Use of extracellular free nucleosome as biomarker

Abstract

To provide a simple and highly cost-effective diagnostic method for vascular cancer or hematological cancer, in particular, a method enabling discrimination from patients who suffer from other kinds of diseases or non-vascular cancer or non-hematological cancer but showing the same symptoms.SOLUTION: Provided is the use of extracellular free nucleosome as a biomarker in a plasma sample for diagnosing or detecting vascular cancer or hematological cancer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention provides extracellular free nucleic acids as biomarkers in plasma samples for vascular or hematological cancers. Regarding leosomes. [Background technology]

[0002] BACKGROUND OF THE INVENTION Hematological cancers are types of cancer that affect the blood, bone marrow, and lymph nodes. Depending on the type of cell, they are called leukemia, lymphoma, and myeloma. Leukemia usually originates in the bone marrow. In leukemia, the bone marrow is suddenly transformed into a cancer of blood cells that develop and travel through the bloodstream. The mutant cells are produced and dispersed into the bloodstream, where they multiply and replace healthy blood cells. It blocks out lymphocytes. Lymphoma affects the cells of the lymphatic system. In this condition, immune cells called lymphocytes multiply uncontrollably and accumulate in the lymph nodes, spleen, and other lymphoid tissues. Myeloma, also called multiple myeloma, develops in the bone marrow and causes sensitization. It affects plasma cells, which produce antibodies that fight infections and diseases. Examples of blood cancers include acute lymphoblastic leukemia (AML), acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML). Myeloid leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma (HL), and non-Hodgkin's lymphoma I have non-Hodgkin lymphoma (NHL).

[0003] Reference to "acute leukemia" refers to a disease that progresses rapidly and aggressively, usually requiring immediate treatment. ALL means cancer. ALL involves the development of large numbers of immature lymphocytes that are unable to fight infection. This leaves less room for healthy white blood cells, red blood cells, and platelets in the patient's circulation. As a result, patients typically experience a weakened immune system and symptoms of anemia, such as fatigue and shortness of breath. Children under the age of 5 are at increased risk of developing ALL. It is the most common type of leukemia affecting children, with the highest risk in children in their mid-twenties. It gradually decreases until age 50 and then starts to gradually increase again after age 50. Overall, 10% of ALL cases Approximately four in every 10 cases are in adults.

[0004] AML affects the bone marrow blasts, causing the accumulation of abnormal monocytes and granulocytes in the bone marrow. It can also affect bone marrow stem cells, causing abnormal red blood cells or platelets. Like ALL, AML reduces the levels of healthy white blood cells, red blood cells, and platelets in a patient's circulation. It is one of the most common types of leukemia in the United States, with an average age at diagnosis of 68 years.

[0005] HL and NHL are the two main types of lymphoma. HL has a specific appearance under a microscope, while While NHL contains cells called De-Sternberg cells (a type of cancerous B lymphocyte), look different under the microscope and do not contain Reed-Sternberg cells. NHL is a cancer that affects lymphocytes, and only about 1 in 5 cases is HL. It usually starts in the lymph nodes or lymphatic tissue. It is common among children, teenagers and young adults. It is one of the more common cancers in the world.

[0006] The current method of diagnosing leukemia and myeloma is to obtain a complete blood count (CBC) test result to identify the leukemia. It involves identifying abnormal levels of white blood cells relative to blood cells and platelets. Elevated white blood cell (WBC) counts are not specific to patients with hematological malignancies, and the event may be due to an infection. or may be the result of an ongoing response to other inflammatory processes. X-rays, CT scans, or PET scans can be used to detect swollen lymph nodes, but these are also particularly It's not unusual.

[0007] A bone marrow or lymph node biopsy is required to confirm the diagnosis of hematologic cancer. Overdiagnosis of hematologic cancers early in the diagnostic process is invasive and potentially dangerous. and may result in unnecessary biopsies that are relatively costly to the healthcare provider. Biochemical analysis and / or immunophenotyping may also be used to confirm a hematological cancer diagnosis; The method is expensive to implement and therefore is typically only used at a later stage in the diagnostic process. can be.

[0008] Human angiosarcoma is a rare tumor that affects the endothelial cells that line the inside of blood vessels. Canine hemangiosarcoma is a rare vascular cancer that develops from the endothelium or wall of blood vessels. It is a common canine cancer that involves proliferation and is difficult to diagnose. is more common than ever, largely due to the genetic effects of inbreeding and the fact that dogs have a shorter lifespan than humans. Cancer detection in animal subjects Cancer detection in non-human animals presents additional challenges compared to the diagnosis of human cancer. often involves scanning, for example with magnetic resonance imaging or MRI scans, Animals will not remain motionless for the time required for scanning and must be anesthetized. Pet medical insurance is rare and may not cover cancer diagnosis or treatment. These tests are financially out of reach for many pet owners. In addition, human blood tests for cancer marker proteins typically do not detect animal proteins. Therefore, there are far fewer blood tests available in veterinary oncology.

[0009] Previously, Holdenrieder et al. (2001) Int J Cancer 95:114-120 reported that benign and malignant diseases It has been described that nucleosome levels can be detected in serum samples from patients with leukemia. However, the results presented for serum samples show that blood No difference was shown between the levels of circulating cells and those of liquid cancers, such as lymphoma. Histone modifications, histone variants, DNA modifications and adducts of extracellular free nucleosomes Epigenetic composition in terms of content is also being studied as a blood-based biomarker for cancer. WO 2005 / 019826, WO 2013 / 030577, WO 2013 / 030579, and WO 2013 / 084002 Please refer to.

[0010] A simple and cost-effective method for diagnosing vascular or blood cancers, especially for other types of cancers distinguishable from patients with vascular or non-hematological cancers who may present with similar symptoms There remains a need in the art to provide a method that can Summary of the Invention

[0011] (Summary of the Invention) According to a first aspect, a method for the diagnosis or detection of vascular or hematological cancers using a biomarker in a plasma sample is provided. The present invention provides the use of extracellular free nucleosomes as targets.

[0012] According to a further aspect, biomarkers in plasma samples for the diagnosis or detection of hematological cancers are provided. The present invention provides the use of extracellular free nucleosomes as a therapeutic agent.

[0013] According to a further aspect, the present invention provides a method for the detection or diagnosis of vascular cancer using a medicament comprising administering to a subject ... The present invention provides for the use of all extracellular free nucleosomes.

[0014] According to a further aspect, there is provided a method for the diagnosis or detection of vascular or hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to obtain extracellular free nucleosomes; detecting or measuring; and (ii) using the detected level of extracellular free nucleosomes to identify the vascular cancer or blood system cancer diagnosing a subject with

[0015] According to a further aspect, a method for determining the prognosis of a subject with vascular or hematological cancer is provided. There is: (i) contacting a plasma sample obtained from the subject with a binding agent to obtain extracellular free nucleosomes; detecting or measuring; and (ii) comparing the detected level of extracellular free nucleosomes with the prognosis of the vascular cancer or blood system cancer. and using the resultant mixture as an index.

[0016] In a further aspect, a patient having, suspected of having, or suffering from vascular or blood cancer is 1. A method for monitoring the effectiveness of a treatment in a susceptible subject, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to obtain extracellular free nucleosomes; detecting or measuring; and (ii) comparing the detected level of extracellular free nucleosomes with a plasma sample previously collected from the subject; and comparing the results of the treatment to determine the effectiveness of the treatment. [Brief explanation of the drawings]

[0017] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Concentrations of extracellular free nucleosomes containing H3.1 in plasma samples obtained from healthy human subjects and human subjects with acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), and non-Hodgkin's lymphoma (NHLym). [Figure 2] Concentrations of extracellular free nucleosomes containing H3.1 in plasma samples obtained from human subjects with different types of cancer. [Figure 3] Comparison of ELISA optical density (OD) results of extracellular free nucleosomes containing H3 citrullinated (H3cit) in plasma samples from healthy human subjects with (A) all types of hematological cancer tested and (B) human subjects with ALL, AML, and NHL. [Figure 4] Comparison of ELISA OD results of extracellular free nucleosomes containing H3K27Me3 in plasma samples from healthy human subjects with (A) results from all types of hematological cancer tested and (B) results from human subjects with ALL, AML, and NHL. [Figure 5] Comparison of ELISA relative light unit (RLU) results of extracellular free nucleosomes containing H4 panacetylation (H4panAc) in plasma samples from healthy human subjects with (A) all types of hematological cancer tested and (B) human subjects with ALL, AML, and NHL. [Figure 6] (A) Comparison of ELISA results for extracellular free nucleosomes containing histone isoform H3.1 (ng / ml) in plasma samples collected from healthy canine subjects with those diagnosed with lymphoma, and (B) ROC curve showing the detection of canine lymphoma using H3.1 results. [Figure 7](A) Comparison of ELISA results for extracellular free nucleosomes containing histone isoform H3.1 (ng / ml) in plasma samples collected from healthy canine subjects with those diagnosed with hemangiosarcoma, and (B) ROC curve showing the detection of canine hemangiosarcoma using H3.1 results. [Figure 8] Results of ELISA for extracellular free nucleosomes containing histone isoform H3.1 (ng / ml) and CRP (μg / ml) in serially collected plasma samples from two canine subjects undergoing treatment for hemangiosarcoma. [Figure 9] Results of ELISA for extracellular free nucleosomes containing histone isoform H3.1 (ng / ml) and CRP (μg / ml) in serially collected plasma samples from two canine subjects undergoing treatment for lymphoma. DETAILED DESCRIPTION OF THE INVENTION

[0018] (Detailed explanation) According to a first aspect, a method for the diagnosis or detection of vascular or hematological cancers by the detection of biomarkers in plasma samples is provided. The present invention provides the use of extracellular free nucleosomes as a marker. In particular, the cancer is a blood cancer. do.

