TWI726009B - Method for analysing cell free neucleosimes of tumor origin from a biological sample by affinity purification, isolating purified tumor dna from a biological sample, detecting an epigenetic epitope of tumor derived nucleosomes in a biological sample, or detecting cancer in an animal or a human subject, or an use of a kit comprising a histone h1 binding agent - Google Patents

Abstract

The present invention relates to the use of a histone H1 binding agent for detecting, isolating and/or purifying cell free nucleosomes of tumor origin from a biological sample. The invention also describes methods of negative or positive selection using histone H1 binding agents, in order to enrich a sample for cell free nucleosomes of tumor origin.

Description

Analysis of tumor-derived cell-free nucleosomes and fractions in biological samples by affinity purification Purified tumor DNA in a biological sample, detection of tumor-derived nucleosome epigenetic epitopes in a biological sample, or a method for detecting cancer in an animal or human subject, or the use of a kit including a histone H1 binding agent

The present invention relates to a method for purifying or increasing tumor-derived cell-free nucleosomes and related tumor DNA from blood, serum and plasma.

When cellular DNA exists in the form of a protein-nucleic acid complex, it is called chromatin. Nucleosomes are the basic unit of chromatin structure and are composed of double-stranded DNA (dsDNA) wrapped around protein complexes. This kind of structure in which DNA is wound into continuous nucleosomes is usually called "beads on a string", and forms the basic structure of open or euchromatin (euchromatin). In compact or heterochromatin, the rope The beads are spirals or super spirals to form a closed and complex structure.

Each nucleosome in chromatin is a protein complex composed of 8 highly conserved core histones (including a pair of histones H2A, H2B, H3, and H4). Approximately 146 base pairs (bp) of DNA surrounds the protein complex. Another histone H1 or H5 located outside the core histone of the nucleosome is responsible for connection and participates in chromatin compression. Cell-free nucleosomes mainly include mononucleosomes and related DNA that decompose chromatin to produce chromatin fragments when cells die.

The normal cell renewal of adults includes about 10 ¹¹ cells produced by cell division every day, and the number of cell deaths is similar mainly through apoptosis. In the process of apoptosis, chromatin is broken down into mononucleosomes and oligonucleosomes, some of which can be found in the blood circulation. Under normal circumstances, the number of circulating nucleosomes found in healthy subjects is not high. Elevated numbers of circulating nucleosomes have been found in subjects with different conditions including many cancers, autoimmune diseases, inflammation, stroke and myocardial infarction (Holdenreider & Stieber, 2009). Nucleosomes from dead cells may also flow into other body fluids, such as urine, stool, or sputum.

DNA abnormalities are characteristic of all cancers. The DNA of cancer cells is different from that of healthy cells in many ways, including but not limited to point mutations, translocations, gene copy numbers, and micro-satellite abnormalities, DNA strand integrity and nucleotide modification (for example, methylation of cytosine carbon position 5). For clinical diagnosis, prognosis and treatment selection purposes, biopsy or surgical removal of cancer cells or tissues, routine research for tumor-related DNA structure or sequence changes. Tumor genetic and epigenetic characteristics are different between different types of tumors and between different patients with the same tumor disease. In addition, these characteristics change over time within the same cancer in the same patient as the disease progresses and the development of acquired resistance to drugs or other therapies. Therefore, for cells removed during surgery or biopsy The continuous study of tumor DNA can help clinicians monitor disease progression and detect any recurrence or acquired resistance at an early stage (possibly a few months earlier than radiological detection), and may make the course of treatment successful.

However, tissue DNA testing has limitations because invasive biopsies cannot be repeated for monitoring purposes. For some patients, biopsy may not be used at all. The biopsy is expensive to perform, causes discomfort to the patient, and poses a risk to the patient, and may lead to surgical complications. In addition, a patient's tumor can be composed of multiple tumor strains located in different areas of the same tumor or at different metastatic locations (in metastatic cancer), and not all tumor strains can be sampled in a biopsy. Therefore, tissue biopsy DNA research provides a snapshot of the tumor in time and space in different tumor strains located in different areas of the tumor at a specific time.

The blood of cancer patients contains circulating tumor DNA (ctDNA), which is believed to be derived from chromatin fragments or nucleosomes released into the circulation by dying or dying cancer cells. Research on blood and tissue samples from cancer patients showed that cancer-related mutations appeared in the patient’s tumors (but not in their healthy cells), and the mutations also appeared in ctDNA in blood samples taken from the same patient ( Newman et al, 2014). Similarly, DNA sequences that are differentially methylated (epigenetic changes caused by methylation of cytosine residues) in cancer cells can be detected as methylated sequences in ctDNA in the blood circulation. In addition, the ratio of cell-free circulating DNA (cfDNA) composed of ctDNA is related to tumor burden, so the quantification of the occurrence ratio of ctDNA and the qualitative analysis of its genetic and/or epigenetic composition can be used to monitor diseases progress. The analysis of ctDNA can generate very useful and clinically accurate data. The data is related to all or many different cells originating from the tumor, so the tumor strain is integrated spatially. In addition, repeated sampling over time is a more practical and economical option. The analysis of ctDNA has the potential to revolutionize the detection and monitoring of tumors, and the study of tumor DNA without invasive tissue biopsy procedures is useful for the recurrence of tumors. And acquired drug-resistant tumor treatment has potential. This type of ctDNA test can be used to study all types of cancer-related DNA abnormalities (for example: point mutations, nucleotide modification status, translocations, gene copy number and microsatellite abnormalities, and DNA strand integrity), and will be suitable for routine use Sexual cancer screening, regular and more frequent monitoring, and regular check-ups for the best treatment plan (Zhou et al, 2012).

Blood, plasma or serum can be used as the material for ctDNA determination, and any DNA analysis method can be used, including but not limited to: DNA sequencing, epigenetic DNA sequencing (for example, for sequences containing 5-methylcytosine) , PCR, bead milk amplification technology (BEAMing), NGS (next-generation sequencing technology) (targeted or whole genome), digital PCR (digital PCR), cold PCR (cold PCR) (under a lower denaturation temperature) Amplification PCR), MAP (MIDI-activated pyrophosphate hydrolysis), personalized analysis of rearranged ends (PARE) and mass spectrometry analysis.

Because DNA abnormality is a characteristic of all cancer diseases, and ctDNA is observed in all cancer diseases in research, ctDNA detection is suitable for all cancer diseases. The cancers studied include but are not limited to bladder cancer, breast cancer, colorectal cancer, ovarian cancer, prostate cancer, lung cancer, skin Skin cancer (e.g. melanoma), pancreatic cancer, liver cancer, endometrial cancer, bowel cancer, lymphoma, oral cancer ( oral cancer, leukaemia, head and neck cancer, and osteosarcoma (Crowley et al, 2013; Zhou et al, 2012; Jung et al, 2010). Three examples (not limited to these three) will be described to illustrate the nature of ctDNA detection.

The first example involves the detection of cancer-related gene sequence mutations in ctDNA. Blood tests involving the detection of single-gene mutations in ctDNA usually have low clinical sensitivity. There are two reasons for this. First, although all cancers have mutations, the frequency of any particular mutation in a particular cancer disease is usually Lower. For example, although K-RAS and p53 mutations are considered to be two of the more common cancer mutations, they have been extensively studied in cancers, including bladder cancer, breast cancer, colon cancer, lung cancer, liver cancer, pancreatic cancer, and uterine cancer. Endometrial cancer and ovarian cancer were detected in 23%-64% and 17%-54% of cancer tissue samples, respectively. Second, even if the patient’s cancer tissue has a mutation, the level or concentration of the mutated ctDNA present in the patient’s blood may be low and difficult to detect. For example, K-RAS and p53 mutations can be detected in 0%-75% of K-RAS and p53 tissue-positive patients' ctDNA. The sum of these two effects is that K-RAS or p53 mutations are detected in the blood of less than 40% of cancer patients (Jung et al, 2010).

The second example involves the detection of multiple cancer-related gene sequence mutations in ctDNA. Although mutations in any specific gene, such as K-RAS or p53, can be present in a small number of cancers, all cancers contain mutations. Therefore, in principle, sufficient mutation studies are required to detect most or even all tumors. Therefore, one way to increase the clinical sensitivity of these tests is to detect a wide range of mutations in many genes. Newman et al. used this method to detect non-small cell lung cancer (NSCLC) and studied 521 exons and 13 intron sequences from 139 recurrent mutant genes. The mutations studied include various types of cancer-related genetic changes, including single nucleotide variants (SNV) and fusion genes, and it is reported that more than 95% of stage II-IV tumors and 50% of stage II-IV tumors and 50% are detected in ctDNA blood testing by this method Of stage I tumors have a specificity of 96% (Newman et al, 2014).

The third example involves the detection of cancer-related epigenetic changes in specific gene sequences in ctDNA. This method can be applied to any DNA or nucleotide modification. The main example of this method is the detection of genes that are differentially methylated at cytosine residues in certain cancers. For this purpose, a large number of genes have been studied in a variety of cancers, several of which are studied in bladder cancer, breast cancer, colorectal cancer, melanoma, ovarian cancer, and prostate cancer. SEPTIN-9, APC, DAPK, GSTP1, MGMT, p16, RASSF1A, T1G1, BRCA1, ERα, PRB, TMS1, MLH1, HLTF, CDKN2A, SOCS1, SOCS2, PAX5, PGR, PTGS2 and RARβ2. An illustrative example of this method is the detection of methylated SEPTIN-9 in ctDNA to detect colorectal cancer (CRC), which is reported to have a clinical specificity of 89% in 68% of CRC cases (Grutzmann et al, 2008 ).

