Use of cell-free nucleosomes as biomarkers

Abstract

The present invention relates to cell-free nucleosomes as biomarkers in plasma samples of vascular or hematological cancers.

Description

Technical Field

[0001] The present invention relates to cell-free nucleosomes as biomarkers in plasma samples of vascular or hematological cancers. Background Art

[0002] Blood cancers are types of cancer that affect the blood, bone marrow, and lymph nodes. They are called leukemias, lymphomas, and myelomas, depending on the type of cells affected. Leukemias are cancers of the blood cells that usually start in the bone marrow and spread through the bloodstream. In leukemias, the bone marrow produces mutated cells and spreads them into the blood, where they grow and crowd out healthy blood cells. Lymphoma diseases affect cells in the lymphatic system. In lymphomas, immune cells called lymphocytes grow out of control and accumulate in the lymph nodes, spleen, other lymphoid tissues, or nearby organs. Myeloma, also known as multiple myeloma, develops in the bone marrow and affects plasma cells, which 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 that the cancer progresses rapidly and aggressively, often requiring immediate treatment. ALL involves the development of large numbers of immature lymphocytes that are unable to resist infection. This results in patients having less room in their circulation for healthy white blood cells, red blood cells, and platelets. As a result, patients typically suffer from a weakened immune system and symptoms of anemia, such as fatigue, difficulty breathing, and an increased risk of heavy bleeding. Children under the age of 5 are at the highest risk of developing ALL, and it is the most common type of leukemia affecting children. The risk slowly decreases until the mid-20s, and then begins to slowly rise again after the age of 50. Overall, approximately 4 out of every 10 cases of ALL occur in adults.

[0004] AML affects myeloblasts, causing abnormal monocytes and granulocytes to accumulate in the bone marrow. AML can also affect myeloid stem cells, producing abnormal red blood cells or platelets. As with ALL, this results in low 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, and the average age of diagnosis is 68.

[0005] HL and NHL are the two main types of lymphoma. HL has a distinctive appearance under a microscope and contains cells called Reed-Sternberg cells (a type of B lymphocyte that has become cancerous), while NHL looks different under a microscope and does not contain Reed-Sternberg cells. Most lymphomas are NHL, and only about one in five are HL. NHL is a cancer that affects lymphocytes and usually begins in the lymph nodes or lymphatic tissue. It is one of the more common cancers among children, adolescents, and young adults.

[0006] Current methods for diagnosing leukemia and myeloma involve performing a complete blood count (CBC) 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 unique to patients with hematologic malignancies; it can also be the result of an ongoing response to an infection or other inflammatory process. For lymphoma, enlarged lymph nodes can be detected using X-rays, CT scans, or PET scans, however, these are also nonspecific.

[0007] To confirm a blood cancer diagnosis, a bone marrow or lymph node biopsy is required. Therefore, overdiagnosing blood cancers early in the diagnostic process can lead to unnecessary biopsies, which are invasive, potentially dangerous, and relatively expensive for healthcare providers. Cytogenetic analysis and / or immunophenotyping can also be used to confirm a blood cancer diagnosis; however, these methods are expensive to perform and are therefore typically reserved for later in the diagnostic process.

[0008] Human angiosarcoma is a rare type of blood vessel cancer that affects the endothelial cells lining the blood vessels. Canine angiosarcoma is also a type of cancer involving the proliferation of the vascular endothelium, or blood vessel walls, and is a common canine cancer that is also difficult to diagnose. Canine cancer is more common than human cancer, often due to the genetic effects of inbreeding and because dogs have shorter lifespans than humans and typically develop cancer at age 8 or older. Detecting and diagnosing cancer in animal subjects presents additional difficulties than diagnosing cancer in humans. Cancer testing in non-human animals often involves scans, such as through magnetic resonance imaging, or MRI, but the animals will not remain still for the time required for the scan and therefore must be anesthetized. Pet health insurance is uncommon and may not cover cancer diagnosis or treatment, making these tests financially out of reach for many pet owners. Additionally, human blood tests for cancer marker proteins generally do not detect animal proteins, so there are fewer blood tests available in veterinary oncology.

[0009] Holdenrieder et al. (2001) Int J Cancer 95: 114–120 previously described the detection of nucleosome levels in serum samples from patients with benign and malignant diseases. However, the results presented by serum samples did not suggest any difference between the levels of hematological cancers (such as lymphomas) compared to other cancer types tested. The epigenetic composition of circulating cell-free nucleosomes has also been studied as a blood-based biomarker in cancer, with respect to its histone modifications, histone variants, DNA modifications, and adduct content, see WO 2005 / 019826, WO 2013 / 030577, WO 2013 / 030579, and WO 2013 / 084002.

[0010] There remains a need in the art for simple, cost-effective diagnostic methods for vascular or hematological cancers, particularly those that can distinguish patients with other types of the disease or non-vascular or non-hematological cancers who may present with similar symptoms. Summary of the Invention

[0011] According to a first aspect, there is provided use of cell-free nucleosomes as biomarkers in a plasma sample for diagnosing or detecting vascular cancer or hematological cancer.

[0012] According to another aspect, there is provided the use of cell-free nucleosomes as a biomarker in a plasma sample for diagnosing or detecting blood cancer.

[0013] According to another aspect, there is provided use of cell-free nucleosomes as a biomarker in a plasma sample for diagnosing or detecting vascular cancer.

[0014] According to another aspect, there is provided a method for diagnosing or detecting vascular cancer or blood cancer, comprising the steps of:

[0015] (i) contacting a plasma sample obtained from a subject with a binding agent to detect or measure cell-free nucleosomes; and

[0016] (ii) using the detected level of cell-free nucleosomes to diagnose a subject with vascular or hematological cancer.

[0017] According to another aspect, there is provided a method for determining the prognosis of a subject having a vascular or hematological cancer, comprising the steps of:

[0018] (i) contacting a plasma sample obtained from a subject with a binding agent to detect or measure cell-free nucleosomes; and

[0019] (ii) using the detected level of cell-free nucleosomes as a prognostic indicator for vascular or hematological cancers.