[0019] Nucleosomes are the basic units of chromatin structure and contain eight highly conserved core His residues. from a protein complex consisting of pairs of histones H2A, H2B, H3, and H4. Around this complex, approximately 146 base pairs of DNA are wrapped. Another histone, H1 or H5 acts as a linker and is involved in chromatin compaction. DNA is often A series of nucleosomes in a structure that is often described as resembling "beads on a string." The chromatin wraps around the chromatin, which forms the basic structure of open, or euchromatin. In compacted or heterochromatin, this string forms coils and supercoils , resulting in a closed complex structure (Herranz and Esteller (2007) Methods Mol. Biol. 36 1:25-62).

[0020] Reference to "nucleosomes" refers to "extracellular free nucleosomes" when detected in a body fluid sample. Throughout this document, the term extracellular free nucleosomes may refer to 1 It is intended to include any extracellular free chromatin fragment containing more than 10 nucleosomes. It will be understood that the "epigenetic" of extracellular free nucleosomes referred to herein is "epigenetic signature," "epigenetic signal signature," or "epigenetic signal signature" "Signal structure" includes, but is not limited to, one or more histone post-translational modifications, histone isoforms, Nucleosomes in nucleosome-protein adducts, modified nucleotides, and / or nucleosomes The antibody may include a protein that binds to the antibody.

[0021] It is understood that extracellular free nucleosomes can be detected by binding to their components. As used herein, the term "component thereof" refers to a part of a nucleosome. The detection of extracellular free nucleosomes is not required, i.e., the entire nucleosome does not need to be detected. The components of the system are: histone proteins (i.e., histone H1, H2A, H2B, H3, or H4) ), histone post-translational modifications, histone variants or isoforms, nucleosomes proteins bound to nucleosomes (i.e., nucleosome-protein adducts), from a group consisting of DNA fragments associated with nucleosomes and / or modified nucleotides associated with nucleosomes For example, the component may be selected from histone (isoform) H3.1 or histone It can be H1 or DNA.

[0022] In the methods and uses of the present invention, the level of (extracellularly free) nucleosomes themselves may be measured. The reference to "the nucleosome itself" does not imply that the nucleosome has any epigenetic characteristics. Nucleic acids present in the sample, whether or not they contain The total level or concentration of nucleosomes refers to the total level or concentration of nucleosomes. This involves the detection of histone proteins common to all nucleosomes, such as histone H4. Therefore, the nucleosome itself is able to detect core histone proteins, e.g., histone H4. As described herein, histone proteins can be measured in eukaryotic cells. Structures known as nucleosomes that are used to compactly pack DNA in cells In WO 2016 / 067029 (which is incorporated herein by reference), As reported, certain histone variants, such as histone H3.1, H3.2, or H3 can be used to isolate extracellular free nucleosomes originating from tumor cells. Therefore, the total level of extracellular free nucleosomes of tumor origin can be detected.

[0023] Normal cell turnover in adults involves the production of hundreds of billions of cells per day through cell division. with a similar number of deaths, primarily due to apoptosis. chromatin is degraded into mononucleosomes and oligonucleosomes and removed from the cell. Under normal conditions, the circulating nucleosomes found in healthy subjects are released. Low levels have been reported in many cancers, autoimmune diseases, inflammatory conditions, stroke, and Elevated levels have been found in subjects with a variety of conditions, including myocardial infarction (H Oldenreider and Stieber (2009) Crit Rev Clin Lab Sci, 46(1):1-24).

[0024] Currently, nucleosome ELISA is generally used as a method to detect apoptosis, mainly It is used in cell culture (Salgame et al. (1997) Nucleic Acids Res, 25(3):680-681; H Oldenrieder et al. (2001) supra; van Nieuwenhuijze et al. (2003) Ann Rheum Dis, 62 :10-14), and is also used to measure circulating extracellular free nucleosomes in serum and plasma (Holden (2001)). Extracellular release of serum and plasma released into the circulation by dying cells. Nucleosome levels have been measured by ELISA in studies of many different cancers. Its use as a potential biomarker is being evaluated. The levels were reported to be high in most, but not all, cancers tested. However, patients with malignant tumors have significantly different serum nucleosome concentrations. It has been reported that some patients with advanced tumor disease have Some were found to have low circulating nucleosome levels within the range specified. (Holdenrieder et al., 2001).

[0025] The extracellular free nucleosomes may be mononucleosomes or oligonucleosomes, or can be a mixture thereof.

[0026] Mononucleosomes and oligonucleosomes were analyzed by enzyme-linked immunosorbent assay (ELISA). and can be detected by several reported methods (e.g., Salgame et al. (1997); Holdenrieder et al. (2001); van Nieuwenhuijze et al. (2003)). The assay typically uses anti-histone antibodies (e.g., anti-H2B, anti-H3, or anti-H1, H2A, H2B, H3 and H4) were used as capture antibodies, and anti-DNA or anti-H2A-H2B-DNA complex antibodies were used as detection antibodies. do.

[0027] Circulating nucleosomes are not a homogeneous group of protein-nucleic acid complexes. are a heterogeneous group of chromatin fragments resulting from chromatin digestion during cell death, Specific histone isoforms (or variants), post-translational histone modifications, and nucleotides or modified nucleotides, and protein adducts. Those skilled in the art will appreciate that increased nucleosome levels may be due to the presence of specific histone isoforms. nucleosomes containing specific post-translational histone modifications (or variants) nucleosomes containing specific nucleotides or modified nucleotides, and specific proteins nucleosomes containing protein adducts, containing specific epigenetic signals, These findings are likely related to the elevation of a subset of circulating nucleosomes. Assays for various types of chromatin fragments are known in the art (see, e.g., the references herein). WO 2005 / 019826, WO 2013 / 030579, WO 2013 / 030578, WO 2013 / (See 084002).

[0028] The biomarkers used in the uses and methods of the present invention include extracellular free nucleosomes themselves and This may be at the level of epigenetic signatures of extracellular free nucleosomes. The terms "epigenetic signal structure" and "epigenetic signature" are used in this It will be understood that these terms are used interchangeably in the specification. In one embodiment, the term "nucleosomal epigenetic feature" refers to a specific detectable feature of a nucleosomal gene. Tic features include: post-translational histone modifications, histone variants, specific nucleotides, and and protein adducts.

[0029] In one embodiment, the epigenetic features of the nucleosome include one or more histidines. Epigenetic analysis of extracellular free nucleosomes A key feature is the histone isoforms, e.g., the core nucleosomal histone isoforms. The histone H3 isoform may be a histone H4 isoform, in particular a histone H3 isoform.

[0030] The terms "histone variant" and "histone isoform" are used herein. Nucleosome structures can also be used interchangeably to identify different genes or splices. alternative histone isoforms that are synthesis products and have different amino acid sequences or Many histone isoforms can be identified by their variants. Histone variants are known in the art. Numerous histone variants can be classified into families. The nucleotide sequences of many histone variants are known, For example, the National Human Genome Research Institute (NHGRI) histone database (Marino-Ramirez et al., " Histone Database: An integrated resource for histones and histone fold-containing proteins ( The Histone Database: an integrated resource for histones and histone fold-conta "Histones Database Vol. 2011" and http: / / genome.nhgri.nih.gov / histones / com plete.shtml), GenBank (NIH gene sequence) database, EMBL nucleotide sequence database These are publicly available at the DNA Data Bank of Japan (DDBJ) and at the Variants of peptide H2 include H2A1, H2A2, mH2A1, mH2A2, H2AX, and H2AZ. In the present study, the histone isoforms of H3 include H3.1, H3.2, and H3t.

[0031] In one embodiment, the histone isoform is H3.1. As shown in the examples, H3.1 distinguishes subjects with vascular or hematological cancers from healthy subjects. H3.1 was effective in distinguishing 84% of patients with NHL from healthy controls with a specificity of 90%. This was particularly useful in identifying patients with lymphoma because it differentiated between the two groups (see Table 1).

[0032] Nucleosome structure can vary depending on post-translational modifications (PTMs) of histone proteins PTMs of histone proteins typically occur on the tails of core histones, and common modifications include acetylation, methylation, or ubiquitination of lysine residues, and methylation of arginine residues. Many histone modifications are known in the art, including cleavage, dehydrogenation, and phosphorylation of serine residues. known, and the number is increasing as new modifications are identified (Zhao and Garcia, Reference, 2015 Cold Spring Harb Perspect Biol, 7: a025064). Therefore, in one embodiment, Therefore, the epigenetic characteristics of extracellular free nucleosomes are characterized by histone post-translational modifications (PTMs). The histone PTM can be a core nucleosome, such as H3, H2A, H2B, or H4, particularly The histone PTM may be a histone H3, H2A, or H2B PTM. In particular, the histone PTM is a histone H3 PTM. Examples of such PTMs are described in WO 2005 / 019826.

