Use of extracellular free nucleosomes as biomarkers

Abstract

The present invention relates to extracellular free nucleosomes as biomarkers in plasma samples for vascular or hematological cancers.

Description

Technical Field

[0001] (Field of the Invention) The present invention relates to extracellular free nucleosomes as biomarkers in plasma samples of vascular cancer or hematological cancer.

Background Art

[0002] (Background of the Invention) Hematological cancer is a type of cancer that affects the blood, bone marrow, and lymph nodes. Hematological cancers are called leukemia, lymphoma, and myeloma depending on the type of affected cells. Leukemia usually starts in the bone marrow and is a cancer of blood cells that move through the bloodstream. In leukemia, the bone marrow produces mutant cells, which are spread in the blood, where the mutant cells proliferate and crowd out healthy blood cells. Lymphoma diseases affect cells of the lymphatic system. In lymphoma, immune cells called lymphocytes grow uncontrollably and collect in lymph nodes, the spleen, other lymphoid tissues, or adjacent organs. Myeloma, also called multiple myeloma, develops in the bone marrow and affects plasma cells that produce antibodies that attack infections and diseases. Examples of blood cancers include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin lymphoma (HL), and non-Hodgkin lymphoma (NHL).

[0003] References to "acute leukemia" mean cancers that progress rapidly and aggressively and usually require immediate treatment. ALL is associated with the development of a large number of immature lymphocytes that cannot fight infections. This leaves less room for healthy white blood cells, red blood cells, and platelets in the patient's circulation. As a result, patients usually suffer from a weakened immune system and symptoms of anemia, such as fatigue, shortness of breath, and an increased risk of excessive bleeding. The risk of developing ALL is highest in children under 5 years old and is the most common type of leukemia affecting children. Thereafter, the risk gradually decreases until the mid-20s and then gradually begins to increase again after the age of 50. Overall, about 4 out of 10 cases of ALL are in adults.

[0004] AML affects myeloblasts and accumulates abnormal monocytes and granulocytes in the bone marrow. AML also affects bone marrow stem cells and can produce abnormal red blood cells or platelets. Similar to ALL, AML reduces the levels of healthy white blood cells, red blood cells, and platelets in the patient's circulation. AML is one of the most common types of leukemia in adults, with an average age at diagnosis of 68 years.

[0005] HL and NHL are two major types of lymphoma. HL has a specific appearance under the microscope and contains cells called Reed-Sternberg cells (a type of cancerous B lymphocyte), while NHL has a different microscopic appearance and does not contain Reed-Sternberg cells. Most lymphomas are NHL, with only about 1 in 5 being HL. NHL is a cancer that affects lymphocytes and usually starts in lymph nodes or lymphoid tissue. It is one of the more common cancers among children, teenagers, and young adults.

[0006] Current methods for diagnosing leukemia and myeloma involve obtaining a complete blood count (CBC) test result to identify abnormal levels of white blood cells relative to red blood cells and platelets. However, an elevated white blood cell count (WBC) is not specific to patients with hematologic malignancies, and this event can also be the result of a continuing response to an infection or other inflammatory process. For lymphomas, X-ray, CT, or PET scans can be used to detect swollen lymph nodes, but this is also not specific.

[0007] To confirm a diagnosis of hematologic cancer, a bone marrow or lymph node biopsy is required. Thus, overdiagnosis of hematologic cancer at the initial stages of the diagnostic process can result in unnecessary biopsies that are invasive, potentially risky, and relatively costly for healthcare providers. Cytogenetic analysis and / or immunophenotyping can also be used to confirm a diagnosis of hematologic cancer, but these methods are expensive to perform and are thus typically only used at later stages of the diagnostic process.

[0008] Human angiosarcoma is a rare vascular cancer that affects the endothelial cells lining the inside of blood vessels. Canine hemangiosarcoma is similarly a cancer that involves the proliferation of vascular endothelium or the blood vessel wall and is also a common canine cancer that is difficult to diagnose. Canine cancers are more common than human cancers, generally due to both the genetic effects of inbreeding and the shorter lifespan of dogs, which generally develop cancer at ages eight and older. The detection and diagnosis of cancer in animal subjects presents additional difficulties compared to the diagnosis of human cancers. Detection of cancer in non-human animals often involves scans such as magnetic resonance imaging or MRI scans, but the animals will not remain still for the length of time required for the scan, so they must be anesthetized. Pet medical insurance is rare and may not cover the diagnosis or treatment of cancer, and these tests are economically out of reach for many pet owners. Furthermore, human blood tests for cancer marker proteins usually do not detect animal proteins, and as a result, there are far fewer blood tests available in veterinary oncology.

[0009] In the past, Holdenrieder et al. (2001) Int J Cancer 95:114-120 described detecting levels of nucleosomes in serum samples from patients with benign and malignant diseases. However, the results presented for serum samples did not show any differences in levels compared to other tested cancer types, including for hematological cancers such as lymphoma. The epigenetic composition of circulating cell-free nucleosomes in terms of their histone modifications, histone variants, DNA modifications, and adduct content has also been studied as a blood-based biomarker for cancer. See WO 2005 / 019826, WO 2013 / 030577, WO 2013 / 030579, and WO 2013 / 084002.

[0010] There is still a need in the art for a simple and cost-effective diagnostic method for vascular cancer or hematological cancer, particularly a method that can be distinguished from patients who have other types of diseases or non-vascular cancer or non-hematological cancer but may present similar symptoms.

Summary of the Invention

[0011] (Summary of the Invention) According to a first aspect, there is provided the use of extracellular free nucleosomes as biomarkers in plasma samples for the diagnosis or detection of vascular cancer or hematological cancer.

[0012] According to a further aspect, there is provided the use of extracellular free nucleosomes as biomarkers in plasma samples for the diagnosis or detection of hematological cancer.

[0013] According to a further aspect, there is provided the use of extracellular free nucleosomes as biomarkers in plasma samples for the diagnosis or detection of vascular cancer.

[0014] According to a further aspect, there is provided a method for the diagnosis or detection of vascular cancer or hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes; and (ii) using the level of the detected extracellular free nucleosomes to diagnose the subject as having the vascular cancer or hematological cancer.

[0015] According to a further aspect, there is provided a method for determining the prognosis of a subject having vascular cancer or hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes; and (ii) using the level of the detected extracellular free nucleosomes as an indicator of the prognosis of the vascular cancer or hematological cancer.

[0016] In a further aspect, there is provided a method for monitoring the effectiveness of treatment in a subject having, suspected of having, or prone to having a vascular cancer or a hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes; and (ii) comparing the level of detected extracellular free nucleosomes with a plasma sample previously taken from the subject to determine the effectiveness of the treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] (Brief description of the drawings)

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Mode for Carrying Out the Invention

[0018] (Detailed Description) According to a first aspect, there is provided the use of extracellular free nucleosomes as a biomarker in a plasma sample for the diagnosis or detection of angiosarcoma or hematological cancer. In particular, the cancer is a hematological cancer.

[0019] The nucleosome is the basic unit of chromatin structure and consists of a protein complex of eight highly conserved core histones (composed of pairs of each of histones H2A, H2B, H3, and H4). Approximately 146 base pairs of DNA are wrapped around this complex. Another histone, H1 or H5, acts as a linker and is involved in chromatin compaction. DNA often wraps around consecutive nucleosomes in a structure often described as "beads on a string," which forms the basic structure of open, or euchromatin. In compressed, or heterochromatin, this string forms coils and supercoils, resulting in a closed complex structure (Herranz and Esteller (2007) Methods Mol. Biol. 361:25-62).

[0020] References to "nucleosomes," when detected in a body fluid sample, may refer to "extracellular free nucleosomes." Throughout this document, it will be understood that the term extracellular free nucleosomes is intended to include any extracellular free chromatin fragment containing one or more nucleosomes. As used herein, "epigenetic features," "epigenetic signal features," or "epigenetic signal structure" of extracellular free nucleosomes may include, without limitation, one or more histone post-translational modifications, histone isoforms, modified nucleotides, and / or proteins that bind to nucleosomes in nucleosome-protein adducts.