The ctDNA fragments of tumor-derived cfDNA circulate as small DNA fragments less than 200bp in length, which is consistent with the expectation that the DNA fragments circulate in the form of mononucleosomes (Newman et al, 2014). Cancer patients are reported to have higher cfDNA levels than healthy subjects. Workers in this field pointed out that the cfDNA range of healthy subjects falls within 0-100ng/ml (average 30ng/ml), while the cfDNA range of cancer subjects falls within 0-1000ng/ml (average 180ng/ml) (Schwarzenbach et al, 2011). Circulating cfDNA is composed of DNA molecules of different sizes composed of base pairs of different lengths, with a length of up to 20,000 base pairs (Zhou et al, 2012). Consistent with the hypothesis that ctDNA mainly circulates with mononucleosomes, the detection amount of circulating cell-free nucleosomes in cancer patients, like the amount of DNA, is higher than in healthy subjects (Holdenrieder et al, 2001). However, because nucleosomes are non-specific products of cell death, and in many diseases, such as acute trauma, an increase in nucleosome mass can be observed, so the increase in circulating nucleosome mass itself is not regarded as a clinical cancer. Biomarkers (Holdenrieder and Stieber, 2009). As a product of cell death, the amount of circulating nucleosomes is significantly increased when treated with cytotoxic drugs or radiotherapy. However, nucleosomes are also cleared from the circulation, so their levels may peak with treatment and then drop (Holdenrieder et al, 2001).

Although the amount of circulating acellular nucleosomes has not been clinically used as a blood-based biomarker in cancer, the epigenetic composition of circulating acellular nucleosomes is related to histone modifications and histone variants. Variant), DNA modification and adduct content are studied as blood cancer biomarkers (WO 2005/019826; WO 2013/030577; WO 2013/030579; WO 2013/084002) .

The biological source of cfDNA is not clearly understood. The fragmentation of chromatin to produce mononucleosomes and oligonucleosomes is characteristic of apoptotic cell death. Necrotic cells are thought to produce larger DNA molecules that are thousands of base pairs in length, but DNA fragmentation may also occur in some cases of necrosis. In addition, common DNA repetitive sequences (such as ALU or LINE1 sequences) can be used as 200-400 base pair DNA fragments, which are released during non-apoptotic or necrotic cell death (Schwarzenbach et al, 2011). DNA fragments can also be secreted by cells as a form of cell-to-cell communication. The source of ctDNA is believed to be related to the death of cancer cells. DNA fragments can be released as nucleosomes from necrotic and/or apoptotic tumor cells. However, necrotic and apoptotic cells are usually phagocytosed by macrophages or other clearing cells, and DNA can be released from the macrophages that phagocytize necrotic or apoptotic cells (Schwarzenbach et al, 2011).

There are many methods for extracting cfDNA from blood, serum or plasma, and the yield of these extracted DNA has been compared with the efficiency of extracting DNA fragments of different lengths. The phenol-chloroform and sodium iodide extraction methods provide the highest yield and extract small DNA fragments less than 200bp in length. It is reported that other tested methods (including commercially available methods) have lower DNA extraction yields and cannot extract small DNA fragments less than 200 bp in length (Fong et al, 2009).

The extraction of cfDNA from blood, serum or plasma for the analysis of ctDNA is usually performed using commercially available DNA extraction products. These extraction methods have a high recovery rate (>50%) of circulating DNA, and other products (for example, the QIAamp circulating nucleic acid reagent set produced by Qiagen) extract short DNA fragments. The sample volume used is usually serum or plasma in the range of 1-5 mL.

Due to some limitations, there is currently no practice based on ctDNA testing for clinical oncology. The main methodological limitation is the need for high-quality DNA. Due to the nature of the sample, current ctDNA sampling methods produce poor-quality ctDNA samples. The main difficulty is that there is a large amount of non-tumor cfDNA in the circulation, making any analysis of ctDNA more complicated. Estimates from different workers Different, but the ctDNA fragments present in the circulation may be too low to detect or more than 50% cfDNA. However, for most cancer patients, ctDNA fragments are a small part of cfDNA. For example, recent research reports indicate that in lung cancer patients before treatment, ctDNA fragments increase with tumor size. The highest amount found in patients with a large tumor burden was 3.2%, but most patients were found to have ctDNA fragments below 0.1% (Newman et al, 2014). This means that for many patient samples, very low amounts of ctDNA must be analyzed in the presence of high amounts of non-tumor-derived DNA. In addition, the DNA comes from the same subject, so it has a similar sequence, and will interfere with any method used to quantify or analyze ctDNA.

A similar problem occurs when measuring the epigenetic composition of circulating cell-free nucleosomes and/or circulating nucleosomes as biomarkers of cancer, because nucleosomes themselves are non-specific indicators of cell death and serve as normal cell renewal processes Part of the release is associated with conditions related to increased levels of cell death, such as autoimmune diseases, stroke, sepsis, post-traumatic, burns, myocardial infarction, cerebral stroke, during transplant rejection after organ transplantation, and after strenuous exercise. Therefore, tumor-derived nucleosomes circulate with other non-tumor nucleosomes derived from various cells and tissues. These non-tumor nucleosomes will interfere with any quantitative or epigenetic analysis methods of tumor-derived nucleosomes. Similar effects may occur in other body fluids. For example, stool may contain nucleosomes and related DNA derived from colorectal cancer cells, as well as nucleosomes derived from healthy large intestine or rectal cells. Sputum may contain nucleosomes and related DNA derived from lung cancer cells, as well as nucleosomes derived from healthy lung cells. Similar effects can occur in other body fluids.

Therefore, there is a great need for a method of adding tumor-derived nucleosomes and DNA in blood, serum or plasma samples and other body fluid samples. Similarly, there is a need for an analysis method for circulating cell-free nucleosomes to distinguish tumor-derived and non-tumor-derived nucleosomes in order to improve the disease state of cancer Detection.

According to the first technical solution of the present invention, there is provided a method for analyzing tumor-derived cell-free nucleosomes in a biological sample by an affinity purification method, the method comprising the following steps: (i) contacting the sample with a histone H1 binding agent (Ii) Separate nucleosomes from samples that are not bound to the histone H1 binding agent; (iii) Analyze the separated nucleosomes by immunoassay or mass spectrometry.

According to another technical solution of the present invention, a method for separating and purifying tumor DNA from a biological sample is provided, wherein the method includes the following steps: (i) contacting the sample with a histone H1 binding agent; (ii) separating and The nucleosomes bound by the histone H1 binding agent; (iii) extracting DNA from the nucleosomes separated in step (ii); and (iv) analyzing the extracted DNA.

According to another technical solution of the present invention, there is provided an immunoassay method for detecting tumor-derived nucleosome epigenetic epitopes in a biological sample, wherein the method includes the following steps: (i) making the sample and the containing group Contact with the first binding agent of the protein H1 binding agent; (ii) separating the nucleosomes not bound to the first binding agent; (iii) contacting the nucleosomes with the second binding agent to bind to the epitope (Iv) detecting and/or quantifying the binding of the second binding agent to the epitope; and (v) using the presence or degree of this binding as a reference for the specific epitope of tumor-derived nucleosomes in the sample The measurement of existence.

According to another technical solution of the present invention, a method for detecting cancer in an animal or human subject is provided, which includes the following steps: (i) obtaining a biological sample from the subject; (ii) using two combinations of The immunoassay method of the reagent is to analyze the cell-free nucleosomes containing epigenetic characteristics and histone H1 in the sample. One of the binding agents is used to bind the specific epigenetic characteristics, and the other binding agent is used for histone H1; ( iii) Analyze the cell-free nucleosomes with specific epigenetic characteristics in the sample using the immunoassay method using two binding agents, the cell-free nucleosomes are nucleosomes with and without histone H1, wherein One binding agent binds to epigenetic features, and the other binding agent binds to the common core nucleosome epitope, which includes any epitope in histone H2, H3, or H4 epitope, any DNA epitope or exists in chromatin fragments except Any epitope other than histone H1 epitope; (iv) using the immunoassay result obtained in the previous step as a combined biomarker for the cancer in the subject.

According to another technical solution of the present invention, the use of a histone H1 binding agent for detecting, separating and/or purifying tumor-free nucleosomes in a biological sample is provided.

According to another technical solution of the present invention, there is provided the use of a kit containing the histone H1 binding agent in the method described herein.

Figure 1: OD ratio (OD2:OD1) refers to the relative ratio of [tumor-derived and non-tumor-derived nucleosomes]: [mainly non-tumor-derived nucleosomes] in 69 human subjects.

Regarding the composition of its epigenetic signals, the structure of nucleosomes in cancer cells may be different from that in healthy cells. Using antibodies or other binding agents that target binding epigenetic signals (which are more common in cancer cells than healthy cells, or vice versa), a cell-derived mixture containing cell-free nucleosomes that can be collected from a subject Of biological samples, isolate cell-free nucleosomes derived from tumors (that is, by positive or negative selection).

The core of the nucleosome is composed of 8 histone proteins, including a pair of H2A, H2B, H3, and H4 histone proteins. Histone H1 (H1) is not a core histone, but is located outside the core and acts as a linker. In particular, H1 involves compressing the "string bead" substructure into a high-level structure. Without being bound by theory, the inventors have confirmed that tumor-derived cell-free nucleosomes contain less histone H1 than nucleosomes derived from healthy cells. Tumor-derived nucleosomes are therefore more likely to consist of only the 8 core histones H2A, H2B, H3 and H4 (plus DNA and any adducted molecules). Therefore, it can be considered that the circulating cell-free nucleosomes derived from tumor or non-tumor in blood are different in nature, because most tumor-derived nucleosomes contain 8 histones and the core nucleosome structure of related DNA fragments. Most non-tumor-derived nucleosomes contain a similar core nucleosome structure plus additional histone H1 protein parts and related DNA fragments. This difference in properties can be used as a basis for a negative selection method to add tumor-derived cell-free nucleosomes from body fluid samples collected from subjects or patients.