[0033] For example, post-translational modifications can be acetylation, mono-, di-, or tri-methylation. Methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation Cosylation, nitrosylation, glutamination, and / or isomerization may occur (A (See Usio (2001) Biochem Cell Bio 79:693). In a further embodiment, the stone PTM is selected from methylation or citrullination. In still further embodiments, the stone PTM is H3K27me3 or H3 citrulline (H3cit). The histone PTM is H3cit. As shown in the examples provided herein, H3cit is Histone PTMs most effective in distinguishing subjects with vascular or hematological cancers from healthy subjects It was.

[0034] Groups or classes of related histone post-translational modifications (rather than single modifications) can also be detected. Typical examples include, but are not limited to, a single antibody directed to binding to a nucleosome. a histone modification targeting antibody or other selective binding agent and a targeting antibody directed to bind to the group of histone modifications of interest. Such histones include two-site immunoassays utilizing antibodies or other selective binding agents. Examples of antibodies directed to binding to a group of modifications include, but are not limited to, antibodies to pan-acetylated antibodies (e.g., pan-acetyl H4 antibodies [H4panAc]), anti-citrullinated antibodies, or anti There are ubiquitin antibodies.

[0035] In one embodiment, the epigenetic characteristics of the nucleosome include one or more DNA modifications Epigenetic regulation mediated by the composition of nucleosomal histone isoforms and PTMs In addition to genetic signaling, nucleosomes are involved in the transport of their nucleotides and modified nucleosomes. Global DNA hypomethylation is a hallmark of cancer cells, Some nucleosomes contain more 5-methylcytosine residues (or is a 5-hydroxymethylcytosine residue or other nucleotide or modified nucleotide) In one embodiment, the DNA modification may include 5-methylcytosine or 5-hydroxymethyl cytosine.

[0036] In one embodiment, the epigenetic signature of the nucleosome comprises one or more proteins. Further types of circulating nucleosomes include nucleosome-nucleosome adducts or complexes. A subset is a nucleosome-protein adduct of which chromatin is a component. It has long been known that DNA contains numerous non-histone proteins bound to DNA and / or histones. These chromatin-associated proteins are extremely diverse and have a variety of functions. , transcriptional regulators, transcriptional enhancers, transcriptional repressors, histone regulatory enzymes, DNA damage repair proteins Nucleosomes and other non-histone chromatin proteins or DNA These chromatin fragments, including histones and other non-histone chromatin proteins, are described in the art. It is described in the field.

[0037] In one embodiment, the nucleosomes are attached (and thus used as biomarkers). The proteins that may be used include: transcriptional regulators, high mobility group proteins, or chromatin regulatory enzymes. Reference to a "transcriptional regulator" refers to a gene that binds to DNA and promotes transcription (i.e. They regulate gene expression by either activating (i.e., activators) or suppressing (i.e., repressors). Transcriptional factors are proteins that regulate specific DNA sequences adjacent to the genes they regulate. All of the circulating Nucleosomes and parts, types or subgroups of nucleosomes are referred to in the present invention as It can be useful.

[0038] Two or more epigenetic characteristics of extracellular free nucleosomes can be determined by the methods and methods of the present invention. It will be appreciated that multiple biomarkers can be detected in combination. Thus, in one embodiment, the use Two or more epigenetic variables of extracellular free nucleosomes as a combined biomarker Epigenetic features include homologous (e.g., PTMs, histone isoforms, etc.) form, nucleotide, or protein adducts) or heterologous (e.g., histone isoforms) For example, post-translational histone modifications and histone variants. (i.e., detecting two or more types of epigenetic features) Alternatively, two or more types of post-translational histone modifications may be detected, or two or more types of histone modifications may be detected. In one embodiment, the use is to detect vascular or hematological cancers. or post-translational histone modifications as combined biomarkers in plasma samples for the detection of - Patents.com and histone isoforms. In one embodiment, the combined biomarkers include H3 In an alternative embodiment, the combined biomarkers are H3.1 and H3K27 It's Me3.

[0039] The term "biomarker" refers to a distinctive biological or physiological signal that indicates a process, event, or condition. means a biologically derived indicator. A biomarker is a diagnostic method, e.g., a clinical screening test. in the assessment of prognosis and outcome, as well as in monitoring treatment outcomes, determining which patients are most responsive to a particular therapeutic treatment, It can be used in identifying patients likely to develop the disease, drug screening, and drug discovery. Biomarkers and their use can be used to identify new drug treatments and to develop new targets for drug treatment. This is useful for target identification.

[0040] The methods and uses described herein may be used to test body fluid samples, particularly blood, serum or plasma samples. Preferably, a plasma sample is used. The plasma sample may contain one or more anticoagulants, For example, ethylenediaminetetraacetic acid (EDTA), heparin, or sodium citrate, especially EDTA. The cells can be collected in a collection tube containing

[0041] (blood cancer) Hematological cancers are cancers of the blood, so they can also be called "blood cancers." There are three main types of hematological cancers: Leukemia caused by the rapid production of normal white blood cells; lymphoma caused by abnormal lymphoma cells lymphoma, a cancer of the plasma cells; and myeloma, a cancer of the plasma cells.

[0042] In one embodiment, the hematological cancer is lymphoma, leukemia, myeloma, chronic myeloproliferative disorder, Monoclonal gammopathy of undetermined significance, myelodysplastic syndrome, and amyloidosis In a further embodiment, the hematological cancer is selected from leukemia or lymphoma. will be done.

[0043] Leukemia affects white blood cells, and the type of white blood cell affected (myeloid or lymphoid) and the progression of the disease vary. They can be classified by the way they are treated (acute or chronic), including but not limited to acute lymphoblastic leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia. acute myeloid leukemia (ALL; also known as acute lymphocytic leukemia), acute myeloid leukemia (AML), acute megalomaniac leukemia (AML), Myeloid leukemia (AMKL), acute promyelocytic leukemia (APL), childhood acute myeloid leukemia (C-AML), small intestine Childhood acute lymphocytic leukemia (C-ALL), chronic eosinophilic leukemia (CEL), chronic lymphocytic leukemia (CLL) ), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia, hairy - Cell leukemia, juvenile myelomonocytic leukemia (JMML), large granular lymphocytic leukemia (LGLL), T cell Several types of leukemia are identified, including acute lymphoblastic leukemia and prolymphocytic leukemia. It has been done.

[0044] In one embodiment, the leukemia is an acute leukemia, such as acute lymphocytic leukemia (ALL), acute lymphocytic leukemia (AL ... Myeloid leukemia (AML), acute megakaryoblastic leukemia (AMKL), or acute promyelocytic leukemia (APL) In further embodiments, the leukemia is acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). Alternatively, in one embodiment, the leukemia is selected from chronic leukemia, e.g., acute myeloid leukemia (AML). For example, chronic eosinophilic leukemia (CEL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Chronic myelomonocytic leukemia (CMML) or chronic neutrophilic leukemia.

[0045] Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL) are both lymphomas. The majority of patients with Hodgkin lymphoma are over 55 years old at the time of initial diagnosis, whereas The median age at diagnosis is 39 years. In one embodiment, the lymphoma is non-Hodgkin's lymphoma. NHL can occur in lymph nodes anywhere in the body, whereas HL typically occurs in It usually occurs on the upper body, such as the neck, chest, or armpits.

[0046] Hodgkin's lymphoma is often diagnosed early and is therefore considered one of the most treatable cancers. Non-Hodgkin's lymphoma is typically not diagnosed until it has reached a more advanced stage. Therefore, the method of the present invention provides a solution to the need to detect patients at an early stage of the disease and improve treatment outcomes. It finds particular use in the diagnosis of NHL.

[0047] (other blood cancers) Angiosarcoma and hemangiosarcoma are tumors that grow in the lining of the vascular system. Because it is a soft tissue cancer involving the proliferation of cells, it can be called a "vascular cancer." They are closely associated with the vasculature, and the affected cells are in direct contact with the circulating blood. They found that these blood cancers had very high levels of leukemia, similar to those observed in lymphomas. We show that the nucleosomes are associated with the circular nucleosomes.

[0048] (Detection and Diagnostic Methods) The present invention provides methods that can be used to detect or diagnose patients with vascular or hematological cancers. Thus, according to a further aspect, there is provided a method for diagnosing or detecting vascular or hematological cancer. The law is: (i) contacting a plasma sample obtained from the subject with a binding agent to obtain extracellular free nucleosomes; detecting or measuring; and (ii) using the detected level of extracellular free nucleosomes to identify the vascular cancer or blood system cancer diagnosing a subject with

[0049] According to a further aspect, there is provided a method for diagnosing or detecting hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to obtain extracellular free nucleosomes; detecting or measuring; and (ii) Using the detected level of extracellular free nucleosomes, subjects with hematological cancer and diagnosing the disease.

[0050] Alternatively, according to a further aspect, there is provided a method for diagnosing or detecting vascular cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to obtain extracellular free nucleosomes; detecting or measuring; and (ii) using the detected levels of extracellular free nucleosomes to identify subjects with vascular cancer; diagnosing the disease.

[0051] According to a further aspect, a method for determining the prognosis of a subject with vascular or hematological cancer is provided. There is: (i) contacting a plasma sample obtained from the subject with a binding agent to obtain extracellular free nucleosomes; detecting or measuring; and (ii) comparing the detected level of extracellular free nucleosomes with the prognosis of the vascular cancer or blood system cancer. and using the resultant mixture as an index.

[0052] Even if a subject is determined not to have vascular or hematological cancer, the present invention still provides a method for detecting disease progression. For example, the use may be to monitor vascular or blood cancer. If the sample includes samples from subjects determined to not have the biomarker, the measurement of the biomarker level is repeated. and at other time points to establish whether biomarker levels have changed. do.