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

[0022] In the methods and uses of the present invention, the level of (extracellular free) nucleosomes themselves can be measured. The reference to "nucleosomes themselves" refers to the total level or total concentration of nucleosomes present in a sample, whether or not any epigenetic features are included in the nucleosomes. Detection of the total level of nucleosomes typically involves detection of a histone protein common to all nucleosomes, such as histone H4. Thus, nucleosomes themselves can be measured by detecting a core histone protein, such as histone H4. As described herein, histone proteins form a structural unit known as a nucleosome, which is used to package DNA in eukaryotic cells. As previously reported in WO 2016 / 067029 (incorporated herein by reference), extracellular free nucleosomes of tumor cell origin can be isolated using specific histone variants, such as histone H3.1, H3.2, or H3t. Thus, the total level of extracellular free nucleosomes of tumor origin can be detected.

[0023] Normal cell turnover in adults involves the generation of hundreds of billions of cells per day by cell division and the death of a comparable number, mainly by apoptosis. During the process of apoptosis, chromatin is degraded into mononucleosomes and oligonucleosomes and released from the cell. Under normal conditions, circulating nucleosome levels found in healthy subjects have been reported to be low. Elevated levels have been found in subjects with various conditions, including many cancers, autoimmune diseases, inflammatory conditions, stroke, and myocardial infarction (Holdenreider and Stieber (2009) Crit Rev Clin Lab Sci, 46(1):1-24).

[0024] Current nucleosome ELISA methods are mainly used in cell culture as a method for detecting apoptosis (Salgame et al. (1997) Nucleic Acids Res, 25(3):680-681; Holdenrieder et al. (2001) supra; van Nieuwenhuijze et al. (2003) Ann Rheum Dis, 62:10-14), but are also used for measuring circulating extracellular free nucleosomes in serum and plasma (Holdenrieder et al. (2001)). The levels of extracellular free nucleosomes in serum and plasma released into the circulation by dead cells have been measured by ELISA in studies of many different cancers and their potential use as biomarkers has been evaluated. The mean levels of circulating nucleosomes have been reported to be elevated in most, but not all, of the cancers tested. However, patients with malignancies have been reported to have widely different nucleosome concentrations in serum, and some patients with advanced tumor disease have been found to have low circulating nucleosome levels within the range measured for healthy subjects (Holdenrieder et al. (2001)).

[0025] Extracellular free nucleosomes can be mononucleosomes or oligonucleosomes, or mixtures thereof.

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

[0027] Circulating nucleosomes are not a homogeneous group of protein-nucleic acid complexes. Rather, they are a heterogeneous mixture of chromatin fragments resulting from the digestion of chromatin during cell death, containing a very diverse epigenetic structure including specific histone isoforms (or variants), post-translational histone modifications, nucleotides or modified nucleotides, and protein adducts. It will be apparent to those skilled in the art that an increase in nucleosome levels is associated with an increase in a subset of some circulating nucleosomes that contain specific epigenetic signals, including nucleosomes containing specific histone isoforms (or variants), nucleosomes containing specific post-translational histone modifications, nucleosomes containing specific nucleotides or modified nucleotides, and nucleosomes containing specific protein adducts. Assays for these types of chromatin fragments are known in the art (see, e.g., WO 2005 / 019826, WO 2013 / 030579, WO 2013 / 030578, WO 2013 / 084002, which are incorporated herein by reference).

[0028] The biomarkers used in the use and method of the present invention can be at the level of the extracellular free nucleosome itself and / or the epigenetic characteristics of the extracellular free nucleosome. It will be understood that the terms "epigenetic signal structure" and "epigenetic characteristics" are used interchangeably herein. These terms refer to specific detectable characteristics of the nucleosome. In one embodiment, the epigenetic characteristics of the nucleosome are selected from the group consisting of post-translational histone modifications, histone variants, specific nucleotides, and protein adducts.

[0029] In one embodiment, the epigenetic characteristics of the nucleosome include one or more histone variants or isoforms. The epigenetic characteristics of the extracellular free nucleosome can be histone isoforms, such as histone isoforms of the core nucleosome, particularly histone H3 isoforms.

[0030] The terms "histone variant" and "histone isoform" may be used interchangeably herein. Also, the structure of the nucleosome can differ by including alternative histone isoforms or variants that are different genes or splicing products and have different amino acid sequences. Many histone isoforms are known in the art. Histone variants can be classified into several families that are subdivided into individual types. The nucleotide sequences of a number of histone variants are known and are publicly available, for example, in the National Human Genome Research Institute (NHGRI) Histone Database (Marino-Ramirez et al., "The Histone Database: an integrated resource for histones and histone fold-containing proteins.", Database Vol. 2011, and http: / / genome.nhgri.nih.gov / histones / complete.shtml), the GenBank (NIH gene sequence) database, the EMBL nucleotide sequence database, and the DNA Data Bank of Japan (DDBJ). For example, variants of histone H2 include H2A1, H2A2, mH2A1, mH2A2, H2AX, and H2AZ. In another example, 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 presented herein, H3.1 was effective in discriminating subjects with angiosarcoma or hematological cancer from healthy subjects. H3.1 was particularly effective in identifying patients with lymphoma because it discriminated 84% of patients with NHL from healthy subjects with 90% specificity (see Table 1).

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

[0033] For example, post-translational modifications can include acetylation, methylation, which can be mono-, di-, or tri-methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination, and / or isomerization (see Ausio (2001) Biochem Cell Bio 79:693). In one embodiment, histone PTMs are selected from methylation or citrullination. In a further embodiment, histone PTMs are H3K27me3 or H3 citrulline (H3cit). In yet a further embodiment, histone PTMs are H3cit. As shown in the examples presented herein, H3cit was the most effective histone PTM for discriminating between subjects with healthy controls and those with vascular or hematological cancers.

[0034] Groups or classes of related post-translational histone modifications (not a single modification) can also be detected. Typical examples include, without limitation, two-site immunoassays that utilize one antibody or other selective binding agent directed to binding to a nucleosome and one antibody or other selective binding agent directed to binding to a group of target histone modifications. Examples of antibodies directed to binding to a group of such histone modifications include, without limitation for purposes of illustration, anti-pan-acetylation antibodies (e.g., pan-acetyl H4 antibody [H4panAc]), anti-citrullination antibodies, or anti-ubiquitin antibodies.

[0035] In one embodiment, the epigenetic features of a nucleosome include one or more DNA modifications. In addition to epigenetic signaling mediated by the composition of nucleosome histone isoforms and PTMs, nucleosomes also differ in their nucleotide and modified nucleotide composition. Global DNA hypomethylation is a characteristic of cancer cells, and some nucleosomes may contain more 5-methylcytosine residues (or 5-hydroxymethylcytosine residues or other nucleotides or modified nucleotides) than other nucleosomes. In one embodiment, the DNA modification is selected from 5-methylcytosine or 5-hydroxymethylcytosine.

[0036] In one embodiment, the epigenetic features of a nucleosome include one or more protein-nucleosome adducts or complexes. A further subset of circulating nucleosomes are nucleosome protein adducts. It has been known for many years that chromatin contains a number of non-histone proteins bound to its components, DNA and / or histones. These chromatin-related proteins are of a very diverse variety and have various functions, including many such as transcription regulators, transcription enhancers, transcription repressors, histone regulatory enzymes, DNA damage repair proteins. These chromatin fragments containing nucleosomes and other non-histone chromatin proteins or DNA and other non-histone chromatin proteins are described in the art.