We previously reported an epigenetic analysis method for cell-free nucleosomes in blood and other body fluids (WO2013/030577, WO2013/030578, WO2013/030579, WO2013/084002). As mentioned above, tumor-derived cell-free nucleosomes are released into the circulation of subjects with tumors, but due to normal cell renewal, they are diluted by cell-free nucleosomes already present in the circulation. Any such non-tumor-derived nucleosomes will interfere with the genetic or epigenetic analysis of tumor nucleosomes. Removal of non-tumor-derived cell-free nucleosomes from body fluid samples will result in higher-purity tumor-derived cell-free nucleosomes, thereby improving the results of genetic or epigenetic analysis of tumor nucleosomes or chromatin fragments in the sample.

In the preferred technical scheme of the present invention, circulating cell-free nucleosomes derived from diseases are purified and their epigenetic characteristics are analyzed by immunoassay or mass spectrometry. In this technical solution, blood, serum, plasma or other body fluid samples are collected from the subject, and by removing the cell-free nucleosomes containing the histone H1 composition, the disease-derived cell-free nucleosomes are added to the sample. The epigenetic features of the remaining cell-free nucleosomes are analyzed using immunoassay or mass spectrophotometric, which epigenetic features are indicative of disease. The epigenetic nucleosome features that can be analyzed include any or all: specific histone post-translational modifications, specific histone isoforms, specific nucleotides or modified nuclei Glycolic acid (for example, methylated, hydroxyl-methylated, or other nucleotide modifications). The advantages of immunoassay and mass spectrometry include low cost, high analytical sensitivity and specificity, and high yield.

In one embodiment, a separation method is provided, which utilizes the affinity purification method and epigenetic analysis of disease-derived cell-free nucleosomes in a biological sample. The method includes the following steps: (i) making the sample and the group Contact with the protein H1 binding agent; (ii) separating the nucleosomes that are not bound to the histone H1 binding agent (ie, unbound nucleosomes) from the sample; and (iii) Analyze isolated nucleosomes with modified nucleotides by an immunoassay method using antibodies or other binding agents that bind to modified nucleotides.

In another embodiment, there is provided a separation method by means of affinity purification and epigenetic analysis of disease-derived cell-free nucleosomes in a biological sample. The method includes the following steps: (i) making the sample and Contact with the histone H1 binding agent; (ii) separating nucleosomes that are not bound to the histone H1 binding agent (ie, unbound nucleosomes) from the sample; and (iii) binding to the post-translational modification group by using Protein (post translationally modified histone) antibody or other binding agent immunoassay method to analyze isolated nucleosomes with post-translationally modified histones.

In another embodiment, there is provided a separation method by means of affinity purification and epigenetic analysis of disease-derived cell-free nucleosomes in a biological sample. The method includes the following steps: (i) making the sample and Contact with the histone H1 binding agent; (ii) separating the nucleosomes that are not bound to the histone H1 binding agent (ie, unbound nucleosomes) from the sample; and (iii) by using the binding to the histone isoforms An immunoassay method for antibodies to histone isoforms or other binding agents to analyze isolated nucleosomes with histone isoforms.

In another embodiment, there is provided a separation method by means of affinity purification and epigenetic analysis of disease-derived cell-free nucleosomes in a biological sample. The method includes the following steps: (i) making the sample and Contact with the histone H1 binding agent; (ii) separating the nucleosomes that are not bound to the histone H1 binding agent (ie, unbound nucleosomes) from the sample; and (iii) Analyze the separated nucleosomes that bind to non-histone proteins by immunoassay methods using antibodies or other binding agents that bind to non-histone proteins.

In another embodiment, there is provided a separation method by means of affinity purification and detection of disease-derived cell-free nucleosomes from a biological sample collected from a patient, the method comprising the following steps: (i) making the sample Contact with histone H1 binding agent; (ii) separate nucleosomes that are not bound to histone H1 binding agent (ie unbound nucleosomes) from the sample; (iii) quantify the separated nucleosomes by immunoassay And (iv) the presence or amount of cell-free nucleosomes that do not include the histone H1 portion as an indicator of the disease state of the subject.

In the preferred analysis scheme of the present invention, in (i) nucleosomes containing histone H1 and (ii) all nucleosomes with and without histone H1, the circulating cell-free nucleosomes present in the sample are analyzed or measured. Epigenetic characteristics of the body. Measuring the epigenetic characteristics of nucleosomes containing histone H1 (ie, mainly non-tumor nucleosomes) can be performed by methods known in the art, including but not limited to mass spectrometry or by using two antibodies (or other The immunoassay of the binding agent), in which one antibody binds to the epigenetic feature of interest, and the other antibody binds to histone H1. Preferably, the affinity purification method described herein is used to remove H1-related nucleosomes for addition, and then directly quantify the epigenetic characteristics of nucleosomes that do not contain histone H1 (that is, mainly tumor nucleosomes) . However, it is possible to analyze or measure (all) circulating cell-free nucleosomes (i.e. tumor and non-tumor nucleus) with and without histone H1 by mass spectrometry or by immunoassay using two antibodies (or other binding agents). Body), in which one antibody binds to the epigenetic feature of interest, and the other antibody binds to any histone H2, H3 or H4 epitope, any DNA epitope or any existing It lies in the common core nucleosome epitope in chromatin fragments except histone H1 epitope. In this technical solution of the present invention, circulating cell-free nucleosomes (ie non-tumor nucleosomes) containing epigenetic characteristics and binding to histone H1 can be measured, as well as all circulating cell-free nucleosomes containing epigenetic characteristics, including those with And nucleosomes that do not have H1 histones (ie tumor and non-tumor nucleosomes). These two analysis or measurement methods can be combined for indicators of the amount or proportion of circulating acellular nucleosomes in a sample, which have specific epigenetic characteristics that are not related to histone H1. The amount or ratio of the aforementioned nucleosomes is derived from the tumor. For example, the difference between two measurements can be used, or the ratio of the two measurements can be used.

In a preferred embodiment of the technical solution of the present invention, a method for measuring the proportion of circulating cell-free nucleosomes containing epigenetic epitopes in a biological sample taken from a subject of tumor origin is provided. It includes the following steps: (i) obtaining a body fluid sample from a subject, (ii) performing a first immunoassay, which includes the following steps: (1A) combining the sample with a first binding agent that binds to the epigenetic epitope Contact; (1B) contacting the nucleosomes or sample with a second binding agent that binds to histone H1; and (1C) detecting or quantifying the binding of the second binding agent to the nucleosomes in the sample; (iii) proceeding The second immunoassay, which includes the following steps: (2A) contacting the sample with the first binding agent that binds to the epigenetic epitope; (2B) contacting the nucleosome or sample with the non-histone H1 nucleosome Contact with the third binding agent of the epitope of the body or chromatin fragment; and (2C) detecting or quantifying the binding of the third binding agent to the nucleosomes in the sample; (iv) combining the steps (1C) and (2C) Binding is measured as an indicator of the amount or proportion of acellular nucleosomes that have the epigenetic epitope in the sample and are tumor-derived.

In another preferred embodiment of the technical solution of the present invention, a method for measuring the proportion of circulating cell-free nucleosomes containing epigenetic epitopes in a biological sample taken from a subject from a tumor is provided, the The method includes the following steps: (i) obtaining a body fluid sample from a subject, (ii) performing a first immunoassay, which includes the following steps: (1A) contacting the sample with a first binding agent that binds to histone H1; 1B) contacting a nucleosome or a sample with a second binding agent that binds to the epigenetic epitope; and (1C) detecting or quantifying the binding of the second binding agent to the nucleosome in the sample; (iii) Performing a second immunoassay, which includes the following steps: (2A) contacting the sample with a first binding agent that binds to a non-histone H1 nucleosome or chromatin fragment epitope; (2B) contacting the nucleosome or sample with Contact with the third binding agent that binds the epigenetic epitope; and (2C) detect or quantify the binding of the third binding agent to the nucleosomes in the sample; (iv) combine steps (1C) and (2C) In combination with the measurement, the sample has the epigenetic epitope and is an indicator of the amount or proportion of acellular nucleosomes derived from tumors.

Those with ordinary knowledge in the field should understand that the above-mentioned "first" and "second" immunoassays can be performed in any order or in parallel or in multiple formats. It should also be clear that the arrangement of the first and second or first and third binding agents used in the two immunoassays does not need to be symmetrical as described above.

In a preferred embodiment, the epigenetic epitope or feature is selected from (i) isoforms of histone H2, H3 or H4, (ii) present in histone H2, H3 or H4 The post-translational modification of (iii) nucleotides including modified nucleotides, (iv) non-histone proteins that bind or add to cell-free chromatin fragments. In another embodiment, the epigenetic epitope is a common epitope that exists in the nucleosome itself, and the two This immunoassay provides an indicator of the presence of tumor nucleosomes themselves.

In a preferred embodiment of the technical solution of the present invention, the amount or proportion of acellular nucleosomes containing specific epigenetic epitopes derived from the disease can be used as a biomarker for the existence of the disease in the subject, or used To assess the subject’s disease state or the suitability of the treatment regimen.

We have successfully developed the analysis program as described above and in Example 15, in which non-histone nucleosome adducts are measured by two immunoassays, and the ratio is obtained: [Non-histone-containing nucleosomes themselves]: [Contains Histone H1 and non-histone nucleosomes]. Therefore, the ratio obtained is an indicator of [all nucleosome-protein adducts]: [non-tumor nucleosome protein adducts]. Therefore, for blood, serum or plasma samples obtained by subjects, the greater the ratio of the samples, the greater the likelihood that some nucleosomes are derived from tumors. In 29 healthy subjects, the average immunoassay output (Optical Density) ratio was found to be 8.7. Among the 29 subjects diagnosed with colorectal cancer, the average ratio was 15.9. Among the 11 subjects diagnosed with pancreatic cancer, the average ratio was 17.1. This shows that the increased ratio is indeed related to cancer-derived nucleosomes.