[0020] According to another aspect, there is provided a method for monitoring the efficacy of a therapy in a subject having, suspected of having, or susceptible to a vascular or hematological cancer, comprising the steps of:

[0021] (i) contacting a plasma sample obtained from a subject with a binding agent to detect or measure cell-free nucleosomes; and

[0022] (ii) comparing the levels of cell-free nucleosomes detected with earlier plasma samples taken from the subject to determine the efficacy of the therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1: Concentration of cell-free nucleosomes containing H3.1 in plasma samples obtained from healthy human subjects and human subjects with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and non-Hodgkin's lymphoma (NHLym).

[0025] Figure 2 : Concentration of cell-free nucleosomes containing H3.1 in plasma samples obtained from human subjects with different types of cancer.

[0026] Figure 3 Figure : ELISA optical density (OD) results of cell-free nucleosomes containing H3 citrullination (H3cit) in plasma samples from healthy human subjects compared with (A) results from all types of hematological cancer tested and (B) results from human subjects with ALL, AML, and NHL.

[0027] Figure 4 Figure 3: ELISA OD results for cell-free nucleosomes containing H3K27Me3 in plasma samples from healthy human subjects compared with (A) results from all types of hematological cancer tested and (B) results from human subjects with ALL, AML, and NHL.

[0028] Figure 5 Figure 3: ELISA relative light unit (RLU) results for cell-free nucleosomes containing H4 panacetylation (H4panAc) in plasma samples from healthy human subjects compared with (A) results from all types of hematological cancer tested and (B) results from human subjects with ALL, AML, and NHL.

[0029] Figure 6 : (A) ELISA results of cell-free nucleosomes containing histone isoform H3.1 (ng / ml) in plasma samples from healthy canine subjects compared to canine subjects diagnosed with lymphoma, and (B) shows the ROC curve for canine lymphoma detection using H3.1 results.

[0030] Figure 7 : (A) ELISA results for cell-free nucleosomes containing the histone isoform H3.1 (ng / ml) in plasma samples from healthy canine subjects compared to canine subjects diagnosed with angiosarcoma, and (B) shows the ROC curve for canine angiosarcoma detection using the H3.1 results.

[0031] Figure 8 : ELISA results for cell-free nucleosomes containing histone isoform H3.1 (ng / ml) and CRP (μg / ml) in plasma samples obtained serially from two canines treated for angiosarcoma.

[0032] Figure 9 : ELISA results for cell-free nucleosomes containing histone isoform H3.1 (ng / ml) and CRP (μg / ml) in plasma samples obtained serially from two canines treated for lymphoma. DETAILED DESCRIPTION

[0033] According to a first aspect, there is provided a use of cell-free nucleosomes as a biomarker in a plasma sample for diagnosing or detecting vascular cancer or blood cancer, in particular, the cancer is a blood cancer.

[0034] The nucleosome is the basic unit of chromatin structure and is composed of a protein complex of eight highly conserved core histones (one pair each of histones H2A, H2B, H3, and H4). Around this complex is wrapped approximately 146 base pairs of DNA. Another histone, H1 or H5, acts as a linker and participates in chromatin compaction. DNA is wound around continuous nucleosomes, a structure often described as being similar to "beads on a rope," and this forms the basic structure of open or euchromatin. In compacted or heterochromatin, this rope is coiled and supercoiled into a closed and complex structure (Herranz and Esteller (2007) Methods Mol. Biol. 361: 25-62).

[0035] When detected in a bodily fluid sample, reference to "nucleosomes" may refer to "cell-free nucleosomes". It should be understood that the term cell-free nucleosomes throughout this document is intended to include any cell-free chromatin fragment that includes one or more nucleosomes. As referred to herein, "epigenetic signatures," "epigenetic signal signatures," or "epigenetic signal structures" of cell-free nucleosomes may include, but are not limited to, one or more histone post-translational modifications, histone isoforms, modified nucleotides, and / or proteins bound to nucleosomes in nucleosome-protein adducts.

[0036] It should be understood that cell-free nucleosomes can be detected by binding to their components. As used herein, the term "components thereof" refers to a portion of a nucleosome, i.e., the entire nucleosome does not need to be detected. The components of cell-free nucleosomes can be selected from the group consisting of: histones (i.e., histone H1, H2A, H2B, H3, or H4), histone post-translational modifications, histone variants or isoforms, proteins bound to nucleosomes (i.e., nucleosome-protein adducts), DNA fragments associated with nucleosomes, and / or modified nucleotides associated with nucleosomes. For example, its components can be histone (isoform) H3.1 or histone H1 or DNA.

[0037] The methods and uses of the present invention can measure the level of (cell-free) nucleosomes themselves. Mention of "nucleosomes themselves" refers to the total nucleosome level or concentration present in a sample, regardless of whether the nucleosomes may include or not include any epigenetic features. The detection of total nucleosome levels generally involves detecting histones common to all nucleosomes, such as histone H4. Therefore, nucleosomes themselves can be measured by detecting core histones (such as histone H4). As described herein, histones form structural units called nucleosomes, which are used to package DNA in eukaryotic cells. As previously reported in WO 2016 / 067029 (incorporated herein by reference), specific histone variants, such as histone H3.1, H3.2 or H3t, can be used to separate cell-free nucleosomes derived from tumor cells. Therefore, the total level of cell-free nucleosomes of tumor origin can be detected.

[0038] Normal cell turnover in adults involves the production of approximately 10 11 Cells die in large numbers and a similar number die primarily through apoptosis. During apoptosis, chromatin breaks down into mononucleosomes and oligonucleosomes, which are released from the cell. It is reported that under normal circumstances, circulating nucleosomes are found at low levels in healthy subjects. Elevated levels have been found in subjects with a variety of 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).

[0039] Current nucleosome ELISA methods are primarily used in cell culture, typically 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 have also been used to measure circulating cell-free nucleosomes in serum and plasma (Holdenrieder et al. (2001)). In studies of many different cancers, the levels of cell-free serum and plasma nucleosomes released into the circulation by dying cells have been measured by ELISA to evaluate their use as potential biomarkers. Average circulating nucleosome levels have been reported to be high in most, but not all, cancers studied. However, serum nucleosome concentrations in patients with malignancies have been reported to vary widely, and some patients with advanced neoplastic disease have been found to have low circulating nucleosome levels within the range measured in healthy subjects (Holdenrieder et al. (2001)).