[0037] In one embodiment, the protein added to the nucleosome (and thus which can be used as a biomarker) is selected from a transcription regulator, a high mobility group protein, or a chromatin regulatory enzyme. Reference to a "transcription regulator" refers to a protein that binds to DNA and regulates gene expression by promoting (i.e., an activator) or suppressing (i.e., a repressor) transcription. A transcription regulator includes one or more DNA binding domains (DBDs) that bind to a specific sequence of DNA adjacent to the gene it regulates. All circulating nucleosomes and portions, types, or subgroups of nucleosomes described herein may be useful in the present invention.

[0038] It will be understood that two or more epigenetic features of extracellular free nucleosomes can be detected in the methods and uses of the present invention. Multiple biomarkers can be used as a combined biomarker. Thus, in one embodiment, the use includes two or more epigenetic features of extracellular free nucleosomes as a combined biomarker. Epigenetic features can be of the same type (e.g., PTM, histone isoform, nucleotide, or protein adduct) or different types (e.g., PTM in combination with a histone isoform). For example, post-translational histone modifications and histone variants can be detected (i.e., two or more types of epigenetic features are detected). Alternatively or additionally, two or more types of post-translational histone modifications can be detected, or two or more types of histone isoforms can be detected. In one aspect, the use includes post-translational histone modifications and histone isoforms as a combined biomarker in a plasma sample for the diagnosis or detection of vascular cancer or hematological cancer. In one embodiment, the combined biomarker is H3.1 and H3cit. In an alternative embodiment, the combined biomarker is H3.1 and H3K27Me3.

[0039] The term "biomarker" means a differential biological or biogenic indicator of a process, event, or condition. Biomarkers can be used in diagnostic methods, such as clinical screening and prognosis evaluation, and in monitoring treatment outcomes, identifying patients most likely to respond to a particular therapeutic treatment, drug screening, and drug discovery. Biomarkers and their use are beneficial for identifying new drug therapies and new targets for drug therapy.

[0040] The methods and uses described herein can be assayed in a body fluid sample, particularly a blood, serum, or plasma sample. Preferably, a plasma sample is used. Plasma samples can be collected in a collection tube containing one or more anticoagulants, such as ethylenediaminetetraacetic acid (EDTA), heparin, or sodium citrate, particularly EDTA.

[0041] (Hematological cancer) Since hematological cancer is a cancer of the blood, it can also be referred to as "blood cancer". There are mainly three types of hematological cancer: leukemia caused by the rapid production of abnormal white blood cells; lymphoma caused by abnormal lymphoma cells; and multiple myeloma, which is a cancer of plasma cells.

[0042] In one embodiment, the hematological cancer is selected from lymphoma, leukemia, multiple myeloma, chronic myeloproliferative disorders, monoclonal gammopathy of undetermined significance, myelodysplastic syndromes, and amyloidosis. In a further embodiment, the hematological cancer is selected from leukemia or lymphoma.

[0043] Leukemia affects white blood cells and can be classified by the type of white blood cell affected (myeloid or lymphoid) and the way the disease progresses (acute or chronic). Some types of leukemia that have been identified include, but are not limited to, acute lymphoblastic leukemia (ALL; also referred to as acute lymphocytic leukemia), acute myeloid leukemia (AML), acute megakaryoblastic leukemia (AMKL), acute promyelocytic leukemia (APL), childhood acute myeloid leukemia (C-AML), childhood acute lymphoblastic 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 acute lymphoblastic leukemia, and prolymphocytic leukemia.

[0044] In one embodiment, the leukemia is acute leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute megakaryoblastic leukemia (AMKL), or acute promyelocytic leukemia (APL). In a further embodiment, the leukemia is selected from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Alternatively, in one embodiment, the leukemia is chronic leukemia, such as chronic eosinophilic leukemia (CEL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), or chronic neutrophilic leukemia.

[0045] Both Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) are lymphomas. The majority of NHL patients are over 55 years old at the time of initial diagnosis, whereas the median age at which Hodgkin lymphoma is diagnosed is 39 years. In one embodiment, the lymphoma is non-Hodgkin lymphoma (NHL). NHL can occur in lymph nodes in any part of the body, whereas HL typically occurs in the upper body, such as the neck, chest, or armpit.

[0046] Hodgkin lymphoma is often diagnosed early and is thus considered one of the most treatable cancers. Non-Hodgkin lymphoma is typically not diagnosed until it reaches a more advanced stage, so the method of the present invention has a special application in the diagnosis of NHL, where it is desired to detect patients early in the disease and improve treatment outcomes.

[0047] (Other blood cancers) Angiosarcoma and hemangiosarcoma are soft tissue cancers that involve the growth of cells lining the vasculature and can thus be referred to as "vascular cancers." Similar to hematological cancers, these are closely associated with the vasculature, and the cells affected are in direct contact with the circulating blood. The inventors herein show that these blood cancers are associated with very high levels of circulating nucleosomes comparable to those observed in lymphomas.

[0048] (Detection and diagnostic methods) The present invention provides a method that can be used for the detection or diagnosis of a patient having a vascular cancer or a hematological cancer. Thus, in a further aspect, a method for diagnosing or detecting a vascular cancer or a hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes; and (ii) using the level of the detected extracellular free nucleosomes to diagnose a subject having the vascular cancer or the hematological cancer, is provided.

[0049] In a further aspect, a method for diagnosing or detecting a hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes; and (ii) using the level of the detected extracellular free nucleosomes to diagnose a subject having a hematological cancer, is provided.

[0050] Alternatively, in a further aspect, a method for diagnosing or detecting a vascular cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes; and (ii) diagnosing a subject having angiosarcoma using the level of the detected extracellular free nucleosomes, wherein the method is provided.

[0051] In a further aspect, a method for determining the prognosis of a subject having or suspected of having or being predisposed to angiosarcoma or hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes; and (ii) using the level of the detected extracellular free nucleosomes as an indicator of the prognosis of the angiosarcoma or hematological cancer, wherein the method is provided.

[0052] Even if it is determined that the subject does not have angiosarcoma or hematological cancer, the present invention can still be used for the purpose of monitoring disease progression. For example, if the use includes samples from subjects determined to not have angiosarcoma or hematological cancer, the measurement of biomarker levels can be repeated at another time point to establish whether the biomarker levels have changed.

[0053] In a further aspect, a method for monitoring the efficacy of treatment in a subject having or suspected of having or being predisposed to angiosarcoma or hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes; and (ii) comparing the level of the detected extracellular free nucleosomes with a plasma sample previously taken from the subject to determine the efficacy of the treatment, wherein the method is provided.

[0054] Detection and / or quantification can be performed directly on a purified or concentrated nucleosome sample, or indirectly on an extract or dilution thereof from the sample. Quantification of the amount of biomarker present in the sample can include determining the concentration of the biomarker present in the sample. The methods of use, detection, monitoring, and diagnosis according to the invention described herein are useful for confirming the presence of a disease, monitoring the development of the disease by assessing onset and progression, or assessing improvement or regression of the disease. Also, the methods of use and detection, monitoring, and diagnosis are useful for evaluation of clinical screening, prognosis, selection of treatment, evaluation of treatment benefit, i.e., methods of drug screening and drug discovery.

[0055] Detection or measurement can include immunoassays, immunochemistry, mass spectrometry, chromatography, chromatin immunoprecipitation, or biosensor methods. In particular, detection and / or measurement can include a two-site immunoassay method for nucleosome moieties. Such methods utilize two anti-nucleosome binding agents or a combination of an anti-nucleosome binding agent with an anti-histone modification or anti-histone variant or anti-DNA modification or anti-accessory protein detection binding agent for the measurement of nucleosomes or nucleosomes incorporating epigenetic features in situ. Also, detection and / or measurement can include a two-site immunoassay utilizing a combination of a labeled anti-nucleosome detection binding agent with an immobilized anti-histone modification or anti-histone variant or anti-DNA modification or anti-accessory protein binding agent.

