 10 patients with colon cancer. The nucleosomes containing 5-methylcytosine were measured in these samples and the cancer results were uniformly elevated in relation to the results obtained in healthy individuals, as shown in Figure 16. EXAMPLE 7
[00168] The levels of 5-methylcytosine associated with the nucleosomes of human plasma samples with EDTA collected from patients with lung cancer and colon cancer were measured. The detected levels were correlated with the patients' disease progression. The results shown in Figure 19 indicate that nucleosome-associated 5-methylcytosine levels increase with disease severity, in terms of size, staging, and nasal spread, and nucleosome-associated 5-methylcytosine levels can be used , alone or as part of a diagnostic panel, as indicators of disease progression. REFERENCES • Allen et al., A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Research: 32 (3) e38DOI: 10.1093 / nar / gnh032 • Bawden et al., Detection of histone modification in cell-free nucleosomes. WO 2005/019826, 2005 • Boulard et al., Histone variant macroH2A1 deletion in mice causes female-specific steatosis. Epigenetics & Chromatin: 3 (8), 1-13, 2010 Cell Biolabs, Inc. Product Manual for ”Global DNA Methylation ELISA Kit (5'-methyl-2'-deoxycytidine Quantitation”, 2011 • Dai et al., Detection of Post-translational Modifications on Native Intact Nucleossomass by ELISA. Http://www.jove.com/details.php id=2593 doi: 10.3791 / 2593. J Vis Exp. 50 (2011). • Deligezer et al., Sequence -Specific Histone Methylation Is Detectable on Circulating Nucleossomass in Plasma.Clinical Chemistry 54 (7), 1125-1131, 2008 • Epigentek Group Inc, Methylamp ™ Global DNA Methylation Quantification Kit, User Guide, Version 2.0802, 2009 • Esteller, Cancer epigenomics: DNA methylomes and histone- modification maps Nature Reviews Genetics: 8, 286-298, 2007 • Feinberg and Vogelstein, Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature: 301, 89-92, 1983 • Grutzmann et al ., Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin 9 DNA Methylation Assay. 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权利要求:
Claims (14)
[0001]
1. Use of a DNA base associated with a cell-free nucleosome, CHARACTERIZED by the fact that it is used as a biomarker, in a sample of body fluid, for the diagnosis of cancer, cardiomyopathy, systemic lupus erythematosus, colitis, lung disease chronic obstructive, Crohn's disease and rheumatoid arthritis, in which the DNA base is selected from: 5-methylcytosine or 5-hydroxymethylcytosine.
[0002]
2. Use according to claim 1, CHARACTERIZED by the fact that the cell-free nucleosome is a mononucleosome or an oligonucleosome.
[0003]
3. Use according to claim 1 or 2, CHARACTERIZED by the fact that the DNA base associated with a cell-free nucleosome is measured in a blood sample.
[0004]
4. Use according to any one of claims 1 to 3, CHARACTERIZED by the fact that the cancer is a cancer of the bladder, breast, colon, cervix, esophagus, kidney, large intestine, lung, oral cavity, ovary, pancreas, prostate, rectum, skin or stomach.
[0005]
5. Use, according to claim 4, CHARACTERIZED by the fact that the cancer is a cancer of the colon, lung, oral cavity or pancreas.
[0006]
6. Method for detecting the presence of a DNA base associated with a cell-free nucleosome in a sample of body fluid, CHARACTERIZED by the fact that it comprises the steps of: (i) putting the sample of body fluid in contact with a first binding agent that binds to nucleosomes; (ii) placing the nucleosomes linked in step (i) in contact with a second binding agent that binds to the DNA base; (iii) detecting or quantifying the binding of said second DNA-based binding agent in the body fluid sample; and (iv) using the presence or degree of such a bond as a measure of the presence of nucleosomes containing the DNA base in the body fluid sample, where the DNA base is selected from: 5-methylcytosine or 5-hydroxymethylcytosine.
[0007]
7. Method for detecting the presence of a DNA base associated with a cell-free nucleosome in a sample of body fluid, CHARACTERIZED by the fact that it comprises the steps of: (i) putting the sample of body fluid in contact with a first binding agent that binds to DNA; (ii) placing the sample bound in step (i) in contact with a second binding agent that binds to the nucleosomes; (iii) detecting or quantifying the binding of said second binding agent to the nucleosomes in the body fluid sample; and (iv) using the presence or degree of such a bond as a measure of the presence of nucleosomes containing the DNA base in the body fluid sample, where the DNA base is selected from: 5-methylcytosine or 5-hydroxymethylcytosine.
[0008]
8. Method according to claim 6 or 7, CHARACTERIZED by the fact that the binding agent is an antibody.
[0009]
9. Method according to any of claims 6 to 8, CHARACTERIZED by the fact that the sample of body fluid is blood or serum or plasma.
[0010]
10. In vitro method for detecting a disease state in an animal or a human being, CHARACTERIZED by the fact that it comprises the steps of: (i) detecting or measuring a DNA base associated with a cell-free nucleosome in a body fluid sample obtained from an individual; and (ii) use the detected level of DNA base associated with the nucleosomes to identify the individual's disease state, in which the DNA base is selected from: 5-methylcytosine or 5-hydroxymethylcytosine, and in which the disease is selected from among : cancer, cardiomyopathy, systemic lupus erythematosus, colitis, chronic obstructive pulmonary disease, Crohn's disease and rheumatoid arthritis.
[0011]
11. In vitro method for assessing the adaptation to medical treatment of an animal or a human being, CHARACTERIZED by the fact that it comprises the steps of: (i) detecting or measuring nucleosomes containing a DNA base in a body fluid sample from individual; and (ii) use the detected level of DNA base associated with nucleosomes as a parameter for the selection of an appropriate treatment for the individual, in which the DNA base is selected from: 5-methylcytosine or 5-hydroxymethylcytosine, and in which treatment is for a disease selected from: cancer, cardiomyopathy, systemic lupus erythematosus, colitis, chronic obstructive pulmonary disease, Crohn's disease and rheumatoid arthritis.
[0012]
12. In vitro method for monitoring a treatment of an animal or a human being, CHARACTERIZED by the fact that it comprises the steps of: (i) detecting or measuring nucleosomes containing a DNA base in a sample of the individual's body fluid; (ii) repeat the detection or measurement of nucleosomes containing a base of DNA in a sample of the individual's body fluid on one or more occasions; and (iii) use any changes in the detected level of DNA base associated with nucleosomes as a parameter for any changes in the individual's condition, where the DNA base is selected from: 5-methylcytosine or 5-hydroxymethylcytosine, and where treatment is for a disease selected from: cancer, cardiomyopathy, systemic lupus erythematosus, colitis, chronic obstructive pulmonary disease, Crohn's disease and rheumatoid arthritis.
[0013]
13. In vitro method according to any of claims 10 to 12, CHARACTERIZED by the fact that the disease is cancer.
[0014]
14. In vitro method according to any of claims 10 to 13, CHARACTERIZED by the fact that the DNA base associated with the nucleosome is detected or measured as one of a measurement panel.

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法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-08-13| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

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2020-06-23| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|

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2020-10-13| B09A| Decision: intention to grant|

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2020-12-22| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 31/08/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161530295P| true| 2011-09-01|2011-09-01|

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GBGB1115095.0A|GB201115095D0|2011-09-01|2011-09-01|Method for detecting nucleosomes containing nucleotides|

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US61/530,295|2011-09-01|

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GB1115095.0|2011-09-01|

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PCT/GB2012/052128|WO2013030577A1|2011-09-01|2012-08-31|Method for detecting nucleosomes containing nucleotides|

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