[0040] The cell-free nucleosomes can be mononucleosomes or oligonucleosomes, or a mixture thereof.

[0041] Mononucleosomes and oligonucleosomes can be detected by enzyme-linked immunosorbent assay (ELISA), and several methods have been reported (e.g., Salgame et al. (1997); Holdenrieder et al. (2001); van Nieuwenhuijze et al. (2003)). These assays typically use anti-histone antibodies (e.g., anti-H2B, anti-H3, or anti-H1, anti-H2A, anti-H2B, anti-H3, and anti-H4) as capture antibodies and anti-DNA or anti-H2A-H2B-DNA complex antibodies as detection antibodies.

[0042] Circulating nucleosomes are not a group of homogeneous protein-nucleic acid complexes. On the contrary, they are a group of heterogeneous chromatin fragments derived from the digestion of chromatin during cell death and include a wide variety of epigenetic structures, including specific histone isoforms (or variants), post-translational histone modifications, nucleotides or modified nucleotides and protein adducts. It will be clear to those skilled in the art that the rise in nucleosome levels will be associated with the rise in some circulating nucleosome subpopulations containing specific epigenetic signals, and the nucleosome subpopulations include containing specific histone isoforms (or variants), including specific post-translational histone modifications, including specific nucleotides or modified nucleotides and nucleosomes comprising specific protein adducts. The determination of these types of chromatin fragments is known in the art (e.g., see WO 2005 / 019826, WO2013 / 030579, WO2013 / 030578, WO 2013 / 084002, which are incorporated herein by reference).

[0043] The biomarkers used in the uses and methods of the present invention can be the level of cell-free nucleosomes themselves and / or the epigenetic characteristics of cell-free nucleosomes. It should be understood that the terms "epigenetic signal structure" and "epigenetic signature" are used interchangeably herein. They refer to specific characteristics of nucleosomes that can be detected. In one embodiment, the epigenetic signature of nucleosomes is selected from the group consisting of: post-translational histone modifications, histone variants, specific nucleotides and protein adducts.

[0044] In one embodiment, the epigenetic signature of a nucleosome comprises one or more histone variants or isoforms. The epigenetic signature of a cell-free nucleosome can be a histone isoform, such as a histone isoform of a core nucleosome, in particular a histone H3 isoform. The terms "histone variant" and "histone isoform" are used interchangeably herein. The structure of a nucleosome can also vary by comprising alternative histone isoforms or variants that are different genes or splice products and have different amino acid sequences. Many histone isoforms are known in the art. Histone variants can be divided into multiple families, which are further subdivided into individual types. The nucleotide sequences of a large number of histone variants are known and are, for example, available in the National Human Genome Research Institute NHGRI Histone Database ( -Ramírez et al. The Histone Database: an integrated resource for histones and histone fold-containing proteins. Database 2011 volume and http: / / genome.nhgri.nih.gov / histones / complete.shtml), GenBank (NIH gene sequence) database, EMBL nucleotide sequence database and the DNA Data Bank of Japan (DDBJ) are publicly available. 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.

[0045] In one embodiment, the histone isoform is H3.1. As shown in the examples presented herein, H3.1 can effectively distinguish subjects with vascular or hematological cancers from healthy subjects. H3.1 is particularly effective in identifying patients with lymphoma, as it distinguishes 84% ​​of NHL patients from healthy subjects with 90% specificity (see Table 1).

[0046] The structure of nucleosomes can change due to post-translational modification (PTM) of histones. Histone PTMs usually occur in the tail of core histones and common modifications include acetylation, methylation or ubiquitination of lysine residues and methylation of arginine residues and phosphorylation of serine residues. Many histone modifications are known in the art and the number increases with the identification of new modifications (Zhao and Garcia, 2015Cold Spring Harb Perspect Biol, 7: a025064). Therefore, in one embodiment, the epigenetic signature of cell-free nucleosomes can be histone post-translational modification (PTM). Histone PTM can be histone PTM of core nucleosomes, such as H3, H2A, H2B or H4, especially H3, H2A or H2B. Specifically, histone PTM is histone H3 PTM. The example of this type of PTM is described in WO2005 / 019826.

[0047] For example, post-translational modifications can include acetylation, methylation (which can be monomethylation, dimethylation or trimethylation), phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamylation and / or isomerization (see Ausio (2001) Biochem Cell Bio 79:693). In one embodiment, the histone PTM is selected from methylation or citrullination. In another embodiment, the histone PTM is H3K27me3 or H3 citrulline (H3cit). In yet another embodiment, the histone PTM is H3cit. As shown in the examples presented herein, H3cit is the most effective histone PTM for distinguishing subjects with vascular cancer or hematological cancer from healthy subjects.

[0048] It is also possible to detect a group or class of related histone post-translational modifications (rather than a single modification). A typical example, but not limited to, would involve a two-site immunoassay using an antibody or other selective binding agent directed to bind to nucleosomes and an antibody or other selective binding agent directed to bind to the histone modification group in question. Examples of such antibodies directed to bind to histone modification groups would include, for illustrative purposes but not limited to, anti-panacetylation antibodies (e.g., panacetyl H4 antibody [H4panAc]), anti-citrullination antibodies, or anti-ubiquitin antibodies.

[0049] In one embodiment, the epigenetic signature of the nucleosome includes one or more DNA modifications. In addition to the epigenetic signaling mediated by nucleosomal histone isoforms and PTMs, the nucleotides and modified nucleotide compositions of the nucleosome are also different. Global DNA hypomethylation is a hallmark 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.

[0050] In one embodiment, the epigenetic signature of nucleosomes includes one or more protein-nucleosome adducts or complexes. Another type of circulating nucleosome subpopulation is a nucleosome protein adduct. For many years, it has been known that chromatin contains a large number of non-histone proteins that are bound to its constituent DNA and / or histones. These chromatin-associated protein types are diverse and have multiple functions, including transcription factors, transcription enhancer factors, transcription repressors, histone modifying enzymes, DNA damage repair proteins, etc. These chromatin fragments are described in the art, including nucleosomes and other non-histone chromatin proteins or DNA and other non-histone chromatin proteins.