[0056] Detection or measurement of the level of biomarker(s) can be performed using one or more reagents such as suitable binding agents. For example, the one or more binding agents can include ligands or binding agents specific for a desired biomarker such as a nucleosome or a component part thereof, an epigenetic feature of a nucleosome, a structural / shape mimic of a nucleosome or a component part thereof, optionally in combination with one or more interleukins.

[0057] As will be apparent to those skilled in the art, although the terms "antibody", "binding agent", or "ligand" as used herein are not intended to be limiting, they are intended to include any binding agent capable of binding to a specific molecule or entity, and it will be apparent that any suitable binding agent can be used in the methods of the present invention. Also, it will be apparent that the term "nucleosome" is intended to include mononucleosomes and oligonucleosomes that can be analyzed in a fluid medium, as well as any protein-DNA chromatin fragment.

[0058] Methods for detecting biomarkers are known in the art. The reagent can include one or more ligands or binding agents capable of specific binding to a desired target, such as natural compounds or chemically synthesized compounds. The ligand or binding agent can include peptides, antibodies, or fragments thereof capable of specific binding to a desired target, or synthetic ligands such as plastic antibodies, or aptamers, or oligonucleotides. The antibody can be a monoclonal antibody or a fragment thereof. When using an antibody fragment, it will be understood that it retains the ability to bind to the biomarker and thus (in accordance with the present invention) can detect the biomarker. The ligand / binding agent can be labeled with a detectable marker, such as a luminescent, fluorescent, enzyme, or radioactive marker; alternatively, the ligand according to the present invention can further be labeled with an affinity tag, such as biotin, avidin, streptavidin, or a His (e.g., hexahistidine) tag. Alternatively, ligand binding can be determined using label-free techniques, such as the label-free technique of ForteBio.

[0059] As used herein, the terms "detect" or "diagnose" include the identification, confirmation, and / or characterization of a disease state. The methods of detection, monitoring, and diagnosis according to the present invention are useful for monitoring the development of a disease by confirming the presence of the disease and evaluating its onset and progression, or for evaluating the improvement or regression of the disease. Further, the methods of detection, monitoring, and diagnosis are useful for the evaluation of clinical screening, prognosis, treatment selection, evaluation of treatment benefit, i.e., drug screening and drug discovery methods.

[0060] In one embodiment, the methods described herein are repeated at multiple times. This embodiment provides the advantage of enabling the monitoring of detection results over a period of time. Such a scheme provides the benefit of monitoring and / or evaluating the effectiveness of treatment of a disease state. Using such a monitoring method of the present invention, onset, progression, stabilization, improvement, recurrence, and / or remission can be monitored.

[0061] In the monitoring method, the test sample can be taken at two or more times. The method may further include comparing the level(s) of biomarker(s) present in the test sample with one or more controls and / or one or more past test samples taken previously from the same test subject, e.g., before treatment initiation and / or from the same test subject at an earlier stage of treatment. The method may include detecting changes in the nature or amount of biomarker(s) in test samples taken at different times.

[0062] A change compared to the level in a past test sample taken previously from the same test subject of the level of a biomarker in the test sample can indicate a beneficial effect of its treatment on a disorder or suspected disorder, e.g., stabilization or improvement. Further, once treatment is completed, the methods of the present invention can be repeated periodically to monitor for recurrence of the disease.

[0063] Using methods for monitoring the effectiveness of treatment, the therapeutic effectiveness of existing and novel therapies in human subjects and non-human animals (e.g., animal models) can be monitored. These monitoring methods can be incorporated into the screening of novel drug substances and combinations of substances.

[0064] In further embodiments, monitoring of more rapid changes due to rapidly acting treatments can be performed at shorter time or day intervals.

[0065] A diagnostic or monitoring kit (or panel population) is provided for practicing the methods of the invention. Such kits preferably include one or more ligands for the detection and / or quantification of biomarkers according to the invention, and / or biosensors and / or arrays described herein, optionally together 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, said kit comprising a biosensor capable of detecting and / or quantifying one or more biomarkers as defined herein. As used herein, the term "biosensor" means anything capable of detecting the presence of a biomarker. Examples of biosensors are described herein. The biosensor can include a ligand binding agent or ligand described herein capable of specific binding to a biomarker. Such biosensors are useful for the detection and / or quantification of the biomarkers of the invention.

[0067] Suitably, a biosensor for the detection of one or more biomarkers combines the recognition of a biomolecule with suitable means for converting the detection or quantification of the presence of the biomarker in a sample into a signal. The biosensor can be adapted for use in "alternate sites" for diagnostic testing, such as hospital wards, outsubjects’ departments, operating rooms, homes, fields, and workplaces. Biosensors for detecting one or more biomarkers of the present invention include acoustic, plasmon resonance, holography, biolayer interferometry (BLI), and microengineering sensors. Imprint recognition elements, thin film transistor technology, magnetoacoustic resonator devices, and other novel acousto-electric systems can be utilized in biosensors for detecting one or more biomarkers.

[0068] Biomarkers for detecting the presence of a disease are extremely important targets for discovering novel targets and drug molecules that slow or halt the progression of a disorder. Since the levels of biomarkers indicate disorders and drug responses, biomarkers are useful in the identification of novel therapeutic compounds in in vitro and / or in vivo assays. The biomarkers described herein can be utilized in methods for screening compounds that modulate the activity of a biomarker.

[0069] Accordingly, in a further aspect of the invention, there is provided the use of a binder or ligand, which can be a peptide, antibody, or fragment thereof, or an aptamer, or an oligonucleotide, directed to a biomarker according to the invention as described; or the use of a biosensor, or an array, or a kit according to the invention for identifying substances that can promote and / or inhibit the production of a biomarker.

[0070] The immunoassays described herein include any method that utilizes one or more antibodies or other specific binding agents that are directed to bind to a biomarker as defined herein. Immunoassays include two-site immunoassays or immunometric assays (e.g., ELISA) that utilize enzyme detection methods, fluorescent-labeled immunometric assays, time-resolved fluorescent-labeled immunometric assays, chemiluminescent immunometric assays, immunoturbidimetric assays, microparticle-labeled immunometric assays, and immunoradiometric assays, as well as one-site immunoassays, reagent-limited immunoassays, competitive immunoassay methods that include labeled antigens and labeled antibodies, and single antibody immunoassay methods that use various types of labels including radioactivity, enzymes, fluorescence, time-resolved fluorescence, and microparticle labels. All of the above immunoassay methods are well known in the art. See, for example, the references by Salgame et al. (1997) and van Nieuwenhuijze et al. (2003).

[0071] Identification, detection, and / or quantification can be carried out by any method suitable for identifying the presence and / or amount of a specific protein in a biological sample derived from a subject, or a purified product, extract, or dilution thereof. In particular, quantification can be carried out by measuring the concentration of the target in one or more samples. Biological samples that can be examined in the methods of the present invention include the biological samples defined above herein. Samples can be prepared by conventional methods and, for example, appropriately diluted or concentrated and stored. The present invention finds particular use in plasma samples obtainable from a subject.

[0072] Identification, detection, and / or quantification of a biomarker can be carried out by detection of the biomarker or a fragment thereof, such as a fragment with a cleaved C-terminus or a cleaved N-terminus. The fragment preferably has a length of more than 4 amino acids, for example, a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In particular, note that peptides having a sequence identical or related to the sequence of the histone tail are particularly useful fragments of histone proteins.

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

[0074] The method involving detection and / or quantification of one or more biomarkers of the present invention can be performed on laboratory equipment or incorporated into a disposable diagnostic or monitoring platform that can be used in an out-of-laboratory environment, such as a physician's office or beside the subject's bed. A biosensor suitable for performing the method of the present invention includes a "credit" card equipped with an optical or acoustic reader. The biosensor is configured to electronically transmit the collected data for interpretation by a physician and thus can form the basis of e-medicine.