In addition, the individual results of each subject are used as cancer biomarkers based on the rising threshold. When the cut-off value is 12, this ratio gives positive results to 73% (8/11) of the subjects diagnosed with pancreatic cancer and 41% (12/29) of the subjects diagnosed with colorectal cancer, which is clinically specific It was 79% (6 false positive results in 29 healthy subjects). When the cut-off value is 22, this ratio gives a positive result for 36% (4/11) of subjects diagnosed with pancreatic cancer and 34% (10/29) of subjects diagnosed with colorectal cancer, which is clinically specific Sex was 93% (2 false positive results out of 29 healthy subjects). These results demonstrate that the ratio described herein can be used as a biomarker for cancer diseases.

It should be understood that the "histone H1 binding agent" mentioned herein (for example, in the negative selection method) generally refers to a binding agent that binds to histone H1 protein, including any histone H1 variants, homologous Functional isomers or their modifications. Therefore, in one embodiment, the histone H1 binding agent is selected from the group consisting of binding agents that bind to histone H1 protein, variants, isomers or modified.

In the above examples, the presence or amount of the added cell-free nucleosomes derived from the disease itself, or containing modified nucleotides, post-translational histone modifications, histone isoforms, or nucleosome-protein adducts The presence or amount of the added cell-free nucleosomes derived from the disease can be used as an indicator of disease status, disease prognosis, disease monitoring, treatment monitoring, or disease susceptibility to specific treatments or other clinical applications.

We previously reported nucleosome immunoassay methods suitable for the present invention, including the methods described in WO2005/019826, WO2013/030577, WO2013/030578, WO2013/030579, WO2013/084002. Any of these methods can be used in the present invention without limitation.

In addition, the inventors have determined that when tumor-derived nucleosomes are related to H1, nucleosome-related H1 is the target of further modification and/or may contain nucleosomes that are different from those derived from healthy cells. H1 variants or H1 isoforms present in the body.

Major histone H1 variants or isoforms include but are not limited to: H1.0, H1.10 and H1X, which are expressed in proliferating and dormant somatic cells, and H1 variants H1.1, H1.2, H1 .3, H1.4, H.1.5 and H1.6, which are expressed at high levels in dividing cells. In addition, there are germ line specific variants, including H1.8 which is mainly expressed in the testis and H1.7 which is mainly expressed in oocytes. The composition of histone variants of chromatin is altered in cancer cells, and it has been reported that among the common H1 isoforms, the expression of histone H1.0 isoforms is down-regulated in cancer cells. The histone isoforms H1.1, H1.2, H1.3, H1.4 and H1.5 are expressed at high levels in cancer cells (Scaffidi; 2015).

Histone H1 can be post-translationally modified at amino acid residues located at the N- and C-termini and within the globular domain of the protein, and these modifications may be related to cancer (Izzo and Schneider; 2015). It should be understood that the “histone H1 modification” mentioned herein refers to H1 post-translational modifications (PTM), which can include acetylation and methylation, which can be single -(mono-), di-(di-) or tri-(tri-) methylation, phosphorylation, ubiquitination, ADP ribosylation, citrullination, hydroxylation ), glycosylation, nitrosylation, glutamination and/or isomerisation. It has a modified histone amino acid residue, and its modification can be any Ser, Lys, Arg, His, Glu, Pro or Thr residue within the histone amino acid sequence.

For example, the lysine residues in the core histone sequence can be mono-(mono-), di-(di-) or tri-(tri-) methylated, acetylated or ubiquitous Ubiquitinated, the arginine residues in the core histone sequence can be monomethylated, symmetric or asymmetric dimethylated, or converted to citrulline , Serine or threonine residues in the core histone sequence can be phosphorylated and/or proline residues in the core sequence can be isomerized.

Those with ordinary knowledge in the art should understand that the label used to describe a specific histone modification indicates which histone has been modified, the specific amino acid that has been modified, and the type of modification that has occurred. For example, H1K64(Ac) represents the acetylation of histone H1 at position 64 of lysine.

In one example, the binding agent used in the present invention binds histone H1 modification associated with cell-free nucleosomes. In another embodiment, the histone H1 modification is selected from phosphorylation, acetylation, methylation, ubiquitination and/or formylation. H1 modification can include: S2, T4, T11, S/T18, S27, T31, S36, S37, Phosphorylation at positions T39, S41, S44, S107, T138, T142, T146, T147, T154, T155, T165, S172, S173, T180, S/T187, S189; Acetylation at the following positions: S2, K17, K26, K34, K46, K49, K52, K63, K64, K85, K88, K90, K93, K97, K109, K168, K169, K192, K209; methylation at the following positions: K26, K27, K34, K52, K64, K97 , K106, K119, K148, K168, K169, K187; Ubiquitination at the following positions: K17, K21, K34, K46, K47, K64, K65, K75, K76, K85, K86, K90, K91, K97, K98, K106, K107; Carboxylation at the following positions: K17, K34, K46, K63, K64, K67, K75, K85, K88, K90, K97, K110, K140, K141, K160. Therefore, in one embodiment, the histone H1 modification associated with the cell-free nucleosome comprises at least one histone H1 modification as listed herein.

In one embodiment, the binding agent used in the present invention binds histone H1 variants or isoforms associated with cell-free nucleosomes. H1 variants and isoforms can include H1.1, H1.2, H1.3, H1.4, H1.5, or H1.6 that are thought to be expressed during mitosis, and H1 that is expressed in resting somatic cells .0, H1.10 and H1X. Other H1 variants are also distinguished in specific tissues, such as the variants H1t, H1T2, H1LS in the testis, and specific cell types, such as the variant H1.0 in terminally differentiated cells. Therefore, in one embodiment, the histone H1 variants or isoforms associated with cell-free nucleosomes comprise at least one histone H1 variant or isoforms listed herein.

Those with ordinary knowledge in the art will understand that based on quantitative H1 binding or qualitative H1 isomeric and/or H1 post-translational modification composition, the addition of tumor-derived nucleosomes from human or animal subjects Biological samples will likely improve the discrimination of differential diagnosis using nucleosomes or DNA biomarkers. Therefore, in one embodiment, the immunoseparation method of healthy or tumor-derived nucleosomes utilizes a combination of at least one histone H1 modification and/or variants and/or isoforms associated with cell-free nucleosomes. Binding agent.

In one embodiment, the biological fluid sample is taken from the subject, and based on the nucleosome histone H1 composition, tumor-derived cell-free nucleosomes are added to the sample. The body fluid sample can be any biological fluid sample taken from the subject, including but not limited to cerebrospinal fluid (CSF), whole blood, serum, plasma, menstrual blood, endometrial fluid, urine, saliva or other body fluids (feces, Tears, joint fluid, sputum), exhalation, for example as concentrated exhalation, extracts or purifications thereof, or dilutions thereof. Biological samples also include samples from living subjects or samples collected after death. The sample can be prepared, for example, in an appropriately diluted or concentrated manner, and stored in a conventional manner.

In one embodiment, the cell-free nucleosomes of tumor origin are derived from cancers selected from the group consisting of breast cancer, bladder cancer, colorectal cancer, skin cancer ( For example, melanoma, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, bowel cancer, liver cancer, endometrial cancer Endometrial cancer, lymphoma, oral cancer, head and neck cancer, leukaemia and osteosarcoma.

In one embodiment, the nucleosome is a cell-free mononucleosome or oligonucleosome.

method

Negative selection

It will be clear to those with ordinary knowledge in the art that blood samples contain nucleosomes derived from tumor cells and healthy cells, based on histone H1 isoforms or histone H1 post-translational modification patterns, when derived from tumors. When cell-free nucleosomes are retained and H1 protein is contained, positive selection by affinity binding to tumor-derived cell-free nucleosomes is possible. However, most circulating cell-free nucleosomes derived from tumors do not contain the H1 part. Therefore, negative selection is used to add circulating cell-free nuclei derived from tumors The better method for small bodies.

According to the first technical solution of the present invention, there is provided a method for separating/analyzing tumor-derived cell-free nucleosomes from a biological sample by affinity purification, wherein the method includes the following steps: (i) combining the sample with histone Contact with H1 binding agent (including any histone H1 variants, isoforms or modifications); (ii) Separate nucleosomes that are not bound to histone H1 binding agent (ie unbound nucleosomes) from the sample ; And (iii) analyzing the isolated nucleosomes and/or related DNA.

According to this technical solution of the present invention, it is provided that the negative selection containing histone H1 protein, one or more specific histone H1 variants, or one or more specific histone H1 modifications is provided to add tumor-derived nucleosomes in biological samples Methods. In one embodiment, antibodies or other binding agents that bind to histone H1 or histone H1 variants H1.0, H1.10, or H1X (or any binding agent that binds to these combinations) are used to select and isolate H1 variants. Nucleosomes that bind to nucleosomes derived from healthy cells. This method therefore removes healthy cell-derived nucleosomes from the sample, thus adding tumor-derived nucleosomes. In a preferred embodiment, the binding agent is an antibody immobilized on a solid phase for immunological extraction of nucleosomes derived from healthy cells. The genetic sequence, epigenetic signal structure, or other characteristics of the nucleosomes and/or related DNA remaining in the liquid phase can be analyzed.

Therefore, according to a technical solution of the present invention, a method for separating/analyzing tumor-derived cell-free nucleosomes from a biological sample by affinity purification is provided, wherein the method includes the following steps: (i) combining the sample with Contact with a binding agent of histone H1 protein itself or a histone H1 variant selected from H1.0, H1.10 or H1.X; (ii) separating nucleosomes that are not bound to the binding agent (ie, unbound nucleosomes) from the sample; and (iii) analyzing the separated nucleosomes and/or related DNA.