[0053] In a further aspect, a patient having, suspected of having, or suffering from vascular or blood cancer is 1. A method for monitoring the effectiveness of a treatment in a susceptible subject, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to obtain extracellular free nucleosomes; detecting or measuring; and (ii) comparing the detected level of extracellular free nucleosomes with a plasma sample previously collected from the subject; and comparing the results of the treatment to determine the effectiveness of the treatment.

[0054] Detection and / or quantification can be performed directly on purified or enriched nucleosome samples. or indirectly on an extract from the sample or a dilution thereof. Quantifying the amount of biomarker present in a sample involves determining the amount of biomarker present in the sample. The use and detection according to the invention described herein may include determining the concentration of The screening and diagnostic methods confirm the presence of the disease and assess its development and progression. It is useful for monitoring the progression of the disease or for assessing the improvement or reversal of the disease. , uses and methods of detection, monitoring, and diagnosis are used for clinical screening assessment, prognosis, , treatment selection, evaluation of treatment benefit, i.e., methods for drug screening and drug discovery. do.

[0055] The detection or measurement may be performed by immunoassay, immunochemistry, mass spectrometry, chromatography, chromatography, or the like. In particular, the detection and / or measurement may involve nucleolytic immunoprecipitation, or biosensor methods. Such a method may involve a two-site immunoassay of the nucleosome portion. The nucleosome-binding agent or anti-nucleosome-binding agent is combined with an anti-histone modification or anti-histone variant or In situ detection using anti-DNA modification or anti-addition protein detection binders Measurement of nucleosomes or nucleosomes incorporating epigenetic features in vitro In addition, the detection and / or measurement is preferably performed by immobilizing a labeled anti-nucleosome detection binding agent. Anti-histone modification or anti-histone variant or anti-DNA modification or anti-addition protein binding The assay may include a two-site immunoassay utilizing a combination of an immunoassay agent and an immunoassay reagent.

[0056] The detection or measurement of the level of the biomarker(s) may be accomplished by one or more reagents, such as suitable binding agents. For example, one or more binding agents may be used to identify a desired biomarker, For example, nucleosomes or their component parts, epigenetic characteristics of nucleosomes, a structural / shape mimic of a nucleosome or its component parts, optionally one or more of these It may include a ligand or binding agent specific for the interleukin in combination.

[0057] Those skilled in the art will understand the terms "antibody," "binding agent," or "ligand" as used herein. is any bond that can bind to a specific molecule or entity, including but not limited to: It is intended that the binder be a binder of any suitable type, and any suitable binder may be used in the method of the present invention. It will be apparent that the term "nucleosome" can be used in various ways. mono- and oligo-nucleosomes, which can be analyzed in a fluid medium It is clear that the term is intended to include any protein-DNA chromatin fragment. It will be clear.

[0058] Methods for detecting biomarkers are known in the art. one or more ligands or binding agents, e.g., naturally occurring compounds or chemically synthesized compounds, capable of specific binding to Ligands or binding agents may include peptides capable of specific binding to a desired target, Antibodies, or fragments thereof, or synthetic ligands such as plastic antibodies, or aptamers The antibody may be a monoclonal antibody or a fragment thereof. If an antibody fragment is used, it may retain the ability to bind to the biomarker. It will be understood that a biomarker can be detected (in accordance with the present invention). / The binding agent is labeled with a detectable marker, such as a luminescent, fluorescent, enzymatic, or radioactive marker Alternatively or additionally, the ligands according to the invention may be provided with an affinity tag, e.g. Label with biotin, avidin, streptavidin, or His (e.g., hexa-His) tags. Alternatively, ligand binding can be achieved using label-free techniques, such as ForteBio's label-free technology. This can be determined using techniques.

[0059] As used herein, the terms "detect" or "diagnose" refer to the identification, confirmation, or determination of a disease state. The detection, monitoring, and diagnostic methods according to the present invention include recognition, and / or characterization. confirm the presence of disease and monitor disease evolution by assessing onset and progression or assessing the improvement or reversal of disease. and diagnostic methods are used for clinical screening evaluation, prognosis, treatment selection, evaluation of treatment benefit, i.e. i.e., in methods of drug screening and drug discovery.

[0060] In one embodiment, the methods described herein are repeated multiple times. This offers the advantage of allowing the detection results to be monitored over a period of time. Such a regimen may be beneficial for monitoring or evaluating the effectiveness of treatment of a disease state. Such monitoring methods of the present invention can be used to monitor the onset, progression, stabilization, improvement, and progression of a disease. Improvement, relapse, and / or remission can be monitored.

[0061] In the monitoring method, test samples may be taken at two or more occasions. The level of biomarker(s) present in the sample is compared with one or more control and / or treatment Previously from the same test subject before initiation and / or from the same test subject at an earlier stage of treatment The method may further comprise comparing the results of one or more previous test samples taken at different times. Detecting changes in the nature or quantity of biomarker(s) in a test sample collected by the machine may include:

[0062] Previous tests taken from the same subject with the same level of biomarkers in the test sample A change in the level compared to the level in the sample indicates the benefit of the treatment for the disorder or suspected disorder. Furthermore, once treatment is complete, the patient may show a significant effect, such as stabilization or improvement of the disease. The methods of the invention can be repeated periodically to monitor for recurrence.

[0063] The methods for monitoring the effectiveness of treatment can be used to monitor the efficacy of treatment in human subjects and non-human animals (e.g., To monitor the therapeutic efficacy of existing and new therapies in animal models These monitoring methods are useful for screening new drug substances and substance combinations. It can be incorporated into the

[0064] In a further embodiment, monitoring of more rapid changes due to fast acting treatments can be performed at intervals of shorter hours or days.

[0065] Diagnostic or monitoring kits (or panel populations) are provided for carrying out the methods of the invention. Such kits are preferably used for the detection and / or analysis of biomarkers according to the present invention. or one or more ligands for quantification, and / or a biosensor and / or The kit includes an array, optionally along with instructions for use of the kit.

[0066] A further aspect of the invention is a kit for detecting the presence of a disease state, comprising the steps of: a biosensor capable of detecting and / or quantifying one or more biomarkers as defined above; The term "biosensor" as used herein means a biosensor. Anything that can detect the presence of a biomarker. Examples of biosensors are described herein. The biosensor is a sensor capable of specifically binding to a biomarker, as described herein. Such a biosensor may comprise a ligand binding agent or ligand according to the present invention. It is useful for detecting and / or quantifying biomarkers.

[0067] Preferably, the biosensor for the detection of one or more biomarkers comprises a biomolecule recognition and suitable means for converting the detection or quantification of the presence of a biomarker in a sample into a signal. Biosensors can be used in "alternate sites" where diagnostic tests are performed, e.g., hospital wards, outpatient departments, etc. Can be adapted to subjects' departments, operating rooms, homes, fields, and workplaces The biosensor for detecting one or more biomarkers of the present invention includes both acoustic and plasmonic sensors. Acoustics, Holography, Biolayer Interferometry (BLI), and Micro-Engineering Imprint recognition elements, thin film transistor technology, magnetoacoustic resonators and other novel acoustoelectric systems for detecting one or more biomarkers. It can be used for sensors.

[0068] Biomarkers to detect the presence of disease, slow or stop the progression of the disorder It is a very important target for the discovery of novel targets and drug molecules. Biomarkers are used in vitro and / or in vivo to indicate disorders and drug responses. The biomarkers described herein are useful for identifying novel therapeutic compounds in assays. can be used in screening methods for compounds that modulate the activity of biomarkers. .

[0069] Thus, in a further aspect of the invention, a biomarker according to the invention is provided as described. peptides, antibodies or fragments thereof, or aptamers, or oligonucleotides directed against the the use of binding agents or ligands, which may be oligonucleotides; or the generation of biomarkers. A biosensor according to the present invention for identifying substances capable of promoting and / or inhibiting Provide for the use of a lei or kit.

[0070] The immunoassays described herein include immunoassays that bind to the biomarkers defined herein. Immunization is any method that utilizes one or more antibodies or other specific binding agents directed against the target. Assays include two-site immunoassays or immunometric assays that utilize enzyme detection methods (e.g. , ELISA), fluorescently labeled immunoassay, time-resolved fluorescently labeled immunoassay, chemiluminescent immunoassay Immunoassays, immunoturbidimetric assays, microparticle-labeled immunoassays, and immunoradiometric assays assays, as well as one-site immunoassays, reagent-limited immunoassays, labeled antigens and labeled antibodies. Competitive immunoassays, including radioactive, enzymatic, fluorescent, time-resolved fluorescent, and particulate labels. There are single antibody immunoassays that use a variety of labels. All of the above immunoassays are well known in the art, see, for example, Salgame et al. (1997) and van Nieuwenhuis. See Ijze et al. (2003).

[0071] Identification, detection, and / or quantification may be performed using a biological sample from a subject, or a purified or purified version of the biological sample. to identify the presence and / or amount of specific proteins in the extracts or dilutions thereof. In particular, quantification can be performed by any method suitable for determining the level of a target in one or more samples. This can be carried out by measuring the concentration of Biological samples include those defined hereinabove. Samples are prepared by conventional methods. The present invention can be carried out by preparing a compound obtained from a subject, for example, by diluting or concentrating the compound appropriately, and then storing the compound. It finds particular use in plasma samples where

[0072] Identification, detection, and / or quantification of biomarkers may involve the use of biomarkers or fragments thereof, e.g. For example, this can be carried out by detecting C-terminal truncated fragments or N-terminal truncated fragments. The pieces are preferably more than 4 amino acids in length, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, particularly those identical to or related to the sequence of a histone tail. It should be noted that peptides of sequences corresponding to the sequence of the histone protein fragments are particularly useful. .