[0075] The identification of biomarkers for disease states enables the integration of diagnostic procedures and treatment regimens. Biomarkers provide means of indicating treatment response, failure of response, undesirable side effect profiles, degree of medication compliance, and achievement of appropriate serum drug levels. Biomarkers can be used to provide warnings of adverse drug reactions. Since the evaluation of responses can be used to fine-tune dosages, minimize the number of doses prescribed, reduce delays in achieving effective treatment, and avoid adverse drug reactions, biomarkers are useful in the development of personalized treatment. Thus, by monitoring the biomarkers of the present invention, the care of a subject can be accurately tailored to meet the needs determined by the subject's disorder and pharmacogenomic profile, and for this purpose, the biomarkers can be used to adjust the optimal dosage, predict a positive treatment response, and identify subjects at high risk of severe side effects.

[0076] Biomarker-based tests provide a first-choice assessment of "new" subjects that cannot be achieved using current means and provide an objective means for accurate and rapid diagnosis.

[0077] Also, methods of monitoring biomarkers, biosensors, and kits are important as subject monitoring tools to enable a physician to determine whether a recurrence is due to a worsening of the disorder. If pharmacological treatment is evaluated as being inadequate, treatment can be restarted or increased; treatment can be changed if appropriate. Since biomarkers are sensitive to the state of the disorder, biomarkers provide an indicator of the effect of drug treatment.

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

[0079] In one embodiment, the subject is suspected of having a recurrence of a vascular or hematological cancer. Minimal residual disease (MRD) is the name given to the small number of leukemia cells (bone marrow-derived cancer cells) that remain during treatment of a patient or after treatment when the patient is in remission (i.e., the patient has no symptoms or signs of the disease). However, MRD is the main cause of recurrence in cancer and leukemia. Thus, the methods of the present invention are useful for monitoring patients suspected of recurrence, particularly patients in remission from cancer.

[0080] Subjects tested using the methods described herein may present with symptoms indicative of a hematological cancer, such as anemia, leukocytosis, and / or swollen lymph nodes. In one embodiment, the subject has a high level of leukocytosis. This may also be referred to as a "high white blood cell count." Hematological cancers typically cause an increase in the proliferation of abnormal white or red blood cells, resulting in a high white blood cell count. However, leukocytosis is often a sign of an inflammatory reaction, most commonly as a result of an infection, and thus leukocytosis is not sufficient to diagnose a patient with a hematological cancer (particularly leukemia). Thus, the methods of the present invention may provide a more specific method for detecting patients likely to be suffering from a hematological cancer.

[0081] The detection results and / or quantification results can be compared with a cut-off level. The cut-off value can be determined in advance by analyzing the results from a plurality of patients and controls and determining a value suitable for classifying subjects as having or not having the disease. For example, for a disease in which the biomarker level of patients suffering from the disease is higher, if the detected level is higher than the cut-off, it is indicated that the patient is suffering from the disease. Alternatively, for a disease in which the biomarker level of patients suffering from the disease is lower, if the detected level is lower than the cut-off, it is indicated that the patient is suffering from the disease. The advantages of using a simple cut-off value may include that clinicians can easily understand the test, and any need for software or other assistance in the interpretation of test results is eliminated. The cut-off level can be determined using methods in the art.

[0082] Also, the detection results and / or quantification results can be compared with a control. It will be apparent to those skilled in the art that control subjects, which can include, for example, subjects known not to have the disease or subjects having different diseases (e.g., for different diagnostic studies), can be selected according to various criteria. The "control" can include healthy subjects, non-affected subjects, and / or subjects without vascular cancer or hematological cancer. Comparison with a control is well known in the field of diagnosis.

[0083] Accordingly, in one embodiment, the method further comprises comparing the level of the extracellular free nucleosomes in the plasma sample with one or more controls. For example, the method can include comparing the level of extracellular free nucleosomes present in a plasma sample obtained from a subject with the level of extracellular free nucleosomes present in a plasma sample obtained from a normal subject. The control can be a healthy subject. Alternatively, the control can be an affected subject, such as a subject having an infectious disease.

[0084] Alternatively, the control is a subject having a cancer that is not a vascular cancer or a hematological cancer, i.e., a control subject having a cancer affecting different organs of the body. The data provided herein show that the biomarker of the present invention is significantly elevated in patients with hematological cancer compared to patients with other forms of cancer, and thus the biomarker of the present invention can be used to diagnose patients having a vascular cancer or a hematological cancer separately from patients having other forms of cancer. Thus, in one aspect, the diagnosis involves diagnosing a vascular cancer or a hematological cancer separately from a non-vascular cancer or a non-hematological cancer. "Non-vascular cancer or non-hematological cancer" is a cancer that is not a hematological cancer and is not associated with the proliferation of blood cells or vascular cells, 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 can be a subject having a high level of leukocytosis. As discussed herein, leukocytosis is not a symptom specific to leukemia. Thus, the methods of the present invention can be used to compare controls having a high level of leukocytosis that is not the result of a vascular cancer or a hematological cancer, e.g., controls having inflammation, an infection, and / or undergoing drug treatment.

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

[0087] It will be understood that in no case is it necessary to measure control levels for comparison purposes. For example, for healthy / non-affected controls, once a "normal range" has been established, it can be used as a reference for all subsequent tests. The normal range can be established by obtaining samples from a plurality of control subjects without vascular cancer or hematological cancer and examining the levels of the biomarker. The results (i.e., biomarker levels) of subjects suspected of having a vascular cancer or a hematological cancer can subsequently be examined to determine whether they are within or outside their respective normal ranges. The use of a "normal range" is a standard convention for detecting diseases.

[0088] In one embodiment, the method further comprises determining at least one clinical parameter of the patient. This parameter can be used for the interpretation of the results. Clinical parameters can include any relevant clinical information, such as, without limitation, gender, weight, body mass index (BMI), smoking status, and eating habits. Thus, in one embodiment, the clinical parameter is selected from the group consisting of age, gender, and body mass index (BMI).

[0089] In one embodiment, the method of the present invention is carried out to identify subjects at high risk of having a vascular or hematological cancer and thus in need of further tests (i.e., further cancer investigations). The further tests can include one or more of: biopsy (e.g., bone marrow biopsy or lymph node biopsy), cytogenetic testing, immunophenotyping, CT scan, X-ray (particularly a chest X-ray to identify swollen lymph nodes), and / or lumbar puncture.

[0090] Using the methods and biomarkers described herein, it is possible to identify whether a patient requires a biopsy, particularly a bone marrow biopsy or a lymph node biopsy. Thus, according to a further aspect of the present invention, there is provided a method of identifying a patient in need of a biopsy, comprising obtaining a plasma sample from the patient, detecting the level of extracellular free nucleosomes in the plasma sample, and using the results obtained from the panel test to identify whether the patient requires a biopsy.

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

[0092] (Additional biomarkers) The level of extracellular free nucleosomes can be detected or measured as one of the measurement panels. The panel may include different epigenetic features of nucleosomes (e.g., histone isoforms and PTMs) described hereinabove. In one embodiment, the panel includes one or more cytokines, such as one or more interleukins.

[0093] Interleukins (ILs) are a group of cytokines usually secreted by white blood cells and act as signaling molecules. Interleukins play important roles in immune responses and inflammation stimuli. Interleukins were first identified in the 1970s and have been numbered as additional interleukin species have been discovered. Examples of interleukins include, but are not limited to: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15.

[0094] In one embodiment, the one or more interleukins are selected from the group consisting of interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-1β (IL-1β).

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

[0096] Alternatively, further, the interleukin can be IL-10. Interleukin-10 (IL-10) is an anti-inflammatory cytokine having extremely diverse biological functions. The sequence of human IL-10 is known in the art and is described in UniProt accession number P22301. In a particular embodiment, the interleukin is IL-10, and the measurement panel can include the measurement of histone post-translational modification H3cit and IL-10.