In one embodiment, the isolated nucleosomes are analyzed by immunoassay or mass spectrometry. The level or amount of tumor-derived nucleosomes and/or any molecular genetic or epigenetic characteristics of tumor-derived nucleosomes can be used to detect cancer in a subject or to evaluate any clinical practice in a cancer subject purpose. In another embodiment, the isolated nucleosomes containing epigenetic characteristics are analyzed by immunoassay or mass spectrometry. In another embodiment, the epigenetic characteristics include post-translational histone modifications, histone isoforms, modified nucleotides, or adducted non-histone protein). In another embodiment, the DNA base sequence of the DNA composition of the isolated nucleosome is analyzed.

The "histone H1 protein" mentioned in the negative selection method described herein includes natural or wild-type histone H1 protein. As mentioned earlier, tumor-derived cell-free nucleosomes are less likely to contain histone H1 than nucleosomes derived from healthy cells. Therefore, this example can detect the presence or absence of natural/wild-type histone H1 protein. To isolate cell-free nucleosomes derived from tumors.

Positive choice

In the present invention, the positive selection method is based on the use of an affinity binding agent that binds to post-translationally modified histone H1 protein molecules or binds to the above-mentioned division or proliferation related H1 protein isoforms. Specifically, the post-translational modified histone H1 protein binding target for positive selection includes any one selected from the list above. The H1 isoforms used for positive selection include histone H1 isoforms or variants selected from H1.1, H1.2, H1.3, H1.4, H.1.5 or H1.6.

According to a technical solution of the present invention, there is provided a method for separating/analyzing tumor-derived cell-free nucleosomes from a biological sample by affinity purification, wherein the method includes the following steps: (i) Contact the sample with histone H1 variants, isoforms or modified binding agents that are commonly found in nucleosomes of cancer origin; (ii) Separate and bind the histone H1 binding agent from the sample Nucleosomes; and (iii) analyzing the isolated nucleosomes and/or related DNA.

According to this technical solution of the present invention, a cell-free nucleosome modified by one or more specific histone H1 that is commonly contained in histone H1 protein, one or more specific histone H1 variants, and cancer-derived nucleosomes is provided It is a positive choice to add tumor-derived nucleosomes in biological samples.

In a preferred embodiment, the antibody or other binding agent that binds to the H1 variant H1.1, H1.2, H1.3, H1.4, H1.5 or H1.6 (or any binding agent combined with its modification ) Is used to select and isolate nucleosomes containing H1 variants, thereby adding tumor-derived nucleosomes, while many nucleosomes derived from healthy cells remain unbound and can be isolated. In another preferred embodiment, H1 modified antibodies or other binding agents (or any binding agent combined with a modified combination thereof) are used to select and isolate nucleosomes containing H1 modifications, thereby adding tumor-derived nucleosomes. Nucleosomes, and many nucleosomes derived from healthy cells remain unbound and can be isolated. In a preferred embodiment, the binding agent is an antibody immobilized on a solid phase for immunoextraction of tumor-derived nucleosomes. The epigenetic signal structure or other characteristics of the genetic sequence of the nucleosome and/or related DNA bound to the solid phase

Therefore, according to a technical solution of the present invention, a method for separating/analyzing tumor-derived cell-free nucleosomes from a biological sample by affinity purification is provided, wherein the method includes the following steps: (i) combining the sample with Agent contact, the binding agent binds histone H1 variants or histone H1 modifications selected from H1.1, H1.2, H1.3, H1.4, H.1.5 or H1.6; (ii) Separate the nucleosomes bound to the binding agent from the sample; and (iii) analyze the separated nucleosomes and/or related DNA.

In one embodiment, the isolated nucleosomes are analyzed by immunoassay or mass spectrometry. In another embodiment, the isolated nucleosomes containing epigenetic characteristics are analyzed by immunoassay or mass spectrometry. In another embodiment, the epigenetic characteristics include post-translational histone modifications, histone isomers, modified nucleotides, or adducted non-histone proteins. In another embodiment, the DNA base sequence composed of the DNA of the isolated nucleosome is analyzed.

Tumor-derived cell-free nucleosomes are part of a mixture of nucleosomes of multiple origins, and only a part of the cell-free nucleosomes are present in biological fluids. The addition or isolation of tumor-derived nucleosomes can be performed by the positive or negative selection of the affinity purification separation method described herein.

Due to the sudden increase in cell death due to indefinite numbers and different reasons, circulating nucleosome levels can reach a significant peak within 2-5 days. The causes include trauma, stroke, or treatment with cytotoxic drugs or radiotherapy. The amount of circulating nucleosomes then decreases during 2-3 days (Holdenrieder et al , 2001). This effect is due to the induction of cell death, which is then eliminated in the circulation (Holdenrieder & Stieber, 2009).

The nucleosomes released into the circulation of patients without tumor disease (including, for example, because of surgical trauma) are not derived from tumors. These nucleosomes constitute cfDNA, but do not contain ctDNA.

The term "nucleosome" as used herein is intended to include mononucleosomes and oligonucleosomes as well as any such chromatin fragments that can be analyzed in a liquid medium. In another embodiment, the cell-free nucleosomes are mononucleosomes, oligonucleosomes, or other chromosomal fragments.

Those with ordinary knowledge in the field will know that the isolated nucleosomes exist as part of the The amount of tumor nucleosomes can be used to measure the proportion of DNA containing tumor DNA in the sample. In addition, the relative amount is the proportion of DNA derived from healthy cells in the sample. Therefore, the method described herein can be used to detect the amount or proportion of ctDNA in a sample. This measurement is similar to the allelic frequency measurement of cancer-related mutations in ctDNA, and can be used as a measure of tumor burden and response to treatment.

In one embodiment of the present invention, the preparation of fully or partially purified tumor nucleosomes is separated from a biological fluid sample. In one embodiment, the purification method involves the affinity isolation of cell-free nucleosomes that contain epitopes characteristic of DNA epigenetic signals of histones or healthy tissues, by using a binding agent that binds to the above-mentioned epitopes. Therefore, the extract contains the tumor nucleosome preparation and/or its related ctDNA, which can then be analyzed.

The method of contacting a sample and separating nucleosomes as required herein is a general knowledge in the art, and any suitable separation method can be used. For example, the separation method may include affinity chromatography or magnetic antibody beads. If an affinity column chromatography device is used, for example, the sample may pass through an affinity column containing an H1 binding agent. Depending on the use of the positive or negative selection method as described herein, solid-phase-bound nucleosomes or a collection fluid (ie, unbound nucleosomes) can be collected for analysis of ctDNA.

In a preferred embodiment, tumor nucleosomes and ctDNA are separated by an immunoaffinity purification method using a binding agent that binds to histone H1. Those skilled in the art know that any binding agent that can bind to a specific histone H1 can be used in the affinity purification method of the present invention. Such binding agents may include, but are not limited to, antibodies, aptamers, or binding proteins (e.g., nucleosome binding proteins).

Antibodies can be produced by a variety of methods known in the art, including immunization and protein display library methods such as phage display. An immune response can be induced against the part or antigen of interest, or a display library can be selected to bind to it. Antibodies that bind to histone H1 can be produced against some parts, including the entire H1 The amino acid sequence of the protein can optionally contain post-translational histone modifications. Proteins can be purified or synthesized from living cells. Alternatively, a peptide sequence that represents a part of the H1 amino acid sequence can be used, and this can also optionally contain post-translational histone modifications. Nucleosomes or other chromatin fragments containing histone H1 can also be used.

Those skilled in the art will know that a binding agent that binds to any part or all of histone H1 can be used in the method of the present invention.

Any suitable analysis method can be used to analyze the isolated tumor-derived nucleosomes, many of which are known in the art. These methods include, but are not limited to: the use of secondary antibodies or other binding agents to interact with common nucleosome epitopes such as dsDNA, or epigenetic structures of interest (including histone modifications, histone variants, DNA modifications or adducts) Another molecule of nucleosome) binding immunoassay. These methods include all methods described in documents WO 2005/019826, WO 2013/030577, WO 2013/030579 and WO 2013/084002, in which a histone H1 binding agent is used instead of a general anti-nucleosome epitope binding agent. These methods also include multiple methods for analyzing multiple epitopes of circulating nucleosomes derived from tumors.

The method for analyzing and separating tumor-derived nucleosomes of the present invention may involve proteomics methods known in the art, including but not limited to: including but not limited to electrophoresis method, chromatographic methods and any Methods involving mass spectrometry, including methods involving chromatography and mass spectrometry, and/or mass spectrometry with stable isotope labeling, and/or methods involving protein digestion to produce peptides, for mass spectrometry Identification and/or quantification

In a preferred embodiment of the present invention, the addition of tumor-derived circulating nucleosomes preparations is through affinity purification of circulating nucleosomes from blood, serum or plasma samples obtained from cancer patients, And by involving mass spectrometry analysis methods to analyze the epigenetic composition of nucleosomal preparations. Therefore, in one embodiment, the method includes the following steps: (i) contacting the sample with the histone H1 binding agent; (ii) separating the nucleosome portion that is free or bound to the histone H1 binding agent, and Isolating one of the parts; and (iii) using mass spectrometry to analyze the nucleosomes separated in step (ii).

Those with ordinary knowledge in the art will know that according to the positive or negative selection method described herein, which nucleosome part is to be separated (ie, the bound part of the positive selection method or the free/precipitated part of the negative selection method) will be determined.

According to another technical solution of the present invention, a method for separating and purifying tumor DNA from a biological sample is provided, wherein the method includes the following steps: (i) contacting the sample with a histone H1 binding agent; (ii) separating and The nucleosomes bound by the binding agent; (iii) extracting DNA from the nucleosome sample separated in step (ii); and (iv) analyzing the extracted DNA.

According to another technical solution of the present invention, a method for separating and purifying tumor DNA from a biological sample is provided, wherein the method includes the following steps: (i) contacting the sample with a binding agent, the binding agent being selected from H1 .1, H1.2, H1.3, H1.4, H.1.5 or H1.6 histone H1 variants or histone H1 modifications; (ii) separation of nucleosomes bound to the binding agent; (iii) Extracting DNA from the nucleosome sample separated in step (ii); and (iv) analyzing the extracted DNA.