[0051] In one embodiment, the protein that binds to nucleosomes (and therefore can be used as a biomarker) is selected from the group consisting of a transcription factor, a high-mobility group protein, or a chromatin-modifying enzyme. References to "transcription factors" refer to proteins that bind to DNA and regulate gene expression by promoting (i.e., activators) or repressing (i.e., repressors) transcription. Transcription factors contain one or more DNA binding domains (DBDs) that attach to specific DNA sequences adjacent to the genes they regulate.

[0052] All circulating nucleosomes and nucleosome fractions, types, or subsets described herein find use in the present invention.

[0053] It should be understood that more than one epigenetic signature of cell-free nucleosomes can be detected in the methods and uses of the present invention. A variety of biomarkers can be used as combination biomarkers. Therefore, in one embodiment, the purposes include more than one epigenetic signature of cell-free nucleosomes as combination biomarkers. Epigenetic signatures can be of the same type (e.g., PTM, histone isoforms, nucleotides, or protein adducts) or different types (e.g., a combination of PTM and histone isoforms). For example, post-translational histone modifications and histone variants (i.e., more than one type of epigenetic signatures) can be detected. Alternatively or in addition, more than one type of post-translational histone modifications are detected, or more than one type of histone isoforms are detected. On the one hand, the purposes include post-translational histone modifications and histone isoforms as combination biomarkers in plasma samples for diagnosing or detecting vascular cancer or hematologic cancer. In one embodiment, the combination biomarker is H3.1 and H3cit. In an alternative embodiment, the combination biomarker is H3.1 and H3K27Me3.

[0054] The term "biomarker" means a distinctive biological or biologically derived indicator of a process, event, or condition. Biomarkers are useful in diagnostic methods, such as clinical screening and prognostic assessment, as well as for monitoring treatment outcomes, identifying patients most likely to respond to a specific treatment, and in drug screening and development. Biomarkers and their use are valuable for identifying new drug therapies and discovering new targets for drug therapy.

[0055] The methods and uses described herein can be tested in body fluid samples, especially blood, serum or plasma samples. Preferably, a plasma sample is used. The plasma sample can be collected in a collection tube containing one or more anticoagulants such as ethylenediaminetetraacetic acid (EDTA), heparin or sodium citrate, especially EDTA.

[0056] blood cancer

[0057] Hematological cancers are cancers of the blood, and therefore may also be called “blood cancers.” There are three main types of blood cancer: leukemias, which are caused by the rapid production of abnormal white blood cells; lymphomas, which are caused by abnormal lymphoma cells; and myeloma, which is a cancer of the plasma cells.

[0058] In one embodiment, the blood cancer is selected from lymphoma, leukemia, myeloma, chronic myeloproliferative disease, monoclonal gammopathy of undetermined significance, myelodysplastic syndrome and amyloidosis. In another embodiment, the blood cancer is selected from leukemia or lymphoma.

[0059] Leukemia affects white blood cells and can be classified according to the type of white blood cell affected (myeloid cells or lymphocytes) and the way the disease progresses (acute or chronic). Several types of leukemia have been identified, including but not limited to: acute lymphoblastic leukemia (ALL; also known as acute lymphoblastic leukemia), acute myeloid leukemia (AML), acute megakaryocytic 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 promyelocytic leukemia.

[0060] In one embodiment, leukemia is an acute leukemia, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute megakaryocytic leukemia (AMKL) or acute promyelocytic leukemia (APL). In another embodiment, leukemia is selected from acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). Alternatively, in one embodiment, leukemia is a chronic leukemia, such as chronic eosinophilic leukemia (CEL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) or chronic neutrophilic leukemia.

[0061] Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) are both lymphomas. Most NHL patients are first diagnosed over the age of 55, while the median age of diagnosis for Hodgkin lymphoma is 39. In one embodiment, the lymphoma is non-Hodgkin lymphoma (NHL). NHL can appear in lymph nodes anywhere in the body, while HL usually starts in the upper body, such as the neck, chest, or armpits.

[0062] Hodgkin lymphoma is often diagnosed in its early stages and is therefore considered one of the most treatable cancers. Non-Hodgkin lymphoma is often not diagnosed until it reaches a more advanced stage, so the methods of the present invention are particularly useful in the diagnosis of NHL, where it is desirable to detect patients at an early stage of the disease to improve treatment outcomes.

[0063] Other blood cancers

[0064] Angiosarcoma and hemangiosarcoma are soft tissue cancers that involve the proliferation of cells lining the vasculature, hence the term "vascular cancers." Like hematopoietic cancers, these cancers are closely associated with the vasculature, and the affected cells are in direct contact with the circulating blood fluid. We have shown that these blood cancers are associated with extremely high levels of circulating nucleosomes, similar to levels observed in lymphomas.

[0065] Detection and diagnostic methods

[0066] The present invention provides methods that can be used to detect or diagnose patients with vascular or hematological cancers. Therefore, according to another aspect, a method for diagnosing or detecting vascular or hematological cancers is provided, comprising the steps of:

[0067] (i) contacting a plasma sample obtained from a subject with a binding agent to detect or measure cell-free nucleosomes; and

[0068] (ii) using the detected level of cell-free nucleosomes to diagnose a subject with vascular or hematological cancer.

[0069] According to another aspect, there is provided a method for diagnosing or detecting blood cancer, comprising the steps of:

[0070] (i) contacting a plasma sample obtained from a subject with a binding agent to detect or measure cell-free nucleosomes; and

[0071] (ii) using the detected level of cell-free nucleosomes to diagnose a subject with a blood cancer.

[0072] Alternatively, according to another aspect, there is provided a method for diagnosing or detecting vascular cancer, comprising the steps of:

[0073] (i) contacting a plasma sample obtained from a subject with a binding agent to detect or measure cell-free nucleosomes; and

[0074] (ii) using the detected level of cell-free nucleosomes to diagnose the subject as having vascular cancer.

[0075] According to another aspect, there is provided a method for determining the prognosis of a subject having a vascular or hematological cancer, comprising the steps of:

[0076] (i) contacting a plasma sample obtained from a subject with a binding agent to detect or measure cell-free nucleosomes; and

[0077] (ii) using the detected level of cell-free nucleosomes as a prognostic indicator for said vascular or hematological cancer.