[0097] Alternatively, further, the interleukin can be IL-1β. Interleukin-1β (IL-1β) is a pro-inflammatory cytokine and is involved in various cellular activities including cell proliferation, differentiation, and apoptosis. In a particular embodiment, the interleukin is IL-1β, and the measurement panel can include the measurement of histone isoform H3.1 and IL-1β. More specifically, the panel can include the measurement of histone isoform H3.1 and IL-1β, where the angiosarcoma or hematological cancer is lymphoma, such as NHL.

[0098] In one embodiment, the panel includes the epigenetic features of extracellular free nucleosomes and an interleukin. In another embodiment, the panel includes the epigenetic features of extracellular free nucleosomes and two interleukins. For example, the measurement of extracellular free nucleosomes can be combined with two or more interleukins, such as IL-6 and IL-1β, or IL-10 and IL-1β, or IL-6 and IL-10. In a further embodiment, the epigenetic features of extracellular free nucleosomes are selected from histone isoforms, such as H3.1, and post-translationally modified histones, such as H3cit. In yet a further embodiment, the measurement panel is H3.1, IL-6, and IL-1β. In an alternative embodiment, the measurement panel is H3cit, IL-10, and IL-1β.

[0099] Using the biomarkers of the present invention, a model can be derived. Methods for deriving models or algorithms such as those in Tables 5 and 6 of the examples are well known in the art and suitable software packages are available. Typical software tools for this purpose include SPSS (Statistical Package for the Social Sciences) and "R". These software packages provide linear and non-linear data modeling of clinical data.

[0100] It will be apparent to those skilled in the art that any combination of the biomarkers disclosed herein can be used in panels and algorithms for the detection of angiocarcinoma or hematological cancer, and that additional markers can be added to the panels containing these markers.

[0101] Use of a panel test for detecting a patient having an angiocarcinoma or hematological cancer, said panel test comprising reagents for detecting the measurement of nucleosomes or components thereof and one or more interleukins in a plasma sample obtained from the patient.

[0102] (Method of treatment) In a further aspect, a method of treating an angiocarcinoma or hematological cancer in a subject comprising: (i) detecting or measuring the level of extracellular free nucleosomes in a plasma sample obtained from the subject; (ii) using the level measured in step (i) as an indicator of the presence of an angiocarcinoma or hematological cancer in the subject; and (iii) when it is determined in step (ii) that the subject has the angiocarcinoma or hematological cancer, performing a surgical procedure or administering a therapeutic agent.

[0103] In a further aspect, a method of treating a vascular cancer or a hematological cancer in a subject in need thereof, the method comprising the step of performing a surgical procedure or administering a therapeutic agent to a subject identified as having a different level of extracellular free nucleosomes in a plasma sample obtained from the subject as compared to the level of extracellular free nucleosomes in a plasma sample obtained from a control subject.

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

[0105] The method may comprise: (i) measuring the level of extracellular free nucleosomes in a plasma sample obtained from the subject (optionally in combination with the level of one or more interleukins); (ii) identifying the subject as having a vascular cancer or a hematological cancer based on the level of extracellular free nucleosomes being higher compared to a control; and (iii) treating the subject.

[0106] According to another aspect of the invention, a method of treating a vascular cancer or a hematological cancer, the method comprising identifying a patient in need of treatment for a vascular cancer or a hematological cancer using a panel assay and providing the treatment, the panel assay comprising reagents for detecting the measurement of nucleosomes or components thereof and one or more interleukins. A patient having a vascular cancer or a hematological cancer is expected to have a higher level of extracellular free nucleosomes compared to a control.

[0107] It will be understood that the embodiments described herein can be applied to all aspects of the invention, i.e., embodiments with descriptions of use can be equally applied to methods described in the claims and the like.

[0108] The present 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, whole blood collections were gathered into EDTA vacutainer tubes and the tubes were gently inverted 10 times. Within 2 hours of blood collection, the whole blood was centrifuged at 1500 g for 15 minutes. The plasma was transferred to cryotubes and immediately frozen. Within the cohort, 62 subjects were healthy, 25 subjects had non-Hodgkin lymphoma (NHL), 22 subjects had acute myeloid leukemia (AML), and 7 subjects had acute lymphoblastic leukemia (ALL). Subjects with leukemia or lymphoma could be further classified into patients diagnosed with cancer (a total of 31 subjects, 16 subjects diagnosed with NHL and 15 subjects diagnosed with leukemia), or patients with cancer due to recurrence (a total of 15 subjects; 6 subjects had recurrent NHL and 9 subjects had recurrent leukemia).

[0110] Samples were analyzed for nucleosomes containing H3.1 by ELISA. Briefly, measurements of nucleosomes containing histone isoform H3.1 were performed as follows: 80 μl of assay buffer and 20 μl of plasma sample or standard nucleosome preparation were added to microtiter wells coated with an antibody directed to bind histone H3.1. The microtiter plate was covered and incubated for 2.5 hours at room temperature with gentle shaking. The contents of the microtiter wells were discarded. The wells were washed three times with 200 μl of wash solution, and 100 μl of biotinylated anti-nucleosome antibody was added. The microtiter plate was covered again and incubated for 1.5 hours at room temperature with gentle shaking. The contents of the microtiter wells were discarded. The wells were washed three times with 200 μl of wash solution, and 100 μl of streptavidin-HRP solution was added. The microtiter plate was covered again and incubated for 0.5 hours at room temperature with gentle shaking. The contents of the microtiter wells were discarded. The wells were washed three times with 200 μl of wash solution, and 100 μl of HRP (horseradish peroxidase) substrate solution was added. The microtiter plate was covered and incubated for 20 minutes in the dark at room temperature with gentle shaking. The absorbance (OD) of the wells was measured at 405 nm. The OD levels were either used directly or the plasma levels of nucleosomes containing histone H3.1 were interpolated from a standard curve.

[0111] The OD results were plotted as receiver operating characteristic (ROC) curves. The results were grouped for all patients with cancer versus healthy subjects, as well as for all patients with lymphoma versus healthy subjects, and for all patients with leukemia (i.e., including ALL and AML) versus healthy subjects. The results of the area under the curve (AUC) are shown in Tables 1 - 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 - Recurrence Patients)

Table 3

[0115] As shown in Table 1, the levels of extracellular free nucleosomes containing H3.1 were able to distinguish more than 75% of blood cancer patients from healthy donors with 90% specificity. As shown in Table 2, this result could increase to more than 80% with 90% specificity for patients diagnosed with cancer. Also, since 84% of patients with NHL were distinguished from healthy subjects with 90% specificity, this result indicated that this biomarker was particularly effective in identifying patients with lymphoma (see Table 1).

[0116] The concentrations of H3.1 in plasma samples of all AML, ALL, and NHL patients were derived from OD values using a standard curve for interpolation and compared with the levels in healthy patients. The results are shown in Figure 1. The levels of H3.1 in patients with hematological cancer were clearly elevated compared to healthy subjects.

[0117] This result indicates that the levels of nucleosomes, particularly nucleosomes containing H3.1, can be used for the detection of hematological cancer.

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

[0119] The results are shown in Figure 2. Surprisingly, it was found that using the level of H3.1, it is possible to distinguish subjects with hematological cancer from patients with other forms of cancer. The levels of H3.1 in patients with various hematological cancers (ALL, AML, and NHL) were significantly elevated compared to the levels of H3.1 in plasma samples from patients with bladder cancer, bone cancer, brain cancer, esophageal cancer, head and neck cancer, skin cancer, thyroid cancer, tongue cancer, uterine cancer and cervical cancer, and melanoma.