The study of purified or isolated tumor DNA may involve the analysis of any or all types of cancer-related DNA abnormalities, including but not limited to: epigenetic analysis, including DNA sequence methylation, point mutations, translocations (translocations), gene copy number (gene copy number), micro-satellite abnormalities and DNA strand integrity. In addition, any DNA analysis method can be used, including but not limited to: DNA sequencing, methylated DNA sequencing, PCR, bead magnetization (BEAMing), NGS (targeted or whole genome), digital PCR (digital PCR) PCR), cold PCR (co-amplification PCR at lower denaturation temperature), MAP (MIDI-activated pyrophospholysis), personalized analysis of rearranged ends (PARE) and mass spectrometry analysis.

In an embodiment of the method of the present invention, the biological sample is whole blood, serum, plasma, cerebrospinal fluid, urine, feces, sputum, saliva or other body fluids, or exhalation, concentrated exhalation or extracts or purifications thereof , Or its dilution. In another embodiment, the biological sample is blood, serum or plasma. In another embodiment, the biological sample is serum.

Methods of collecting biological samples are well known in the art, and it should be understood that any such collection method is suitable for use with the methods described herein.

Further epigenetic markers

Once the tumor-derived augmented nucleosome has been isolated, its epigenetic markers ("epigenetic epitopes") can be further analyzed or further augmentations can be made.

Therefore, according to a technical solution of the present invention, an immunoassay method for detecting epigenetic epitopes of tumor-derived nucleosomes in biological samples is provided, wherein the method includes the following steps: (i) Contact the sample with a first binding agent that binds to histone H1 or a histone H1 variant selected from H1.0, H1.10 or H1.X; (ii) separate unbound The nucleosomes of the first binding agent (ie unbound nucleosomes); (iii) contacting the nucleosomes obtained in step (ii) with the second binding agent that binds to the epitope; (iv) detecting and /Or quantify the binding of the second binding agent to the epitope; and (v) use the presence or degree of this binding to determine the presence of a specific epitope of tumor-derived nucleosomes in the sample.

According to another technical solution of the present invention, there is provided an immunoassay method for detecting epigenetic epitopes of tumor-derived nucleosomes in a biological sample, wherein the method includes the following steps: (i) combining the sample with the first A binding agent is contacted, and the first binding agent binds histone H1 variants or histone H1 modifications selected from H1.1, H1.2, H1.3, H1.4, H.1.5 or H1.6; (ii) Separating the nucleosomes bound to the first binding agent; (iii) contacting the nucleosomes obtained in step (ii) with a second binding agent that binds to the epitope; (iv) detection and/or quantification The binding of the second binding agent to the epitope; and (v) using the presence or extent of this binding to determine the presence of a specific epitope of tumor-derived nucleosomes in the sample.

In one embodiment, the epitope is selected from histone modifications, modified nucleotides, histone variants or isomers, or nucleosome adducts or variants thereof. In another embodiment, the epitope comprises a histone modification. In another embodiment, the histone modification comprises H3K27Ac and/or 5-methylcytosine.

The literature has described a variety of epigenetic modified nucleotides, and it is known that the pattern of epigenetic modification in DNA and/or DNA nucleotide residues can be altered in cancer. The best description includes the methylation of cytosine at position 5. DNA containing 5-methylcytosine (5-methylcytosine) is often referred to as "methylated DNA." Compared with the DNA of healthy cells, the methylation of DNA in cancer cells is estimated to be reduced by about 50% (Guerrero-Preston et al , 2007; Soares et al , 1999). However, it has been reported that cancer-related circulating nucleosomes have increased by an average of 970% (Holdenrieder et al , 2001), and cfDNA has increased by approximately 600% (Schwarzenbach et al , 2011).

Further epigenetic epitope measurement can be performed alone or as part of a measurement group.

In one embodiment, the isolated nucleosome sample obtained by the method described herein is subjected to a further addition step. Therefore, in one embodiment, the method of the present invention further comprises contacting the isolated nucleosomes with histone H3.1 and/or H3.2 and/or H3t binding agents. It has previously been shown that histone 3 variants H3.1, H3.2 and H3t can be used for the addition of ctDNA. In this embodiment, the method steps may include: (i) contacting the sample with the histone H1 binding agent; (ii) separating the part of the nucleosome that is free or bound to the histone H1 binding agent, and separating the part of the nucleosome One; (iii) contact the separated part with histone H3.1 and/or H3.2 and/or H3t binding agent; (iv) separate bound nucleosomes from the sample; and (v) analyze the separated nucleus Body and/or related DNA.

It should be understood that any of the above methods can be used as a standalone method or in combination with existing test methods.

diagnosis method

The methods described herein can be used in conjunction with diagnostic methods. For example, the method described herein can be used to add samples to isolate tumor-derived DNA or cell-free nucleosomes, and then this added sample The product can be used in diagnostic methods by detecting additional epigenetic markers associated with the disease.

Therefore, according to another technical solution of the present invention, a method for diagnosing or detecting cancer in an animal or human subject is provided. The method includes the following steps: (i) obtaining a biological sample from the subject; (ii) making the sample Contact with the histone H1 binding agent; (iii) separating the nucleosome part that is free or bound to the histone H1 binding agent, and separating one of the parts; (iv) making the isolated nucleosome obtained in step (iii) Contact with a second binding agent that binds to the epigenetic epitope of the tumor-derived nucleosome; (v) detecting and/or quantifying the binding of the second binding agent to the epitope; and (vi) using the The measured amount of biomarker serves as an indicator of the presence of the disease in the subject.

Detection and/or quantification can be performed directly on the purified or augmented nucleosome sample, or indirectly on its extract or its dilution. Quantifying the amount of biomarkers present in a sample can include determining the concentration of biomarkers present in the sample. The use and method of detection, monitoring and diagnosis of the present invention can be used to confirm the existence of a disease, monitor the development of the disease by assessing the onset and progression, or evaluate the improvement or regression of the disease. The uses and methods of detection, monitoring, and diagnosis are also useful in methods for evaluating clinical screening, prognosis, treatment options, and treatment effect evaluation, namely drug screening and drug development.

The diagnostic method described herein may further include comparing the level of the second binding agent present in the biological sample with one or more control groups. In one embodiment, one or more biological samples of the control group are taken from healthy (or "normal") patients and/or patients with related benign diseases. In another embodiment, one or more biological samples of the control group are taken from healthy patients.

As described herein, the method of the present invention can use two kinds of immunoassays in combination. therefore, According to another technical solution of the present invention, there is provided a method for diagnosing or detecting cancer in an animal or human subject, the method comprising the following steps: (i) obtaining a biological sample from the subject; (ii) by using The immunoassay of two binding agents analyzes the sample containing epigenetic features and also contains histone H1 cell-free nucleosomes. One of the binding agents is aimed at binding specific epigenetic features, and the other is aimed at binding histone H1; ( iii) Analyze cell-free nucleosomes with specific epigenetic characteristics in the sample by immunoassay using two binding agents, the epigenetic characteristics with and without histone H1, one of which binds to the apparent Genetic characteristics, another binding agent involves binding to include any histone H2, H3 or H4 epitope, any DNA epitope or any common core nucleosome epitope existing in chromatin fragments except histone H1 epitope; And (iv) using the combination of immunoassay results obtained in steps (ii) and (iii) as a combined biomarker indicating the presence of the cancer in the subject.

treatment method

According to another technical solution of the present invention, there is provided a method of treating cancer in an animal or human subject, the method comprising the following steps: (i) contacting a biological sample obtained from the subject with a histone H1 binding agent (Ii) Separate the part of the nucleosome that is free or bound to the histone H1 binding agent, and separate one of the parts; (iii) combine the isolated nucleosome obtained in step (ii) with the tumor-derived nucleus Contact with the second binding agent of the epitope of the body; (iv) detecting and/or measuring the binding amount of the second binding agent to the epitope; (v) Use the amount of the biomarker measured in step (iv) as an indicator of the presence of the disease in the subject; and (vi) perform surgery on a patient diagnosed with the disease in step (v) Treat or administer a therapeutic agent.

As described herein, determine which part to separate based on the histone H1 binding agent used and the need for negative or positive selection methods.

As described herein, the method of the present invention can use two immunoassays in combination. Therefore, according to another technical solution of the present invention, there is provided a method of treating cancer in an animal or human subject, the method comprising the following steps: (i) obtaining a biological sample from the subject; (ii) by using The immunoassay method of two binding agents analyzes the cell-free nucleosomes containing epigenetic characteristics and also containing histone H1 in the sample. One of the binding agents is used to bind specific epigenetic characteristics, and the other is used to bind histone H1 (Iii) Analyze cell-free nucleosomes with specific epigenetic features in the sample by immunoassay using two binding agents, the epigenetic features with and without histone H1, one of which binds Epigenetic characteristics, another binding agent involves binding to any histone H2, H3, or H4 epitope, any DNA epitope, or any common core nucleosome that exists in chromatin fragments other than histone H1 epitope (Iv) use the amount of biomarkers measured in steps (ii) and (iii) as an indication of the presence of the cancer in the subject; Patients with the diseases are treated surgically or administered therapeutic agents.

The diagnostic methods described herein may further include comparing the amount of biomarkers present in the biological sample with one or more control groups. In one embodiment, one or more biological samples of the control group are taken from healthy (or "Normal") patients and/or patients with related benign diseases. In another embodiment, one or more biological samples of the control group are taken from healthy patients.

Therefore, according to another technical solution of the present invention, there is provided a method of treating cancer for an individual in need, the method comprising administering a therapeutic agent to a patient whose biological sample is identified as having a different amount of biomarker compared to a control group A step of.