[0078] If the subject is determined not to have vascular or hematological cancer, the present invention can still be used for the purpose of monitoring disease progression. For example, if the use includes a sample from a subject determined not to have vascular or hematological cancer, the biomarker level measurement can be repeated at another time point to establish whether the biomarker level has changed.

[0079] According to another aspect, there is provided a method for monitoring the efficacy of a therapy in a subject having, suspected of having, or susceptible to a vascular or hematological cancer, comprising the steps of:

[0080] (i) contacting a plasma sample obtained from a subject with a binding agent to detect or measure cell-free nucleosomes; and

[0081] (ii) comparing the detected levels of cell-free nucleosomes to an earlier plasma sample taken from the subject to determine the efficacy of the therapy.

[0082] Detection and / or quantification can be performed directly on a purified or enriched nucleosome sample, or indirectly on an extract or dilution thereof. Quantifying the amount of a biomarker present in a sample can include determining the concentration of the biomarker present in the sample. The uses and methods of detection, monitoring and diagnosis according to the present invention described herein can be used to confirm the presence of a disease, monitor the development of a disease by assessing onset and progression, or assess improvement or regression of a disease. The uses and methods of detection, monitoring and diagnosis can also be used in methods for evaluating clinical screening, prognosis, therapy selection, and evaluation of therapeutic benefit, i.e., for drug screening and drug development.

[0083] Detection or measurement can include immunoassay, immunochemistry, mass spectrometry, chromatography, chromatin immunoprecipitation or biosensor method. Specifically, detection and / or measurement can include 2-site immunoassay of nucleosome portion. Such method is preferably used for adopting two anti-nucleosome binding agents or anti-nucleosome binding agents and anti-histone modification or anti-histone variant or anti-DNA modification or anti-additive protein detection binding agent combination to measure the epigenetic signature of nucleosome or nucleosome in situ. Similarly, detection and / or measurement can include 2-site immunoassay using labeled anti-nucleosome detection binding agent and immobilized anti-histone modification or anti-histone variant or anti-DNA modification or anti-additive protein binding agent combination.

[0084] The level of a biomarker can be detected or measured using one or more reagents such as suitable binding agents. For example, the one or more binding agents can comprise a ligand or binding agent specific for a desired biomarker, such as a nucleosome or a component thereof, an epigenetic signature of a nucleosome, or a structure / shape mimetic of a nucleosome or a component thereof, optionally in combination with one or more interleukins.

[0085] It will be clear to those skilled in the art that the terms "antibody," "binding agent," or "ligand," as used herein, are not limiting, but are intended to include any binding agent capable of binding to a particular molecule or entity, and any suitable binding agent may be used in the methods of the present invention. It will also be clear that the term "nucleosome" is intended to include mononucleosomes and oligonucleosomes, as well as any protein-DNA chromatin fragment that can be analyzed in a fluid medium.

[0086] Methods for detecting biomarkers are known in the art. The reagent may comprise one or more ligands or binding agents capable of specifically binding to the desired target, such as naturally occurring or chemically synthesized compounds. The ligand or binding agent may comprise a peptide, antibody or fragment thereof, or a synthetic ligand such as a plastic antibody, or an aptamer or oligonucleotide capable of specifically binding to the desired target. The antibody may be a monoclonal antibody or a fragment thereof. It will be understood that if an antibody fragment is used, it retains the ability to bind to the biomarker, thereby allowing detection of the biomarker (according to the present invention). The ligand / binding agent may be labeled with a detectable marker such as a luminescent, fluorescent, enzyme or radioactive marker; alternatively or in addition, the ligand according to the present invention may be labeled with an affinity tag such as biotin, avidin, streptavidin or a His (e.g., hexa-His) tag. Alternatively, label-free techniques, such as those of ForteBio Inc., may be used to determine ligand binding.

[0087] As used herein, the terms "detection" or "diagnosis" encompass the identification, confirmation, and / or characterization of a disease state. The methods of detection, monitoring, and diagnosis according to the present invention can be used to confirm the presence of a disease, monitor the development of a disease by assessing onset and progression, or assess improvement or regression of a disease. The methods of detection, monitoring, and diagnosis can also be used in methods for evaluating clinical screening, prognosis, therapy selection, and therapeutic benefit assessment, i.e., for drug screening and drug development.

[0088] In one embodiment, the methods described herein are repeated multiple times. This embodiment provides the advantage of allowing the test results to be monitored over a period of time. Such an arrangement would provide the benefit of monitoring or assessing the therapeutic efficacy of a disease state. Such monitoring methods of the present invention can be used to monitor onset, progression, stabilization, improvement, relapse, and / or remission.

[0089] In monitoring methods, samples may be collected two or more times. The method may further comprise comparing the level of the biomarker present in the sample with one or more controls and / or with one or more previous samples taken from the same test subject earlier, for example, before the start of therapy, and / or taken from the same test subject in an early stage of therapy. The method may comprise detecting changes in the nature or amount of the biomarker in the samples collected at different times.

[0090] A change in the level of a biomarker in a sample relative to the level in a previous sample taken earlier from the same test subject can indicate a beneficial effect of the therapy on the condition or suspected condition, such as stabilization or improvement. In addition, once treatment is completed, the methods of the present invention can be repeated periodically to monitor for recurrence of the disease.

[0091] Methods for monitoring therapeutic efficacy can be used to monitor the therapeutic effectiveness of existing and new therapies in human subjects and non-human animals (e.g., in animal models). These monitoring methods can be incorporated into the screening of new drug substances and combinations of substances.

[0092] In another embodiment, monitoring for more rapid changes due to fast-acting therapies can be performed at shorter intervals of hours or days.

[0093] Diagnostic or monitoring kits (or panels) for performing the methods of the invention are provided. Such kits will suitably comprise one or more ligands for detecting and / or quantifying biomarkers according to the invention, and / or biosensors, and / or arrays as described herein, optionally together with instructions for use of the kit.

[0094] Another aspect of the present invention is a kit for detecting the presence of a disease state, 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 comprise a ligand binding agent or ligand as described herein that is capable of specifically binding to a biomarker. Such biosensors can be used to detect and / or quantify the biomarkers of the present invention.