[0073] For example, detection and / or quantification can be performed using: SELDI(-TOF), MALDI(-TOF), 1-D gel-based analysis, 2-D gel base analysis, mass spectrometry (MS), reversed-phase (RP) LC, size permeation (gel filtration), ion exchange, affinity chromatography one or more selected from the group consisting of: community, HPLC, UPLC, and other LC or LC MS based techniques. Suitable LC MS techniques include ICAT® (Applied Chromatography). Biosystems, CA, USA) or iTRAQ® (Applied Biosystems, CA, USA). Chromatography (e.g., high pressure liquid chromatography (HPLC) or low pressure liquid chromatography) LPLC), thin layer chromatography, and NMR (nuclear magnetic resonance) spectroscopy can also be used. .

[0074] Methods involving the detection and / or quantification of one or more biomarkers of the present invention can be performed using benchtop equipment. The test can be performed in a laboratory setting, such as a doctor's office or the subject's bed. Integrate into a disposable diagnostic or monitoring platform that can be used at the side Biosensors suitable for carrying out the methods of the present invention include those equipped with optical or acoustic readers. Biosensors transmit collected data electronically. It is designed to allow doctors to interpret the information, thus forming the basis for e-medicine. obtain.

[0075] The identification of biomarkers of disease states allows for the integration of diagnostic procedures and treatment regimens. Biomarkers can be used to predict treatment response, failure to respond, undesirable side effect profiles, and medication compliance. Provides a means of indicating the degree of compliance and achievement of adequate serum drug levels. The monitor can be used to provide warning of adverse drug reactions. , fine-tuning medication dosage, minimizing the number of prescribed doses, and delaying the achievement of effective treatment. Biomarkers are a valuable tool for customized medicine because they can reduce risk and avoid adverse drug reactions. Therefore, monitoring the biomarkers of the present invention is useful for the development of anti-cancer drugs. to meet the needs dictated by the subject's disorder and pharmacogenomic profile. Care can be precisely tailored to the patient, using biomarkers to identify optimal outcomes. Adjust doses, predict positive treatment responses, and identify subjects at high risk for serious side effects It is possible.

[0076] Biomarker-based testing is aimed at identifying “novel” targets that are not achievable using current tools. It provides a first-line evaluation and provides an objective means for accurate and rapid diagnosis.

[0077] In addition, the method, biosensor, and kit for monitoring biomarkers are Targeted monitoring to enable physicians to determine whether the condition is due to a worsening of the disability If pharmacological treatment is assessed as inadequate, treatment should be restarted. and treatment can be altered if appropriate. Because biomarkers are sensitive to the state of the disorder, they provide an indication of the impact of drug treatment.

[0078] References to "subject" or "patient" are used interchangeably herein. The subject may be a human or animal subject. In one embodiment, the subject is a human. In one embodiment, the subject is a (non-human) animal. The animal may be a dog, mouse, rat, or horse, particularly a dog. The methods may be performed in vitro, in vivo, or ex vivo.

[0079] In one embodiment, the subject is suspected of recurrence of vascular or hematological cancer. RD) is a condition in patients undergoing treatment or when the patient is in remission (i.e., the patient is free of disease symptoms). The name given to the small number of leukemia cells (cancer cells derived from the bone marrow) that remain after treatment in patients with leukemia who have no symptoms However, MRD is a major cause of relapse in cancer and leukemia. The methods of the present invention are useful for monitoring patients suspected of recurrence, particularly those in remission from cancer. It is useful.

[0080] Subjects tested using the methods described herein may have symptoms indicative of hematological cancer, such as anemia, In one embodiment, the subject may present with high levels of leukocytosis, and / or swollen lymph nodes. Hematologic cancers typically have Bell's leukocytosis, which may also be called "high white blood cell count." Typically, it causes an increased proliferation of abnormal white or red blood cells, resulting in a high white blood cell count. However, leukocytosis is a result of an inflammatory response, most commonly as a result of infection. Leukocytosis is often a sign of hematologic cancer (especially leukemia), so it is important to diagnose patients with hematologic cancers. Therefore, the method of the present invention is effective in treating patients who are likely to suffer from blood cancer. It may provide a more specific method for detecting patients.

[0081] The detection and / or quantification results can be compared with a cut-off level. The values ​​are calculated by analyzing results from multiple patients and controls and determining whether they have the disease or not. This can be determined in advance by determining the values ​​that are suitable for classifying the object as unsuitable. For example, patients with a disease may have higher levels of a biomarker. If the detected level is higher than the cutoff, the patient is diagnosed with the disease. Alternatively, patients with a disease may have lower levels of the biomarker. For a disease, if the detected level is below the cutoff, the patient is considered to have the disease. The advantages of using a simple cutoff value include making the test easier for clinicians. understandable to the user and any need for software or other aids in interpreting test results. The cutoff level may be determined using methods known in the art. It is possible.

[0082] The detection and / or quantification results can also be compared with a control. For example, subjects known to be disease-free may be included, or ... Control subjects, who may be subjects for different diagnostic studies, may be selected by a variety of criteria. It will be apparent that "controls" include healthy subjects, non-diseased subjects, and / or subjects with vascular cancer or other conditions. Comparison with a control may include subjects with or without hematological cancer. Comparison with a control is a well-known method in the field of diagnostics. It is being done.

[0083] Thus, in one embodiment, the method comprises: For example, the method may further comprise comparing the level of The levels of extracellular free nucleosomes present in plasma samples obtained from normal subjects were analyzed. The method may include comparing the level of extracellular free nucleosomes present in a plasma sample obtained by the method with the level of extracellular free nucleosomes present in a plasma sample obtained by the method. The control may be a healthy subject. Alternatively, the control may be a diseased subject, e.g., a subject with an infectious disease. It could be an elephant.

[0084] Alternatively, the control may be a subject with a cancer that is not a vascular or hematological cancer, i.e., a subject in a different part of the body. The data provided herein are based on the results of the biomarker study of the present invention. The markers were significantly elevated in patients with blood cancer compared with patients with other forms of cancer. Thus, the biomarkers of the present invention can be used to identify patients with vascular or hematological cancers. It can be diagnosed separately from patients with other forms of cancer. Diagnosis includes distinguishing vascular or blood cancer from non-vascular or non-blood cancer. "Non-vascular or non-blood cancers" are not blood cancers and do not involve proliferation of blood cells or vascular cells Cancer, such as bladder cancer, bone cancer, brain cancer, esophageal cancer, head and neck cancer, skin cancer (e.g., melanoma), thyroid cancer , tongue cancer, uterine cancer, and / or cervical cancer.

[0085] The control may be a subject with a high level of leukocytosis. Leukocytosis is not specific to leukemia. Therefore, the methods of the present invention can be used to treat vascular cancer or controls with high levels of leukocytosis that are not the result of hematologic cancer, e.g., inflammation, infection The subjects can be compared to controls who have the disease and / or are receiving drug treatment.

[0086] In one embodiment, the level of extracellular free nucleosomes is increased compared to a control.

[0087] It is not necessary in all cases to measure a control level for comparison purposes. It will be understood that, for example, once a "normal range" is established for healthy / non-diseased controls, and can be used as the reference for all subsequent examinations. The method involves obtaining samples from multiple control subjects without hematological cancer and analyzing the biomarker levels. This can be established by examining the blood vessels. The results (i.e., biomarker levels) of the subjects are then examined to determine whether they are in line with their respective normal ranges. You can see if you are within range or outside of range. The use of "normal range" It is standard practice to detect the disease.

[0088] In one embodiment, the method comprises determining at least one clinical parameter of the patient. This parameter can be used to interpret the results. The data may include any relevant clinical information, such as, but not limited to, sex, weight, body mass, The clinical parameters may include body mass index (BMI), smoking status, and dietary habits. The metrics are selected from the group consisting of: age, sex, and body mass index (BMI).

[0089] In one embodiment, the methods of the present invention are directed to patients at high risk of having vascular or blood cancer, and This is done to identify subjects who require further testing (i.e., further cancer surveillance). Further testing may include one or more of: biopsy (e.g., bone marrow biopsy or lymph node biopsy), cytogenetic testing, immunohistochemistry, or immunohistochemistry. Immunophenotyping, CT scan, X-ray (especially chest X-ray to identify enlarged lymph nodes), and / or lumbar May include a vertebral puncture.

[0090] Using the methods and biomarkers described herein, patients may undergo biopsy, particularly bone marrow biopsy or Therefore, it is possible to identify whether a lymph node biopsy is necessary. According to one embodiment, there is provided a method for identifying a patient in need of a biopsy, comprising obtaining a plasma sample from the patient. obtaining a plasma sample, detecting the level of extracellular free nucleosomes in the plasma sample, and Use the results from the Nel test to identify whether a patient needs a biopsy and

[0091] According to a further aspect of the present invention there is provided a method of identifying a patient in need of a biopsy, the method comprising: obtaining a plasma sample from the subject, applying the sample to a panel test as defined herein; and Use results from the panel to identify whether a patient needs a biopsy and

[0092] (additional biomarkers) The level of extracellular free nucleosomes can be detected or measured as part of a panel of assays. The panel can be a set of panels that can be used to identify different epigenetic patterns of nucleosomes, as described hereinabove. In one embodiment, the target gene may include a gene encoding a target protein, a gene encoding a target protein, or a gene encoding a target protein. The panel includes one or more cytokines, for example, one or more interleukins.