In one embodiment, the cancer is selected from: breast cancer, bladder cancer, colorectal cancer, skin cancer (e.g. melanoma), ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, bowel cancer, liver cancer, endometrial cancer, Lymphoma, oral cancer, head and neck cancer, leukemia and osteosarcoma.

The therapeutic agents and surgical methods used to treat the diseases are known to those with ordinary knowledge in the art. Methods of treating cancer include, but are not limited to: surgery, chemotherapy, radiation therapy or other therapeutic agents (such as drugs or biological therapies, such as monoclonal antibodies).

use

According to another technical solution of the present invention, the use of a histone H1 binding agent for detecting, separating and/or purifying tumor-derived cell-free nucleosomes in a biological sample is provided. Alternatively, according to another technical solution of the present invention, a use of a histone H1 binding agent for detecting, separating and/or purifying a cell-free nucleosome derived from healthy tissue in a biological sample is provided. As described herein, the presence or absence of histone H1 or the presence of certain histone H1 variants and modifications are associated with cell-free nucleosomes derived from tumors or healthy tissues, so histone H1 binding agents can be used as biomarkers The nucleosomes are detected.

In one embodiment, the cell-free nucleus system is isolated and/or purified.

Set

According to another aspect of the present invention, there is provided the use of a reagent kit comprising a histone H1 binding agent in any of the methods described herein.

It should be understood that the embodiments described herein can be applied to all technical solutions of the present invention. In addition, all works cited in this specification, including but not limited to patents and patent applications, are incorporated by reference as described herein.

The following non-limiting examples will illustrate the invention.

Example 1

Use the method recommended by the manufacturer to biotinylate (biotinylate) and immobilize antibodies that specifically bind to histone H1 variants H1.0, H1.10, or H1.X on streptavidin-coated magnetic beads (Dynal) on. A magnetic separation system was used to wash the beads with loading buffer several times. The serum or plasma taken from the cancer patient is diluted in the loading buffer and added to the beads. Nucleosomes derived from healthy cells with histone H1 variants are adsorbed to the beads. Tumor-derived nucleosomes (which do not contain histone H1 variants) remain in solution. The beads are removed by magnetic separation, leaving a solution containing tumor-derived nucleosomes. The ctDNA associated with nucleosomes is extracted by the phenol/chloroform method or other standard extraction methods. The extracted DNA can be analyzed for cancer genetic or epigenetic characteristics.

Example 2

Fix histone H1 variant H1.0, H1.10 or H1.X specific binding antibody on the solid support by the method recommended by the solid phase column manufacturer to produce affinity purification chromatography column. The serum or plasma taken from the cancer patient is diluted in the loading buffer and added to the column. Nucleosomes derived from healthy cells containing histone H1 variants are adsorbed onto the column. Tumor-derived nucleosomes (which do not contain histone H1 variants) remain in the solution and pass through the column to be collected as a eluate. With phenol/chloroform Method (phenol/chloroform method) or other standard extraction methods to extract ctDNA associated with nucleosomes. Analyze the genetic or epigenetic characteristics of the cancer from the extracted DNA.

Example 3

The solid phase immobilized anti-H1 variant antibody (anti-H1 variant antibody) as in Example 1 or Example 2 was used to isolate a solution of tumor-derived nucleosomes. The isolated nucleosomes present in the solution are analyzed by immunoassay or proteomics methods (including mass spectrometry).

Example 4

Use the method recommended by the manufacturer to biotinylate and fix histone H1 variants H1.1, H1.2, H1.3, H1.4, H1.5, or H1.6 with antibodies that specifically bind to it. On magnetic beads (Dynal) with streptavidin (streptavidin). A magnetic separation system was used to wash the beads with loading buffer several times. The serum or plasma taken from the cancer patient is diluted in the loading buffer and added to the beads. Tumor-derived nucleosomes with histone H1 variants are adsorbed to the beads. Separate the beads from the solution by magnetic separation. The ctDNA associated with the nucleosome is extracted from the solid-phase magnetic beads by the phenol/chloroform method or other standard extraction methods. Analyze the extracted DNA for cancer genetic or epigenetic characteristics.

Example 5

Fix histone H1 variants H1.1, H1.2, H1.3, H1.4, H1.5 or H1.6 specific binding antibodies on the solid-phase carrier by the method recommended by the solid-phase column manufacturer , To produce affinity purification chromatography column. The serum or plasma taken from the cancer patient is diluted in the loading buffer and added to the column. Tumor-derived nucleosomes containing histone H1 variants are adsorbed to the column. Nucleosomes derived from healthy cells (which do not contain histone H1 variants) remain in solution and pass through the column. The column is washed and ctDNA associated with nucleosomes is extracted by the phenol/chloroform method or other standard extraction methods. Analysis by The genetic or epigenetic characteristics of the cancer in the extracted DNA.

Example 6

Preparation of solid-phase-bound nucleosomes derived from tumors using solid-phase anti-H1 variant antibodies as in Example 4 or Example 5. The isolated nucleosomes are analyzed by immunoassay or by proteomics methods including mass spectrometry.

Example 7

By the method recommended by the manufacturer, biotinylate (biotinylate) and immobilize the antibody that specifically binds to histone H1 itself on magnetic beads (Dynal) coated with streptavidin. A magnetic separation system was used to wash the beads with a loading buffer several times. The serum or plasma taken from the cancer patient is diluted in the loading buffer and added to the beads. Nucleosomes with histone H1 are adsorbed to the beads. Tumor-derived nucleosomes (not containing histone H1) remain in solution. Separate the beads from the solution by magnetic separation. Once the beads are removed, the separated nucleosomes are analyzed by immunoassay or proteomics methods including mass spectrometry.

Example 8

Use the method recommended by the solid-phase column manufacturer to immobilize the antibody specifically bound to histone H1 itself on a solid-phase carrier to produce an affinity purification chromatography column. The serum or plasma taken from the cancer patient is diluted in the loading buffer and added to the column. Nucleosomes containing histone H1 are adsorbed onto the column. Tumor-derived nucleosomes (which do not contain histone H1) remain in solution and pass through the column. Collect the eluate containing nucleosomes. The isolated nucleosomes are analyzed by ELISA or proteomics methods including mass spectrometry.

Example 9

The tumor-derived nucleosome solution was prepared as in Example 7 or Example 8. By phenol/chloroform Method or other standard extraction methods to extract nucleosome-related ctDNA from the solution. It can analyze the cancer genetic or epigenetic characteristics of the extracted DNA.

Example 10

The solid-phase immobilized anti-H1 antibody as described in any one of Examples 1-9 was used to isolate tumor-derived nucleosomes from a body fluid sample of a subject. The isolated nucleosomes and/or related DNA present in the solution are analyzed by methods known in the art.

Example 11

By the method recommended by the manufacturer, biotinylate and immobilize antibodies that specifically bind to histone H1 itself or histone H1 variants on magnetic beads (Dynal) coated with streptavidin. Use a magnetic separation system to wash the beads with loading buffer several times. The serum or plasma taken from the cancer patient is diluted in the loading buffer and added to the beads. Nucleosomes derived from healthy cells with histone H1 variants are adsorbed to the beads. Tumor-derived nucleosomes (not containing histone H1 variants) remain in solution. The beads are separated from the solution by magnetic separation to leave a solution of nucleosomes mainly derived from the tumor.

Then use the ELISA method to determine the nucleosome-associated nucleotide methylated DNA in the tumor nucleosome solution, which uses solid phase anti-histone H1, H3 and/or H4 tables that bind intact nucleosomes Position or anti-nucleosome (anti-nucleosome) capture antibody (capture antibody), and biotinylated monoclonal antibody anti-5-methylcytosine (anti-5-methylcytosine) detection antibody (detection antibody), its steps As described below:

Add a 0.1M phosphate buffer solution (pH 7.4) containing anti-histone H2, H3, or H4 antibodies to a micro multi-well plate (100 microliters (μL)/well), and incubate at 4°C overnight to coat the wells. Cover with capture antibody. Slowly pour out excess anti-histone antibody. The bovine serum albumin (20g/L) solution was added to the well plate (200μL/well) and incubated at room temperature for 30 minutes to block the excess protein binding sites on the well.

The excess bovine serum albumin solution was removed, and the wells were washed three times with washing buffer (200 μL/well, 0.05M Tris/HCl buffer pH 7.5 containing 1% Tween 20). Add sample (10μL/well) and buffer (50μL/well, containing 0.9% NaCl, 0.05% sodium deoxycholate, and 1% Nonidet P40 substituted 0.05M Tris/HCl pH 7.5) to the well In, shake gently for 90 minutes at room temperature. Slowly remove the mixture of sample and buffer, and wash the wells with washing buffer (200 μL/well) three times.

Add biotinylated anti-5-methylcytosine antibody solution (50 μL/well), and gently shake at room temperature for 90 minutes. Excess antibody was removed, and the wells were washed again three times with washing buffer (200 μL/well). Add a solution (50 μL/well) containing streptavidin-horse radish peroxidase conjugate, and shake gently for 30 minutes at room temperature.

The excess conjugate was removed, and the well was washed again with buffer (200 μL/well) three times. Add the colored matrix solution (100μL/well, 2,2-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (2,2'-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid) ]-diammonium salt)), and gently shake at room temperature for 20 minutes to react. The optical density (OD) of the wells was measured at a wavelength of 405nm using a standard microplate reader.

Observe the dose-response curve that increases the color due to the increased nucleosome-related anti-5-methylcytosine (anti-5-methylcytosine) concentration, which is observed in the control without 5-methylcytosine (fetal bovine serum) Low background signal. A positive ELISA signal indicates that 5-methylcytosine has been detected in intact histones including histones and DNA except histone H1 by ELISA; because (i) the nucleosomes containing histone H1 have been removed by affinity purification , (Ii) capture the antibody binding histone H2, H3 and/or H4 epitope in the sample, and (iii) detect the 5-methylcytosine portion of the antibody binding DNA.