[0095] Suitably, biosensors for detecting one or more biomarkers combine biomolecular recognition with appropriate means to convert the detection or quantification of the presence of the biomarker in a sample into a signal. Biosensors can be used for "point-of-care" diagnostic testing, such as in hospital rooms, outpatient clinics, consulting rooms, homes, field sites, and workplaces. Biosensors for detecting one or more biomarkers of the present invention include acoustic sensors, plasmon resonance sensors, holographic sensors, biolayer interferometry (BLI) sensors, and microengineered sensors. Imprinted recognition elements, thin-film transistor technology, magnetoacoustic resonator devices, and other novel acoustoelectric systems can be used in biosensors to detect the one or more biomarkers.

[0096] Biomarkers used to detect the presence of disease are essential targets for discovering novel targets and drug molecules that hinder or prevent the progression of the disease. Because the levels of biomarkers indicate disease and drug response, biomarkers can be used to identify novel therapeutic compounds in in vitro and / or in vivo assays. The biomarkers described herein can be used in methods to screen for compounds that modulate the activity of the biomarkers.

[0097] Therefore, in another aspect of the present invention, there is provided the use of a binding agent or ligand as described above for identifying substances that can promote and / or inhibit the production of biomarkers, wherein the binding agent or ligand can be a peptide, antibody or fragment thereof, or aptamer or oligonucleotide directed against the biomarker according to the present invention; or there is provided the use of a biosensor, or array or kit according to the present invention for identifying substances that can promote and / or inhibit the production of biomarkers.

[0098] Immunoassays as described herein include any method using one or more antibodies or other specific binding agents directed to biomarkers as defined herein. Immunoassays include 2-site immunoassays or immunoassays using enzyme detection methods (e.g., ELISA), fluorescent labeling immunoassays, time-resolved fluorescent labeling immunoassays, chemiluminescent immunoassays, immunoturbidimetric assays, microparticle labeling immunoassays, and immunoradiometric assays, as well as single-site immunoassays, reagent-limited immunoassays, competitive immunoassays, including single antibody immunoassays with labeled antigens and labeled antibodies of various label types (including radioactivity, enzymes, fluorescence, time-resolved fluorescence, and microparticle labels). All of these immunoassays are well known in the art, for example, see Salgame et al. (1997) and van Nieuwenhuijze et al. (2003).

[0099] 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 or a purified product or extract or its dilution from a subject. Specifically, quantification can be carried out by measuring the concentration of the target in one or more samples. The biological samples that can be tested in the method of the present invention include those defined above. Samples can be prepared, for example, diluted or concentrated in appropriate circumstances, and stored in a usual manner. The present invention is particularly suitable for plasma samples that can be obtained from a subject.

[0100] The identification, detection and / or quantification of biomarkers can be performed by detecting biomarkers or fragments thereof, such as fragments with C-terminal truncations or N-terminal truncations. The length of the fragment is suitably greater than 4 amino acids, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. It is particularly noted that peptides with sequences identical to or related to histone tail sequences are particularly useful histone fragments.

[0101] 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 permeation (gel filtration), ion exchange, affinity, HPLC, UPLC and other LC or LC MS based techniques. Suitable LC MS techniques include (Applied Biosystems, CA, USA) or (Applied Biosystems, CA, USA) Liquid chromatography (eg, high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin layer chromatography, NMR (nuclear magnetic resonance) spectroscopy may also be used.

[0102] Methods involving detection and / or quantification of one or more biomarkers of the present invention can be performed on a benchtop instrument or can be incorporated into a disposable, diagnostic or monitoring platform that can be used in a non-laboratory setting (e.g., a physician's office or a subject's bedside). Suitable biosensors for performing the methods of the present invention include "credit" cards with optical or acoustic readers. Biosensors can be configured to allow the collected data to be electronically transmitted to a physician for interpretation, thereby forming the basis of digital medicine.

[0103] The identification of biomarkers of disease states allows for the integration of diagnostic procedures and treatment regimens. Biomarkers provide a means of indicating treatment response, failure to respond, adverse side effect profiles, degree of drug compliance, and achieving adequate serum drug levels. Biomarkers can be used to provide warnings of adverse drug reactions. Biomarkers can be used for the development of personalized therapies because response assessments can be used to fine-tune dosages, minimizing the number of prescribed medications, reducing delays in obtaining effective therapies, and avoiding adverse drug reactions. Therefore, by monitoring the biomarkers of the present invention, subject care can be precisely tailored to match the needs determined by the subject's condition and pharmacogenomics profile, so biomarkers can be used to titrate optimal dosages, predict positive treatment responses, and identify those subjects at high risk of severe side effects.

[0104] Biomarker-based tests provide the first line of assessment of 'new' subjects and offer an objective measure for accurate and rapid diagnosis that is not achievable using current measures.

[0105] Biomarker monitoring methods, biosensors, and kits are also crucial as subject monitoring tools, enabling physicians to determine whether a relapse is due to a worsening of the condition. If drug therapy is assessed to be inadequate, therapy can be resumed or increased; if appropriate, therapy can be changed. Because biomarkers are sensitive to the condition's state, they provide an indication of the impact of drug therapy.

[0106] References to "subject" or "patient" are used interchangeably herein. The subject can be a human or animal subject. In one embodiment, the subject is a 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.

[0107] In one embodiment, the subject is suspected of having a relapse of a vascular cancer or a blood cancer. Minimal residual disease (MRD) is the name given to a small amount of leukemia cells (cancer cells from the bone marrow) that remain in the body during 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 relapse of cancer and leukemia. Therefore, the method of the present invention can be used to monitor patients suspected of having relapsed, particularly patients in remission of cancer.

[0108] Subjects tested using the methods described herein may develop symptoms indicative of a blood cancer, such as anemia, leukocytosis, and / or lymphadenopathy. In one embodiment, the subject has a high level of leukocytosis. This may also be referred to as a "high white blood cell count." Blood cancers typically result in increased proliferation of abnormal white blood cells or red blood cells, leading to a high white blood cell count. However, leukocytosis is not sufficient to diagnose a patient with a blood cancer, particularly a leukemia, because it is often a sign of an inflammatory response, most commonly the result of an infection. Therefore, the methods of the present invention can provide a more specific method for detecting patients who may be suffering from a blood cancer.