[0093] Interleukins (ILs) are a group of cytokines normally secreted by leukocytes. Interleukins play an important role in stimulating immune responses and inflammation. Interleukins were first identified in the 1970s, and additional interleukins have been identified. As new interleukins were discovered, they were given numerical names. Examples of interleukins include: including but not limited to: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-1 0, IL-11, IL-12, IL-13, IL-14, and IL-15.

[0094] In one embodiment, the one or more interleukins are interleukin-6 (IL-6), interleukin-2 (IL-2), interleukin-3 (IL-4), interleukin-4 (IL-5), interleukin-5 (IL-6), interleukin-6 (IL-7), interleukin-6 (IL-8), interleukin-6 (IL-9), interleukin-6 (IL-10), interleukin-6 (IL-11), interle Interleukin-10 (IL-10), and interleukin-1β (IL-1β). .

[0095] The interleukin can be IL-6. Interleukin-6 (IL-6) is a very diverse biological IL-6 is a potent inducer of fever and the acute phase response. The sequence of human IL-6 is known in the art and is described in UniProt accession number P05231. In one particular embodiment, the interleukin is IL-6 and the measurement panel is These may include measurements of the IL-6 isoform H3.1 and IL-6.

[0096] Alternatively or additionally, the interleukin can be IL-10. Interleukin-10 (IL-10) IL-10 is an anti-inflammatory cytokine with extremely diverse biological functions. It is known in the art and described in UniProt accession number P22301. In an embodiment, the interleukin is IL-10 and the measurement panel includes histone post-translational modification H3c This may include measuring IL-10 and IL-10.

[0097] Alternatively or additionally, the interleukin can be IL-1β. Interleukin-1β (IL-1 β) is a pro-inflammatory cytokine that mediates various cell proliferation, differentiation, and apoptosis. In one particular embodiment, the interleukin is IL-1β, The measurement panel may include measurements of histone isoforms H3.1 and IL-1β. The panel may include measurement of histone isoforms H3.1 and IL-1β, where vascular cancer or blood The cancer is a lymphoma, such as NHL.

[0098] In one embodiment, the panel comprises a panel of extracellular free nucleosomes. In another embodiment, the panel comprises extracellular free nucleosides. These include the epigenetic signature of the genome and two interleukins. Measurement of isolated nucleosomes can be performed using a combination of two or more interleukins, e.g., IL-6 and IL- IL-1β, or IL-10 and IL-1β, or IL-6 and IL-10. In embodiments, the epigenetic characteristics of the extracellular free nucleosomes are determined by histone identity. isoforms, such as H3.1, and post-translationally modified histones, such as H3cit. In a further embodiment, the panel of measurements is H3.1, IL-6, and IL-1β. In this study, the measurement panel was H3cit, IL-10, and IL-1β.

[0099] The biomarkers of the present invention can be used to derive models. Methods for deriving such models or algorithms are well known in the art and are preferably Suitable software packages are available. Typical software for this purpose are The tools include SPSS (Statistical Package for Social Sciences) and R. The software package provides linear and non-linear data modeling of clinical data.

[0100] Those skilled in the art will appreciate that any combination of the biomarkers disclosed herein may be used to treat vascular cancer or hematologic and that the antibodies can be used in panels and algorithms for the detection of systemic cancers. It is clear that other markers can be added to the panel containing these markers. It would be.

[0101] According to an aspect of the present invention, a panel test for detecting patients with vascular or hematological cancer is The use, wherein the panel test comprises determining whether nucleosomes or their constituents are present in a plasma sample obtained from a patient. and a reagent for detecting the measurement of one or more interleukins. To provide.

[0102] (Treatment method) According to a further aspect, there is provided a method of treating vascular or hematological cancer in a subject, comprising: (i) detecting or measuring the level of extracellular free nucleosomes in a plasma sample obtained from a subject; The process of: (ii) determining the level measured in step (i) as an indication of the presence of vascular or hematological cancer in the subject; and (iii) if the subject is determined to have the vascular or blood cancer in step (ii), performing a surgical procedure or administering a therapeutic agent.

[0103] In a further aspect, treating vascular or hematological cancer in a subject in need thereof. The method comprises determining the level of extracellular free nucleosomes in a plasma sample obtained from the subject by: When compared to the levels of extracellular free nucleosomes in plasma samples obtained from control subjects, performing a surgical procedure or administering a therapeutic agent to the subject identified as different; The method is provided.

[0104] In one embodiment, the treatment comprises one or more of: chemotherapy, immunotherapy, hormonal therapy, biological therapy. chemotherapy, radiation therapy, leukapheresis, and stem cell transplantation.

[0105] The method comprises: (i) measuring the level of extracellular free nucleosomes in a plasma sample obtained from a subject (optionally 1 or more) in combination with interleukin levels; (ii) based on higher levels of extracellular free nucleosomes compared with the control; Identifying the subject as having vascular or blood cancer; and (iii) administering treatment to the subject.

[0106] According to another aspect of the present invention, there is provided a method for treating vascular or hematological cancers using a panel test, and identifying patients in need of treatment for vascular or blood cancers and providing said treatment. and wherein the panel test comprises: The method further comprises measuring the amount of leukin in the blood. Patients with HIV have higher levels of extracellular free nucleosomes compared to controls is expected.

[0107] The embodiments described herein are applicable to all aspects of the present invention, i.e. The embodiments described in the claims apply equally to the methods etc. It will be understood that this is possible.

[0108] The invention will now be described with reference to the following non-limiting examples. [Example]

[0109] (Example) Example 1 Plasma samples were collected from a cohort of 116 human subjects. For each subject, a whole blood draw was performed. The blood was collected in an EDTA vacutainer tube and the tube was gently inverted 10 times. Within 2 hours, whole blood was centrifuged at 1500 g for 15 minutes. Plasma was transferred to cryotubes and immediately Within the cohort, 62 subjects were healthy and 25 subjects were non-Hodgkin's disease. 22 subjects had acute myeloid leukemia (NHL), 22 subjects had acute myeloid leukemia (AML), and 7 subjects had acute lymphoblastic leukemia (ALL). Subjects with leukemia or lymphoma were not diagnosed with cancer. Patients (31 subjects in total, 16 subjects diagnosed with NHL and 15 subjects diagnosed with leukemia) patients with recurrent cancer (15 subjects in total; 6 subjects had recurrent NHL) , and 9 subjects had recurrent leukemia).

[0110] Samples were analyzed for H3.1-containing nucleosomes by ELISA. Measurement of nucleosomes containing histone isoform H3.1 was performed as follows: 80 μl Assay buffer and 20 μl of plasma sample or standard nucleosome preparation were added to the histone H3.1 added to microtiter wells coated with antibodies directed to bind to The microtiter plate was covered and incubated at room temperature with gentle shaking for 2.5 hours. The contents of the microtiter wells were discarded. The wells were washed with 200 μl of washing solution. After washing three times, 100 μl of biotinylated anti-nucleosome antibody was added. The container was recapped and incubated at room temperature with gentle shaking for 1.5 hours. The contents of the titer wells were discarded. The wells were washed three times with 200 μl of wash solution and 100 μl of slides were added. Streptavidin-HRP solution was added. The microtiter plate was then re-covered and allowed to stand at room temperature. The contents of the microtiter wells were discarded and incubated for 0.5 hours with gentle shaking. The wells were washed three times with 200 μl of washing solution and 100 μl of HRP (horseradish peroxide) was added. The microtiter plate was covered and gently shaken at room temperature. The wells were incubated in the dark for 20 minutes. The optical density (OD) of the wells was measured at 405 nm. The level can be used directly or by measuring plasma levels of nucleosomes containing histone H3.1 using a standard curve. I tried interpolating from

[0111] The OD results were plotted as receiver operating characteristic (ROC) curves. Patients vs. healthy controls, and all patients with lymphoma vs. healthy controls, and all patients with leukemia Patients were grouped according to the area under the curve (AUC) of the ALL and AML patients versus healthy controls. The results are shown in Tables 1 to 3.

[0112] Table 1: ROC curve for H3.1 OD in plasma - all patients [Table 1]

[0113] Table 2: ROC curve for H3.1 OD in plasma - diagnosed patients [Table 2]

[0114] Table 3: ROC curve for H3.1 OD in plasma - relapse patients [Table 3]

[0115] As shown in Table 1, the levels of extracellular free nucleosomes containing H3.1 were elevated in over 75% of patients with hematological cancers. The results were as shown in Table 2. The specificity can be increased to over 80% with 90% specificity for patients diagnosed with NHL. This result supports the conclusion that this biomarker was highly effective, as 84% ​​of patients were distinguished from healthy controls with a specificity of 90%. have been shown to be particularly effective in identifying patients with lymphoma (see Table 1). sea ​​bream).

[0116] Standard for interpolating H3.1 concentrations from OD values ​​in plasma samples of all AML, ALL, and NHL patients The curves derived and compared with levels in healthy patients are shown in Figure 1. Levels of H3.1 are significantly elevated in patients with cancer compared to healthy subjects .

[0117] These results suggest that the levels of nucleosomes, especially H3.1-containing nucleosomes, may be related to the level of hematologic cancer. It has been shown that it can be used for detection.