Example 12

The solid phase-immobilized anti-H1 antibody as described in Example 11 was used to isolate tumor-derived nucleosomes in a body fluid sample taken from a subject. The isolated nucleosomes were analyzed by the ELISA method described in Example 11, but using a biotinylated monoclonal anti-modified histone antibody, such as an antibody that binds to H3K9(Me)3.

Example 13

The solid phase-immobilized anti-H1 antibody as described in Example 11 was used to isolate tumor-derived nucleosomes from a body fluid sample taken from a subject. The isolated nucleosomes were analyzed by the ELISA method described in Example 11, but using biotinylated monoclonal anti-histone variant antibodies, such as antibodies that bind H2AZ.

Example 14

The solid phase-immobilized anti-H1 antibody as described in Example 11 was used to isolate tumor-derived nucleosomes from a body fluid sample taken from a subject. The isolated nucleosomes were analyzed by the ELISA method described in Example 11, but using biotinylated monoclonal antibodies that bind to non-histone proteins adducted to the isolated circulating cell-free nucleosomes; for example, binding Antibodies to the androgen receptor. The results show that tumor-derived cell-free nucleosomes added to the protein are present in the blood circulation (or other body fluids) of the subject.

Example 15

Serum samples were taken from 29 healthy subjects, 29 subjects diagnosed with colorectal cancer, and 11 subjects diagnosed with pancreatic cancer. Plastic microwell plates are coated with antibodies that bind to non-histone chromatin protein and blocked according to standard procedures. Biotinylation is used to label antibodies that bind to histone H1 itself and antibodies that bind to the common core histones present on all or most of the nucleosomes. Two types of immunoassays were performed on each sample.

In the first immunoassay, the sample (10μL/well) and analysis buffer (50μL/well, containing 0.9% NaCl, 0.05% sodium deoxycholate and 1% Nonidet P40 substituted 0.05M TRIS/HCl (pH 7.5) were added to the wells, and gently shaken at room temperature for 90 minutes. The mixture of sample and buffer was removed, and the wells were washed three times with washing buffer (200 μL/well). Add biotinylated anti-histone H1 detection antibody solution (50 μL/well), and shake gently for 90 minutes at room temperature. Excess detection antibody was removed, and the well was washed again three times with washing buffer (200 μL/well). Add a solution containing streptavidin-horseradish peroxidase conjugate conjugate (50 μL/well), and gently shake at room temperature for 30 minutes. The excess conjugate is removed, and the wells are washed again three times with washing buffer (200 μL/well). Add the colored matrix solution (100μL/well, 2,2-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (2,2'-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid) ]-diammonium salt)) and gently shake at room temperature for 20 minutes. The optical density 1 (OD1) of the immunoassay of the wells was measured at a wavelength of 405 nm using a standard microplate reader. All measurements are performed twice, and the average result is used.

In the second immunoassay, the sample (10μL/well) and analysis buffer (50μL/well, containing 0.9% NaCl, 0.05% sodium deoxycholate (sodium deoxycholate) and 1% Nonidet P40 substituted 0.05M Tris/ HCl (pH 7.5) was added to the wells, and gently shaken for 90 minutes at room temperature. The sample and buffer mixture was removed, and the wells were washed three times with washing buffer (200 μL/well). Add biotinylated anti-core histone detection antibody solution (50 μL/well), and shake gently for 90 minutes at room temperature. Excess detection antibody was removed, and the well was washed again three times with washing buffer (200 μL/well). Add a solution containing streptavidin-horseradish peroxidase conjugate conjugate (50 μL/well), and gently shake at room temperature for 30 minutes. The excess conjugate is removed, and the wells are washed again three times with washing buffer (200 μL/well). Add the colored matrix solution (100μL/well, 2,2-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (2,2'-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid) ]-diammonium salt)) and gently agitate for 20 minutes at room temperature. The optical density 2 (OD2) of the well was measured at a wavelength of 405 nm using a standard microwell plate reader. All measurements are made twice and the average result is used.

For each sample, calculate the ratio of OD2:OD1. The OD2:OD1 ratio results of all 69 subjects are shown in Figure 1. For 29 healthy subjects, the average immunoassay ratio was found to be 8.7. For 29 subjects diagnosed with colorectal cancer, the average ratio was 15.9. For the 11 subjects diagnosed with pancreatic cancer, the average ratio was 17.1. This shows that the increased ratio is related to cancer-derived nucleosomes.

In addition, the individual results of each subject are used as a cancer biomarker with a rising threshold. When the cut-off value is 12, this ratio gives positive results to 73% (8/11) of the subjects diagnosed with pancreatic cancer and 41% (12/29) of the subjects diagnosed with colorectal cancer, which is clinically specific It was 79% (6 false positive results in 29 healthy subjects). When the cut-off value is 22, this ratio gives a positive result for 36% (4/11) of subjects diagnosed with pancreatic cancer and 34% (10/29) of subjects diagnosed with colorectal cancer, which is clinically specific Sex was 93% (2 false positive results out of 29 healthy subjects). The results in Figure 1 show that the methods described herein can be used to detect cancer. These results also demonstrate that the combined immunoassay results as described herein can be used as a combined biomarker for cancer diseases.

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Claims (20)

A method for analyzing tumor-derived cell-free nucleosomes in a biological sample by affinity purification, comprising: contacting the sample with a histone H1 binding agent; separating the nucleosomes from the sample that is not bound to the histone H1 binding agent ; And analyze the separated nucleosomes by immunoassay or mass spectrometry. The method described in item 1 of the scope of patent application, wherein the histone H1 binding agent is a binding agent that binds to histone H1 protein, variants, isoforms, or modifications thereof. The method described in item 1 or item 2 of the scope of patent application, wherein the histone H1 binding agent is combined with histone H1 or a histone H1 selected from histone H1.0, H1.10 or H1.X Binder for binding of variants. The method described in item 1 of the scope of patent application, wherein the isolated nucleosomes are analyzed for epigenetic characteristics by immunoassay and mass spectrometry methods, and the epigenetic characteristics include histone modifications after translation, and histone similarities and differences Constructs, modified nucleotides, or adducts non-histone proteins. The method according to claim 1, wherein the step of analyzing the isolated nucleosome includes immunoassay. The method according to claim 1, wherein the step of analyzing the separated nucleosomes includes mass spectrometry. A method for separating purified tumor DNA from a biological sample, wherein the method comprises the following steps: contacting the sample with a histone H1 binding agent; separating nucleosomes that are not bound to the histone H1 binding agent; Extracting DNA from the separated nucleosomes; and analyzing the extracted DNA. The method described in item 7 of the scope of patent application, wherein the steps of analyzing the extracted DNA include: DNA sequencing, methylated DNA sequencing analysis, polymerase chain reaction, pearl milk magnetization technology, next-generation sequencing technology ( Target gene or all genes), digital PCR, cold PCR (lower denaturation temperature co-amplification PCR), MIDI-Activated Pyrophosphorolysis (MIDI-Activated Pyrophosphorolysis), Personalized Analysis of Rearranged Ends (PARE), Or mass spectrometry. The method described in item 1 or 7 of the scope of the patent application further comprises contacting the isolated nucleosome with a histone H3.1 and/or H3.2 and/or H3.2 and/or H3t binding agent. An immunoassay method for detecting epigenetic epitopes of nucleosomes derived from tumors in biological samples, wherein the method includes: contacting the sample with a first binding agent containing a histone H1 binding agent; The nucleosome bound by the first binding agent; contacting the nucleosome with the second binding agent to bind to the epitope of the nucleosome; detecting and/or quantifying the binding of the second binding agent to the epitope ; And using the presence or extent of the binding to determine the presence of a specific epitope of tumor-derived nucleosomes in the sample. The method according to claim 10, wherein the epitope includes histone modification. The method described in claim 11, wherein the histone modification includes H3K27Ac and/or 5-methylcytosine. The method according to claim 10, wherein the epitope includes a modified nucleotide. The method according to claim 10, wherein the epitope includes a variant or isoform of histone H2A, H2B, H3, or H4. The method according to item 10 of the scope of patent application, wherein the epitope includes a nucleosome adduct or a variant thereof. The method according to any one of items 1, 7 or 10 in the scope of the patent application, wherein the biological sample includes blood, serum, plasma, menstrual blood, cerebrospinal fluid, endometrial fluid, urine, saliva, feces, tears, Synovial fluid, sputum, or other body fluids, exhalation, extracts, purifications, or dilutions thereof, this breathing includes concentrated exhalation. The method according to claim 16, wherein the biological sample includes blood, serum or plasma. A method for detecting cancer in animal or human subjects, including the following steps: obtaining a biological sample from the subject; using an immunoassay method using two binding agents to analyze that the sample contains epigenetic characteristics and contains histone H1 In the cell-free nucleosomes, one binding agent is used to bind the specific epigenetic characteristics, and the other binding agent is used to bind the histone H1; through the immunoassay method using two binding agents, the analysis sample contains a specific table Circulating cell-free nucleosomes of epigenetic characteristics, the circulating cell-free nucleosomes including nucleosomes with histone H1 and those without histone H1, wherein a binding agent binds the epigenetic characteristics of the circulating cell-free nucleosomes, Another binding agent binds to a common core nucleosome epitope, which includes any epitope in histone H2, H3, or H4 epitope, any DNA epitope, or is present in chromatin fragments other than the group Any epitope other than the protein H1 epitope; and the result of comprehensive immunoassay as a biomarker for the subject's cancer. The method according to claim 18, wherein the cancer is selected from breast cancer, bladder cancer, colorectal cancer, skin cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, colon cancer, liver cancer, and uterus Endometrial cancer, lymphoma, oral cancer, head and neck cancer, leukemia, and osteosarcoma form the group. The skin cancer includes melanoma. A use of a kit including histone H1 binding agent in the method described in item 1, 7, 10 or 18 of the scope of the patent application.

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