[0109] Detection and / or quantification can be compared with the cut-off level. Cut-off value can be predetermined by analyzing the result from multiple patients and controls and determining the appropriate value for classifying the subject as suffering from the disease or not suffering from the disease. For example, for a disease in which the biomarker level is higher in the patient suffering from the disease, if the level detected is higher than the cut-off value, then indicate that the patient suffers from the disease. Alternatively, for a disease in which the biomarker level is higher in the patient suffering from the disease, if the level detected is lower than the cut-off value, then indicate that the patient suffers from the disease. The advantage of using a simple cut-off value includes that the clinician can easily understand the test and eliminate the need for any software or other auxiliary tools when explaining the test result. The cut-off level can be determined using the method in this area.

[0110] Detection and / or quantification can also be compared with a control. It will be clear to those skilled in the art that a control subject can be selected on a variety of bases and can include, for example, a subject known to be free of disease or a subject suffering from a different disease (e.g., for investigation of differential diagnosis). A "control" can include healthy subjects, non-diseased subjects, and / or subjects not suffering from vascular cancer or hematologic cancer. Comparison with a control is well known in the field of diagnosis.

[0111] Thus, in one embodiment, the method further comprises comparing the level of cell-free nucleosomes in the plasma sample with one or more controls. For example, the method can comprise comparing the level of cell-free nucleosomes present in a plasma sample obtained from a subject with the level of cell-free nucleosomes present in a plasma sample obtained from a normal subject. The control can be a healthy subject. Alternatively, the control can be a diseased subject, such as an infected subject.

[0112] Alternatively, the control is a subject with a cancer that is not a vascular or hematological cancer, i.e., the control subject has a cancer that affects a different organ in the body. The data provided herein show that the biomarkers of the present invention are significantly elevated in patients with hematological cancers compared to patients with other forms of cancer, and therefore they can be used to differentially diagnose patients with vascular or hematological cancers from patients with other forms of cancer. Thus, in one aspect, the diagnosis includes the differential diagnosis of vascular or hematological cancers from non-vascular or non-hematological cancers. "Non-vascular or non-hematological cancers" are cancers that are not hematological cancers and do not involve the proliferation of blood cells or vascular cells, such as bladder cancer, bone cancer, brain cancer, esophageal cancer, head and neck cancer, skin cancer (such as melanoma), thyroid cancer, tongue cancer, uterine cancer, and / or cervical cancer.

[0113] The control can be a subject with elevated levels 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 with elevated levels of leukocytosis that are not a result of vascular or hematologic cancers, such as controls with inflammation, infection, and / or taking medication.

[0114] In one embodiment, the level of cell-free nucleosomes is increased compared to a control.

[0115] It will be understood that it is not necessary to measure the control level for the purpose of comparison every time. For example, for a healthy / non-diseased control, once a "normal range" is established, it can be used as a benchmark for all subsequent tests. A normal range can be established by obtaining samples from a plurality of control subjects who do not suffer from vascular cancer or hematological cancer and testing the levels of biomarkers. The results (i.e., biomarker levels) of subjects suspected of having vascular cancer or hematological cancer can then be checked to see if they fall within or outside the corresponding normal range. Using a "normal range" is a standard practice for detecting disease.

[0116] In one embodiment, the method further comprises determining at least one clinical parameter of the patient. This parameter can be used to interpret the results. The clinical parameter can include any relevant clinical information, such as, but not limited to, gender, weight, body mass index (BMI), smoking status, and dietary habits. Thus, in one embodiment, the clinical parameter is selected from the group consisting of: age, gender, and body mass index (BMI).

[0117] In one embodiment, the methods of the invention are performed to identify subjects who are at high risk for developing vascular or hematological cancers and therefore require further testing (i.e., further cancer investigation). Further testing may involve one or more of the following: a biopsy (such as a bone marrow biopsy or a lymph node biopsy), cytogenetic testing, immunophenotyping, CT scan, X-ray (particularly a chest X-ray to identify enlarged lymph nodes), and / or a lumbar puncture.

[0118] The methods and biomarkers described herein can be used to identify patients who require a biopsy, particularly a bone marrow or lymph node biopsy. Thus, according to another aspect of the present invention, there is provided a method for identifying patients who require a biopsy, comprising obtaining a plasma sample from the patient, detecting the level of cell-free nucleosomes in the plasma sample, and using the results obtained from the panel test to identify the patient as requiring a biopsy.

[0119] According to another aspect of the present invention, there is provided a method of identifying a patient requiring 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.

[0120] Other biomarkers

[0121] The level of cell-free nucleosomes can be detected or measured as one of the panel measurements. The panel can include different epigenetic features of nucleosomes as described above (e.g., histone isoforms and PTMs). In one embodiment, the panel includes one or more cytokines, such as one or more interleukins.

[0122] Interleukin (IL) is a class of cytokines, usually secreted by leukocytes, acting as signaling molecules. They play a key role in stimulating immune responses and inflammation. They were first identified in the 1970s and named with numbers along with the discovery of more interleukin types. 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.

[0123] 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 1 beta (IL-1 beta).

[0124] The interleukin may be IL-6. Interleukin 6 (IL-6) is a cytokine with multiple biological functions. It is a potent inducer of fever and acute phase reactions. The sequence of human IL-6 is known in the art and is described in UniProt accession number P05231. In a specific embodiment, the interleukin may be IL-6 and the panel measurement may include measurement of histone isoform H3.1 and IL-6.

[0125] Alternatively or in addition, the interleukin may be IL-10. Interleukin 10 (IL-10) is an anti-inflammatory cytokine with multiple biological functions. The sequence of human IL-10 is known in the art and is described in UniProt accession number P22301. In a specific embodiment, the interleukin may be IL-10, and the panel measurement may include measurement of the histone post-translational modification H3cit and IL-10.