[0118] Example 2 The levels of extracellular free nucleosomes containing H3.1 in the human plasma samples tested in Example 1 The samples were obtained from human patients with other forms of cancer, also collected as described in Example 1. The levels of H3.1 in the plasma samples were further compared.

[0119] The results are shown in Figure 2. Surprisingly, levels of H3.1 could be used to predict the progression of cancer in patients with other forms of cancer. It has been found that the method can distinguish subjects with hematological cancer from those with non-hematological cancer. The levels of H3.1 in patients with ALL, AML, and NHL are significantly higher in patients with bladder cancer, bone cancer, brain cancer, esophageal cancer, and head and neck cancer. From patients with cervical cancer, skin cancer, thyroid cancer, tongue cancer, uterine and cervical cancer, and melanoma It was significantly elevated compared to the level of H3.1 in the plasma samples.

[0120] Example 3 For the human plasma samples described in Example 1, extracellular free nucleosomes with post-translational modifications were analyzed. The ELISA method was performed using an antibody directed against histone H3.1. directed to post-translational histone modifications selected from H3cit, H3K27Me3, and H4panAc. The ELISA was carried out in the same manner as in Example 1, except that the antibody was replaced with H3.1. The results are summarized in Table 4. The results for all blood cancers and for each type of blood cancer are also shown. The results are shown in the figures (see Figures 3 to 5).

[0121] Table 4: ROC curves for PTM nucleosomes in plasma - all hematological cancers [Table 4]

[0122] All nucleosome post-translational modifications tested were significantly higher in patients with hematological cancers than in healthy controls. Even when blood cancers were divided by type (ALL, AML, NHL), H3c The levels of it, H3K27Me3, and H4panAc extracellular free nucleosomes in plasma samples were significantly higher than those of healthy controls. The post-translational modification of nucleosomes is significantly increased compared to the control (see Figures 3B, 4B, and 5B). Levels of IL-1 were most greatly elevated in plasma samples obtained from patients with ALL.

[0123] Example 4 Combining two of the best extracellular free nucleosome markers (H3.1 and H3cit) into one The extracellular free nucleosomes were measured either alone or in combination with the measurement of different interleukins. Blood levels were measured as previously described, and plasma interleukin levels were determined using a commercially available ELISA method. and measured.

[0124] Assay results were modeled by logistic regression analysis to identify human patients with hematological cancers. The model or algorithm was trained using the highest AUC for the comparison versus normal human donors. The results are summarized in Table 5.

[0125] Table 5: ROC curves for combined biomarker panels in plasma - all hematological cancers♯ ) [Table 5] ♯ Cohort size: 54 cancer vs. 62 healthy

[0126] As shown in Table 5, all models identified over 75% of hematological cancer patients with a specificity of 90% compared with healthy donors. The results showed that the nucleosome levels were distinguishable from the interleukin levels. In combination, we have developed an effective assay platform with associated algorithms for the detection of hematological cancers. This indicates that it can be used as a channel.

[0127] Example 5 Plasma samples from only human patients with NHL were analyzed using logistic regression as described in Example 4. The results were analyzed and modeled, and are summarized in Table 6.

[0128] Table 6: ROC curves for combined biomarker panels in plasma - NHL cancer ♯ ) [Table 6] ♯ Cohort size: 25 NHL vs. 62 healthy

[0129] As shown in Table 6, all models identified over 80% of patients with NHL as healthy donors with a specificity of 90%. In particular, the combination of H3.1 and IL-1β resulted in 100% NHL. The results showed that the nucleosome and interleukin-1 (IL-1) levels were significantly higher than those of the control group. The level of IL-1 is used to establish an effective assay with associated algorithms for the detection of NHL. This shows that it can be used as a panel.

[0130] Example 6 Plasma samples were collected from 73 dogs diagnosed with canine hemangiosarcoma and 127 dogs diagnosed with canine lymphoma. Samples were collected from 134 dogs with cancer and 134 control dogs without cancer. Samples were tested for H3.1 by ELISA. Nucleosomes were analyzed.

[0131] Healthy dogs uniformly have a high concentration of circulating nucleosomes containing histone isoform H3.1. were found to be low, less than 67.4 ng / ml (mean 32 ng / ml, median 31 ng / ml).

[0132] Dogs diagnosed with lymphoma have circulating nucleosomes containing histone isoform H3.1 The levels of α-amyloid were found to be significantly elevated (mean 570 ng / ml, median 211 ng / ml). The resulting dot plots and ROC curves for lymphoma are shown in Figures 6A and 6B, respectively. The AUC for lymphoma detection was 87% when a cutoff of 67.4 ng / ml was used. The specificity of the assay was 100% and the sensitivity was 74%. Using the tipoff gave a sensitivity of 81% and a specificity of 90%.

[0133] Additionally, dogs diagnosed with hemangiosarcoma have high levels of circulating nucleosomal proteins containing histone isoform H3.1. Highly elevated levels of thrombosomes (mean 513 ng / ml, median 361 ng / ml) were found. The resulting dot plot and ROC curve for canine hemangiosarcoma are shown in Figures 7A and 7B, respectively. The AUC for angiosarcoma detection was 97.6% using a cutoff of 67.4 ng / ml. When the assay was performed, the specificity of the assay was 100% and the sensitivity was 89%. Using a low cutoff value gave a sensitivity of 95% and a specificity of 90%.

[0134] Also, as found in human disease, dogs diagnosed with lymphoma or hemangiosarcoma The levels of circulating nucleosomes observed in this study were similar to those observed for other canine tumors investigated. It was much higher than the level.

[0135] Most assays for human proteins are not transferable to other animals. However, the structure of the nucleosome is highly conserved among species and even among phyla. The present inventors believe that this fact allows the assay for human H3.1 nucleosomes to be used in dogs. This means that the drug can be used in other species, including dogs and horses. The results observed by the present inventors for vascular and hematopoietic cancers in humans are consistent with those observed in human subjects. The inventors have demonstrated that the method of the present invention is effective in reducing blood vessel damage in both humans and animals. We conclude that this is a highly effective method for detecting cancer and hematopoietic cancer.

[0136] Example 7 Serial plasma samples were collected from two dogs undergoing treatment for hemangiosarcoma and one dog undergoing treatment for lymphoma. This information was collected from two dogs undergoing treatment. To avoid any discrepancies in the results, all samples from the four dogs were analyzed after the last day of treatment in Figure 8. were collected and subsequently analyzed for nucleosomes containing histone isoform H3.1.

[0137] C-reactive protein (CRP) is a well-known biomarker of inflammation. Whether the results simply reflect an inflammatory response or have further implications for the subject's condition, prognosis, and treatment response This protein is also measured in the sample as a control to determine what other information it provides. did.

[0138] Dog 1 (Fig. 8A) was diagnosed with hemangiosarcoma and received CHOP (SEQ ID NO: 1) starting on day 1 and continuing until day 129. 4 known as rofosfamide, doxorubicin, vincristine, and prednisone The treatment was successful, with Dog 1 recovering at day 160. The success of CHOP therapy was determined by the nuclei over the course of treatment. Disease remission was reflected by a downward trend in nucleosome levels, with near-normal nucleosome levels by day 122. This result suggests that nucleosome levels can be used as a prognostic indicator. It is useful in guiding treatment regimens and monitoring subjects during remission to monitor disease progression. CRP analysis was not useful for clinical purposes.

[0139] Dog 2 (Figure 8B) was originally diagnosed with hemangiosarcoma in 2017 and was treated with doxorubicin and immunomodulatory agents. The dog was monitored regularly with whole-body CT scans every 2-3 months. The dog was noted to have relapsed 2 years later, which was attributed to high levels of circulating nucleic acids. This recurrence is reflected in the retinopathy. and have been successfully treated with whole-lung irradiation and stereotactic body radiation therapy (SBRT), Dog 2 was determined to be in remission based on whole-body CT imaging. The success of various treatments was assessed in both dogs. This is reflected in the near-normal nucleosome levels measured in January 2020. Dog 2 was diagnosed as having a complete response based on imaging results, but interestingly , the measured nucleosome levels began to rise in February and then decreased by the next scan in April 2020. Disease progression was detected by imaging in the 2016 study. Nucleosome levels remained high when the treatment was initiated. If the serosome levels had been known, more intensive monitoring could have been performed on this patient in February. This result suggests that nucleosome levels can be used to determine treatment regimens. demonstrate that subjects can be monitored during remission for disease recurrence CRP analysis was not useful for clinical purposes.

[0140] Dog 3 (Fig. 9A) was diagnosed with lymphoma in early 2018 and was successfully treated, resulting in remission. This was reflected in the measured nucleosome levels. vincristine as part of a CHOP chemotherapy regimen on day 1 for progressive disease (PD) in Vincristine treatment improved the patient's clinical condition to stable (SD), and This was reflected in a decrease in nucleosome levels, as shown by the results in Figure 9A. However, CHOP treatment is discouraged due to both toxicity and the lack of robust clinical responses. Treatment with rosmustine plus L-asparaginase (L-spar) was initiated on day 22. This was followed by further lomustine treatments on days 34, 79, and 113. The outcome improved to partial response as determined by tumor measurements on days 34 and 79, followed by partial response on day 113. After 145 days of treatment, the response improved to complete response (CR), and Dog 3 had been in remission up to this point. No further chemotherapy was administered. Nucleosome levels were measured between days 22 and 34. The blood pressure rose to 100°C, then steadily progressed downward from the 34th day, and later became clinically observable on the 113th and 145th days. This predicted the success of lomustine treatment, with an expected complete response. Clinical findings indicated that serum levels had decreased to the range observed in healthy dogs at day 145. CRP levels were always within the normal range and were not useful for clinical purposes.