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

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

[0122] All nucleosome post-translational modifications tested showed a significant increase in patients with hematological cancer compared to healthy controls. Even when hematological cancers were divided by type (ALL, AML, NHL), the levels of H3cit, H3K27Me3, and H4panAc extracellular free nucleosomes in plasma samples were significantly elevated compared to healthy controls (see Figures 3B, 4B, and 5B). The levels of post-translationally modified nucleosomes were most elevated in plasma samples obtained from patients with ALL.

[0123] (Example 4) The two most prominent extracellular free nucleosome markers (H3.1 and H3cit) were combined together, or combined with the measurement of different interleukins. The levels of extracellular free nucleosomes were measured as described above, and the levels of plasma interleukins were measured using a commercially available ELISA method.

[0124] The assay results were modeled by logistic regression analysis and the model or algorithm was trained using the highest AUC for the comparison of human patients with blood cancer against normal human donors. The results are summarized in Table 5.

[0125] (Table 5: ROC curves for the combined biomarker panel in plasma - all blood cancers ♯ )

Table 5

[0126] As shown in Table 5, all models were able to distinguish more than 75% of blood cancer patients from healthy donors with a specificity of 90%. The results indicate that combining the levels of nucleosomes with the levels of interleukins can result in an effective assay panel by a relevant algorithm for the detection of blood cancers.

[0127] (Example 5) Plasma samples from human patients with NHL only were modeled by logistic regression analysis as described in Example 4. The results are summarized in Table 6.

[0128] (Table 6: ROC curves for the combined biomarker panel in plasma - NHL cancer ♯ )

Table 6

[0129] As shown in Table 6, all models were able to distinguish patients with NHL greater than 80% from healthy donors with 90% specificity. In particular, the combination of H3.1 and IL-1β was able to distinguish patients with 100% NHL with 80% specificity. The results indicate that using the levels of nucleosomes and interleukins, an effective assay panel can be made with relevant algorithms for the detection of NHL.

[0130] (Example 6) Plasma samples were collected from 73 dogs diagnosed with canine angiosarcoma, 127 dogs diagnosed with canine lymphoma, and 134 control dogs without cancer. Samples were analyzed for nucleosomes containing H3.1 by ELISA.

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

[0132] Dogs diagnosed with lymphoma were found to have greatly elevated levels of circulating nucleosomes containing histone isoform H3.1 (mean 570 ng / ml, median 211 ng / ml). The dot plot and ROC curve of the results obtained for canine lymphoma are shown in FIGS. 6A and 6B, respectively. The AUC for lymphoma detection was 87%. When using a cut-off of 67.4 ng / ml, the specificity of the assay was 100% and the sensitivity was 74%. When using a relatively low cut-off of 48.1 ng / ml, it gave a sensitivity of 81% and a specificity of 90%.

[0133] In addition, dogs diagnosed with angiosarcoma were found to have a significantly increased level of circulating nucleosomes containing histone isoform H3.1 (average 513 ng / ml, median 361 ng / ml). The dot plot and ROC curve of the results obtained for canine angiosarcoma are shown in FIGS. 7A and 7B, respectively. The AUC for angiosarcoma detection was 97.6%. When a cutoff of 67.4 ng / ml was used, the specificity of the assay was 100% and the sensitivity was 89%. When a relatively low cutoff value of 48.1 ng / ml was used, it gave a sensitivity of 95% and a specificity of 90%.

[0134] Also, as found in human diseases, the levels of circulating nucleosomes observed in dogs diagnosed with lymphoma or angiosarcoma were much higher than those observed for other canine tumors investigated.

[0135] Most assays for human proteins cannot be transferred to other animals. However, the structure of nucleosomes is highly conserved across species and even across phyla. The inventors have shown that this means that the assay for this human H3.1 nucleosome can be transferred to other species including dogs and horses. Furthermore, the results the inventors observed for canine vascular and hematopoietic cancers closely resemble those observed in human subjects. The inventors conclude that the method of the present invention is a very effective method for detecting vascular and hematopoietic cancers in both humans and animals.

[0136] (Example 7) A series of plasma samples were collected from two dogs undergoing treatment for angiosarcoma and two dogs undergoing treatment for lymphoma. To avoid providing information that could influence treatment decisions in a clinical setting based on treatment background, all samples from the four dogs were collected after the last day of treatment in FIG. 8 and then analyzed for nucleosomes containing histone isoform H3.1.

[0137] C-reactive protein (CRP) is a well-known biomarker of inflammation. This protein in the sample was also measured as a control to determine whether the level of nucleosome merely reflects the inflammatory response or provides additional information regarding the subject's condition, prognosis, and treatment response.

[0138] Dog 1 (Figure 8A) was diagnosed with angiosarcoma and received treatment with a chemotherapy regimen of four drugs known as CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), which began on day 1 and continued until day 129. The treatment was successful, and Dog 1 was determined to be in remission for the treatment context on day 160. The success of CHOP treatment was reflected in the downward trend of nucleosome levels over the course of treatment, and the remission of the disease was predicted by nearly normal nucleosome levels by day 122. This result indicates that nucleosome levels are useful as a prognostic indicator and that it can be used to guide treatment regimens and monitor subjects during remission to investigate disease recurrence. CRP analysis was not useful for clinical purposes.

[0139] Dog 2 (Figure 8B) was initially diagnosed with angiosarcoma in 2017 and treated with doxorubicin and immunomodulatory agents. This dog was regularly monitored by whole-body CT scans every 2–3 months. The dog was found to have relapsed after 2 years, which was reflected in high levels of circulating nucleosomes. This relapse was successfully treated with doxorubicin, dacarbazine, and immunomodulatory agents, as well as whole-lung radiation and stereotactic body radiotherapy (SBRT) to the trunk, and Dog 2 was determined to be in remission based on whole-body CT imaging. The success of the various treatments was reflected in the nearly normal nucleosome levels measured in January 2020. In February 2020, Dog 2 was diagnosed with a complete response based on imaging results; interestingly, the measured nucleosome levels began to increase in February and disease progression was detected by imaging at the next scan in April 2020. When disease progression was noted in April, the nucleosome levels remained high. Had the nucleosome levels been known at the time of treatment, more intensive monitoring could have been established for this patient at the February time point. These results indicate that nucleosome levels can be used to guide treatment regimens and monitor subjects during remission to detect disease relapse. CRP analysis was not useful for clinical purposes.

[0140] Dog 3 (Figure 9A) was initially diagnosed with lymphoma in early 2018 and treatment was successful, resulting in remission. This was reflected in the measured nucleosome levels. For this dog's disease progression (PD), treatment with vincristine was performed on day 1 as part of the CHOP chemotherapy regimen. Treatment with vincristine improved the clinical status to stable (SD), which was reflected in the decrease in nucleosome levels as shown by the results in Figure 9A. However, CHOP treatment was discontinued due to both toxicity reasons and the lack of a strong clinical response being observed. Treatment with romustatin + L-asparaginase (L-spar) was initiated on day 22, and further romustatin treatment was performed on days 34, 79, and 113 thereafter. Clinical findings improved to partial remission as determined by tumor measurements on days 34 and 79, and then to complete response (CR) for the treatments on days 113 and 145. By this point, Dog 3 was in remission and no further chemotherapy was performed. Nucleosome levels increased between days 22 and 34, and then steadily declined from day 34 onwards, predicting the success of romustatin treatment, which was clinically observed as complete response on days 113 and 145. Furthermore, nucleosome levels decreased to the range observed for healthy dogs by clinical findings on day 145. CRP levels were always within the normal range and were not useful for clinical purposes.

[0141] Dog 4 (Figure 9B) was diagnosed with abdominal hypercalcemic lymphoma at stage Vb. This dog underwent CHOP chemotherapy regimen with vincristine and doxorubicin from day 1 to day 93, and the response to the treatment was monitored based on the circulating calcium level. The veterinarian clinically observed a certain degree of response to vincristine but no response to doxorubicin. Subsequently, the treatment was switched from CHOP to romustine + L-spar on day 108 and continued until day 164, but still no response was observed. Since a response to vincristine was observed, treatment with COP (CHOP without using doxorubicin) using vincristine was initiated on day 164 (blood samples could not be collected on day 167). Dog 4 responded to the COP therapy and still continues to respond to the treatment at the time of writing. The measured nucleosome levels reflect the observed clinical findings (Figure 9B). At most time points after treatment with vincristine, reflecting the response to this treatment, the nucleosome levels decreased, and after each administration of doxorubicin, reflecting no response to this treatment, the nucleosome levels increased. Also, Dog 4 was found to have a partial response to cyclophosphamide clinically, which was also reflected in the decrease in nucleosome concentration. However, the nucleosome levels still exceeded the normal levels, indicating that continuous further treatment was necessary. The nucleosome measurement results clearly correlated with the clinical findings and could be used to monitor the remission of the disease and guide treatment selection. For example, the nucleosome levels predicted the lack of response to doxorubicin therapy, and if findings based on the nucleosome levels had been obtained, it might have been possible to discontinue it earlier. The CRP analysis showed no significant fluctuations and was not useful for clinical purposes. This application provides an invention in the following aspects. (Aspect 1) Use of extracellular free nucleosomes as biomarkers in plasma samples for the diagnosis or detection of vascular cancer or hematological cancer. (Aspect 2) The use according to Aspect 1, wherein the extracellular free nucleosomes are mononucleosomes or oligonucleosomes. (Aspect 3) The use according to Aspect 1 or 2, wherein the biomarker is the level of the extracellular free nucleosomes and / or the epigenetic characteristics of the extracellular free nucleosomes. (Aspect 4) The use according to Aspect 3, wherein the epigenetic characteristics of the extracellular free nucleosomes are histone isoforms, such as histone isoforms of core nucleosomes, particularly histone H3 isoforms. (Aspect 5) The use according to Aspect 4, wherein the histone isoform is H3.1. (Aspect 6) The use according to Aspect 3, wherein the epigenetic characteristics of the extracellular free nucleosomes are histone post-translational modifications (PTMs), such as histone PTMs of core nucleosomes, particularly histone H3 PTMs. (Aspect 7) The use according to Aspect 6, wherein the histone PTM is selected from citrullination (e.g., H3 citrulline) or methylation (e.g., H3K27me3). (Aspect 8) The use according to any one of Aspects 1 to 7, wherein the hematological cancer is selected from leukemia or lymphoma. (Aspect 9) The use according to Aspect 8, wherein the leukemia is selected from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). (Aspect 10) The use according to Aspect 8, wherein the lymphoma is non-Hodgkin lymphoma (NHL). (Aspect 11) The use according to any one of Aspects 1 to 7, wherein the vascular cancer is selected from angiosarcoma or hemangiosarcoma. (Aspect 12) A method for diagnosing or detecting vascular cancer or hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes; and (ii) using the level of the detected extracellular free nucleosomes to diagnose a subject having the vascular cancer or hematological cancer. (Aspect 13) A method for determining the prognosis of a subject having vascular cancer or hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes; and (ii) using the level of the detected extracellular free nucleosomes as an indicator of the prognosis of the vascular cancer or hematological cancer, the method comprising the above steps. (Aspect 14) A method for monitoring the efficacy of treatment in a subject having, suspected of having, or predisposed to a vascular cancer or hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes; and (ii) comparing the level of the detected extracellular free nucleosomes with a plasma sample previously collected from the subject to determine the efficacy of the treatment, the method comprising the above steps. (Aspect 15) The method according to any one of aspects 12 to 14, wherein the detection or measurement includes an immunoassay, immunochemistry, mass spectrometry, chromatography, chromatin immunoprecipitation, or biosensor method. (Aspect 16) The method according to any one of aspects 12 to 15, wherein the subject is a human or animal subject. (Aspect 17) The method according to any one of aspects 12 to 16, wherein the subject is suspected of having a recurrence of a vascular cancer or hematological cancer. (Aspect 18) The method according to any one of aspects 12 to 17, wherein the subject has a high level of leukocytosis / high white blood cell count. (Aspect 19) The method according to any one of aspects 12 to 18, further comprising comparing the level of the extracellular free nucleosomes in the plasma sample with one or more controls. (Aspect 20) The method according to aspect 19, wherein the control is a healthy subject. (Aspect 21) The method according to aspect 19, wherein the control is a subject having a cancer other than a vascular cancer or hematological cancer. (Aspect 22) The method according to aspect 19, wherein the control is a subject having a high level of leukocytosis. (Aspect 23) The method according to any one of aspects 12 to 22, wherein the level of the extracellular free nucleosomes is elevated compared to the control. (Aspect 24) The method according to any one of aspects 12 to 23, wherein the level of the extracellular free nucleosomes is detected or measured as one of a measurement panel. (Aspect 25) The method according to aspect 24, wherein the panel includes one or more interleukins. (Aspect 26) The method according to aspect 25, wherein the one or more interleukins are selected from the group consisting of IL-6, IL-10, and IL-1β.

Claims

1. In vitro use of the H3.1 histone isoform of extracellular free nucleosomes as a biomarker in a plasma sample for the diagnosis or detection of angiosarcoma or hematological cancer.

2. The use according to claim 1, wherein the extracellular free nucleosomes are mononucleosomes or oligonucleosomes.

3. The use according to claim 1 or 2, wherein the hematological cancer is selected from leukemia, lymphoma or myeloma.

4. The use according to claim 3, wherein the leukemia is selected from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

5. The use according to claim 3, wherein the lymphoma is non-Hodgkin lymphoma (NHL).

6. The use according to claim 1 or 2, wherein the angiosarcoma is selected from angiosarcoma or hemangiosarcoma.

7. A method for obtaining data for diagnosing or detecting angiosarcoma or hematological cancer, comprising: contacting a plasma sample obtained from a subject with a binding agent to detect or measure extracellular free nucleosomes containing histone isoform H3.

1.

8. A method for obtaining data for determining the prognosis of a subject having angiosarcoma or hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes containing histone isoform H3.1; and (ii) using the level of extracellular free nucleosomes containing the detected histone isoform H3.1 as an indicator of the prognosis of the angiosarcoma or hematological cancer.

9. A method for obtaining data for monitoring the effectiveness of treatment in a subject having, suspected of having, or prone to vascular cancer or hematological cancer, comprising: (i) contacting a plasma sample obtained from the subject with a binding agent to detect or measure extracellular free nucleosomes containing histone isoform H3.1; and (ii) comparing the level of extracellular free nucleosomes containing the detected histone isoform H3.1 with a plasma sample previously collected from the subject, the method.

10. The method according to any one of claims 7 to 9, wherein the detection or measurement comprises immunoassay, immunochemistry, mass spectrometry, chromatography, chromatin immunoprecipitation, or biosensor method.

11. The method according to any one of claims 7 to 9, wherein the step of detecting or measuring comprises a two-site immunoassay using a labeled anti-nucleosome detection binding agent in combination with an immobilized anti-histone H3.1 binding agent.

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

13. The method according to any one of claims 7 to 12, wherein the subject is suspected of having a recurrence of vascular cancer or hematological cancer.

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

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

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

17. The method according to claim 15, wherein the control is a subject having a cancer other than vascular cancer or hematological cancer.

18. The method according to claim 15, wherein the control is a subject having a high level of leukocytosis. **Claim 19** The method according to any one of claims 7 to 18, wherein the level of extracellular free nucleosomes containing the histone isoform H3.1 is increased as compared with a control. **Claim 20** The method according to any one of claims 7 to 19, wherein the level of extracellular free nucleosomes containing the histone isoform H3.1 is detected or measured as one of the measurement panels. **Claim 21** The method according to claim 20, wherein the panel comprises one or more interleukins. **Claim 22** The method according to claim 21, wherein the one or more interleukins are selected from the group consisting of IL-6, IL-10, and IL-1β.

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