[0141] Dog 4 (Fig. 9B) was diagnosed with stage Vb intra-abdominal hypercalcemic lymphoma. The patient began a CHOP chemotherapy regimen of vincristine and doxorubicin on day 1 and completed the 9th The response to treatment is monitored based on circulating calcium levels. Veterinarians have reported in clinical practice that some animals respond to vincristine, but not to doxorubicin. There was no response to rubicin. On the 108th day, treatment was switched from CHOP to Romustine. The patient was switched to vincristine + L-spar and continued until day 164, but still showed no response. Because of the response to COP (without doxorubicin) with vincristine, Treatment with CHOP (low-dose CHOP) was initiated on day 164 (blood samples could not be obtained on day 167). Dog 4 responded to COP therapy and continues to respond to treatment at the time of writing. The cleosome levels mirrored the observed clinical findings (Figure 9B). At most time points after treatment, nucleosome levels decreased, reflecting a response to this treatment. , after each dose of doxorubicin, reflecting the lack of response to this treatment. Nucleosome levels were increased. Dog 4 also showed a clinical response to cyclophosphamide. It was found that patients had a partial response to rituximab, which was also reflected in a decrease in nucleosome concentration. However, nucleosome levels were still above normal, Nucleosome measurement results were consistent with clinical findings and indicated the need for further treatment. It correlates clearly and can be used to monitor disease remission and guide treatment selection. For example, nucleosome levels predict lack of response to doxorubicin therapy. However, if we had had knowledge of the nucleosome level, we could have stopped earlier. CRP analysis did not show any significant changes and was not useful for clinical purposes. . The present application provides the following aspects of the invention. (Aspect 1) Cells as biomarkers in plasma samples for the diagnosis or detection of vascular or hematological cancers Use of extravesicular free nucleosomes. (Aspect 2) The extracellular free nucleosome is a mononucleosome or an oligonucleosome. The use according to embodiment 1. (Aspect 3) The biomarker is a level of the extracellular free nucleosomes and / or the extracellular 3. The use according to embodiment 1 or 2, which is an epigenetic signature of free nucleosomes. (Aspect 4) The epigenetic characteristics of the extracellular free nucleosomes include histone isoforms. forms, such as core nucleosomal histone isoforms, especially histone H3 isoforms. 4. The use according to embodiment 3, wherein the composition is a pharmaceutical formulation. (Aspect 5) The use according to embodiment 4, wherein the histone isoform is H3.1. (Aspect 6) The epigenetic features of the extracellular free nucleosomes are involved in histone post-translational modifications. 3. A PTM, e.g., a core nucleosomal histone PTM, particularly a histone H3 PTM. Use as described. (Aspect 7) The histone PTM can be citrullination (e.g., H3 citrulline) or methylation (e.g., H3K2 7me3). (Aspect 8) The method according to any one of aspects 1 to 7, wherein the blood cancer is selected from leukemia and lymphoma. use. (Aspect 9) The leukemia is selected from acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). 9. The use according to embodiment 8. (Aspect 10) The use of embodiment 8, wherein the lymphoma is non-Hodgkin's lymphoma (NHL). (Aspect 11) The vascular cancer is selected from angiosarcoma or hemangiosarcoma. The use according to any one of aspects 1 to 7. (Aspect 12) 1. A method for diagnosing or detecting vascular or blood system cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to obtain extracellular free nucleosomes; detecting or measuring; and (ii) using the detected level of extracellular free nucleosomes to identify the vascular cancer or blood system cancer diagnosing a subject with (Aspect 13) 1. A method for determining the prognosis of a subject having vascular or hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to obtain extracellular free nucleosomes; detecting or measuring; and (ii) comparing the detected level of extracellular free nucleosomes with the prognosis of the vascular cancer or blood system cancer. using the resultant product as an indicator. (Aspect 14) In subjects with, suspected of having, or susceptible to vascular or hematological cancer 1. A method for monitoring the effectiveness of a treatment comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to obtain extracellular free nucleosomes; detecting or measuring; and (ii) comparing the detected level of extracellular free nucleosomes with a plasma sample previously collected from the subject; and comparing the treatment to determine the effectiveness of the treatment. (Aspect 15) The detection or measurement may be performed by immunoassay, immunochemistry, mass spectrometry, chromatography, 15. The method according to any one of aspects 12 to 14, comprising a chromosome immunoprecipitation method or a biosensor method. Law. (Aspect 16) 16. The method of any one of aspects 12 to 15, wherein the subject is a human or animal subject. (Aspect 17) 17. The method of any one of aspects 12 to 16, wherein the subject is suspected of having a recurrence of vascular cancer or blood cancer. How to post. (Aspect 18) Any one of aspects 12 to 17, wherein the subject has a high level of leukocytosis / high white blood cell count. The method described. (Aspect 19) comparing the level of the extracellular free nucleosomes in the plasma sample to one or more controls. 19. The method according to any one of embodiments 12 to 18, further comprising the step of: (Aspect 20) 20. The method of embodiment 19, wherein said control is a healthy subject. (Aspect 21) 20. The method of embodiment 19, wherein the control is a subject with a cancer that is not a vascular or hematological cancer. (Aspect 22) 20. The method of embodiment 19, wherein the control is a subject with a high level of leukocytosis. (Aspect 23) Aspect 12. wherein the level of extracellular free nucleosomes is elevated compared to the control. The method described in any one of items 1 to 22. (Aspect 24) The level of the extracellular free nucleosomes is detected or measured as one of a panel of measurements. The method according to any one of embodiments 12 to 23, wherein (Aspect 25) 25. The method of embodiment 24, wherein the panel comprises one or more interleukins. (Aspect 26) The one or more interleukins are selected from the group consisting of IL-6, IL-10, and IL-1β. 26. The method of embodiment 25.

Claims

1. Cells as biomarkers in plasma samples for the diagnosis or detection of vascular or hematological cancers In vitro use of the H3.1 histone isoform from extravesicularly free nucleosomes.

2. The extracellular free nucleosome is a mononucleosome or an oligonucleosome. The use according to claim 1.

3. The method according to claim 1 or 2, wherein the blood cancer is selected from leukemia, lymphoma, or myeloma. For.

4. The leukemia is selected from acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML).

4. The use according to claim 3.

5. 4. The use of claim 3, wherein the lymphoma is non-Hodgkin's lymphoma (NHL).

6. The vascular cancer is selected from angiosarcoma or hemangiosarcoma.

3. The use according to claim 1 or 2.

7. 1. A method of obtaining data for diagnosing or detecting vascular or blood system cancer, comprising: A plasma sample obtained from the subject is contacted with the binding agent to detect a plasma containing histone isoform H3.

1. The above method, which comprises the step of detecting or measuring extracellular free nucleosomes.

8. A method for obtaining data for determining the prognosis of a subject with vascular or blood cancer. hand: (i) contacting a plasma sample obtained from the subject with a binding agent to detect histone isoform H3.1; detecting or measuring extracellular free nucleosomes comprising: (ii) Levels of extracellular free nucleosomes containing histone isoform H3.1 detected as an indicator of the prognosis of said vascular cancer or blood system cancer.

9. In subjects with, suspected of having, or susceptible to vascular or hematological cancer 1. A method for obtaining data for monitoring the effectiveness of a treatment, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect histone isoform H3.1; detecting or measuring extracellular free nucleosomes comprising: (ii) Levels of extracellular free nucleosomes containing histone isoform H3.1 detected comparing the result with a plasma sample previously taken from the subject.

10. The detection or measurement may be performed by immunoassay, immunochemistry, mass spectrometry, chromatography, The method according to any one of claims 7 to 9, comprising a chromosome immunoprecipitation method or a biosensor method. Law.

11. The detecting or measuring step may be performed in combination with an immobilized anti-histone H3.1 binding agent. Any of claims 7 to 9, comprising a two-site immunoassay utilizing a labeled anti-nucleosome detection binding agent.

1. The method according to any one of the preceding items.

12. The method of any one of claims 7 to 11, wherein the subject is a human or animal subject.

13. 13. The method of claim 7, wherein the subject is suspected of having a recurrence of vascular or hematological cancer. How to post.

14. Any one of claims 7 to 12, wherein the subject has a high level of leukocytosis / high white blood cell count. The method described in section.

15. of extracellular free nucleosomes containing the histone isoform H3.1 in the plasma sample. The method of any one of claims 7 to 14, further comprising comparing the level to one or more controls. Law.

16. 16. The method of claim 15, wherein the control is a healthy subject.

17. 16. The method of claim 15, wherein the control is a subject with a cancer that is not a vascular or blood system cancer.

18. 16. The method of claim 15, wherein the control is a subject with a high level of leukocytosis.

19. The level of extracellular free nucleosomes containing the histone isoform H3.1 was compared with that of the control. The method of any one of claims 7 to 18, wherein the level is increased compared to the level of the serotonin receptor agonist.

20. The level of extracellular free nucleosomes containing the histone isoform H3.1 was measured. The method of any one of claims 7 to 19, wherein the marker is detected or measured as one of the channels.

21. 21. The method of claim 20, wherein the panel comprises one or more interleukins.

22. The one or more interleukins are selected from the group consisting of IL-6, IL-10, and IL-1β.

22. The method of claim 21.

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