[0126] Alternatively or in addition, the interleukin may be IL-1β. Interleukin-1β (IL-1β) is a proinflammatory cytokine and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. In a specific embodiment, the interleukin may be IL-1β and the panel measurement may include measurement of histone isoform H3.1 and IL-1β. More specifically, the panel may include measurement of histone isoform H3.1 and IL-1β, wherein the vascular cancer or blood cancer is a lymphoma, such as NHL.

[0127] In one embodiment, the panel includes epigenetic features of cell-free nucleosomes and one interleukin. In another embodiment, the panel includes epigenetic features of cell-free nucleosomes and two interleukins. For example, cell-free nucleosome measurements can be combined with more than one interleukin measurement, such as IL-6 and IL-1β or IL-10 and IL-1β or IL-6 and IL-10. In another embodiment, the epigenetic features of cell-free nucleosomes are selected from histone isoforms, such as H3.1, and post-translationally modified histones, such as H3cit. In yet another embodiment, the panel measurements are H3.1, IL-6, and IL-1β. In an alternative embodiment, the panel measurements are H3cit, IL-10, and IL-1β.

[0128] The biomarker derivation model of the present invention can be used. Methods for deriving models or algorithms, such as those in Table 5 and Table 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 nonlinear data modeling for clinical data.

[0129] It will be clear to those skilled in the art that any combination of the biomarkers disclosed herein can be used in panels and algorithms for detecting vascular or hematological cancers, and that other markers can be added to panels that include these markers.

[0130] According to one aspect of the present invention, there is provided use of a panel test for detecting a patient suffering from a vascular or hematological cancer, wherein the panel test comprises reagents for detecting the measurement of nucleosomes or components thereof and one or more interleukins in a plasma sample obtained from the patient.

[0131] Treatment

[0132] According to another aspect, there is provided a method of treating vascular or hematological cancer in a subject, comprising the steps of:

[0133] (i) detecting or measuring the level of cell-free nucleosomes in a plasma sample obtained from a subject;

[0134] (ii) using the level measured in step (i) as an indication of the presence of said vascular or hematological cancer in the subject; and

[0135] (iii) if it is determined in step (ii) that the subject has the vascular cancer or blood cancer, performing surgery or administering a therapeutic agent.

[0136] According to another aspect, a method of treating a vascular or hematological cancer in a subject in need thereof is provided, comprising the step of performing surgery or administering a therapeutic agent to a subject identified as having a different level of cell-free nucleosomes in a plasma sample obtained from the subject when compared to the level of cell-free nucleosomes in a plasma sample obtained from a control subject.

[0137] 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.

[0138] The method may include:

[0139] (i) measuring the level of cell-free nucleosomes (optionally in combination with the level of one or more interleukins) in a plasma sample obtained from the subject;

[0140] (ii) identifying the subject as having a vascular or hematological cancer based on an increased level of cell-free nucleosomes compared to a control; and

[0141] (iii) administering the treatment to the subject.

[0142] According to another aspect of the present invention, a method of treating a vascular or hematological cancer is provided, comprising identifying a patient in need of treatment for the vascular or hematological cancer using a panel test, wherein the panel test comprises a reagent for detecting nucleosomes or components thereof and a measurement of one or more interleukins, and providing said treatment. Patients with the vascular or hematological cancer are expected to have elevated levels of cell-free nucleosomes compared to controls.

[0143] It will be understood that the embodiments described herein are applicable to all aspects of the invention, ie embodiments described with respect to the use are equally applicable to the claimed methods and so on.

[0144] The invention will now be illustrated with reference to the following non-limiting examples.

[0145] Example

[0146] Example 1

[0147] Plasma samples were obtained from a cohort of 116 human subjects. For each subject, the whole blood extracted was collected in an EDTA vacuum blood collection tube and the tube was gently inverted 10 times. Whole blood was centrifuged at 1500g for 15 minutes within 2 hours of blood draw. Plasma was transferred to a cryogenic tube and immediately frozen. In this cohort, 62 subjects were healthy, 25 subjects had non-Hodgkin's lymphoma (NHL), 22 subjects had acute myeloid leukemia (AML) and 7 subjects had acute lymphoblastic leukemia (ALL). Subjects with leukemia or lymphoma can be further classified as patients with cancer when diagnosed (31 subjects in total; 16 subjects had NHL when diagnosed, and 15 subjects had leukemia when diagnosed), or patients with cancer relapse (15 subjects in total; 6 subjects had NHL relapse, and 9 subjects had leukemia relapse).

[0148] The samples were analyzed for nucleosomes containing H3.1 by ELISA. Briefly, nucleosomes containing histone isoform H3.1 were measured 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 against histone H3.1. 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 three times with 200 μl of wash buffer and 100 μl of biotinylated anti-nucleosome antibody was added. The microtiter plate was covered again and incubated at room temperature with gentle shaking for 1.5 hours. The contents of the microtiter wells were discarded. The wells were washed three times with 200 μl of wash buffer and 100 μl of streptavidin-HRP solution was added. The microtiter plate was covered again and incubated at room temperature with gentle shaking for 0.5 hours. The contents of the microtiter wells were discarded. The wells were washed three times with 200 μl of washing solution and 100 μl of HRP (horseradish peroxidase) substrate solution was added. The microtiter plate was covered and incubated in the dark at room temperature with gentle shaking for 20 minutes. The absorbance (OD) of the wells was measured at 405 nm. The OD level was used directly or interpolated from a standard curve for plasma levels of nucleosomes containing histone H3.1.

[0149] OD results were plotted as receiver operating characteristic (ROC) curves. Results were grouped for all cancer patients versus healthy controls, all lymphoma patients versus healthy controls, and all leukemia (i.e., including ALL and AML) patients versus healthy controls. The area under the curve (AUC) results are shown in Tables 1 to 3.

[0150] Table 1: Roc curves of H3.1 OD in plasma—all patients

[0151]

[0152] Table 2: Roc curves for H3.1 OD in plasma—patients at diagnosis

[0153]

[0154]

[0155] Table 3: Roc curve of H3.1 OD in plasma - relapse patients

[0156]

[0157] As shown in Table 1, levels of cell-free nucleosomes containing H3.1 were able to distinguish over 75% of blood cancer patients from healthy donors with 90% specificity. This increased to over 80% with 90% specificity for patients diagnosed with cancer, as shown in Table 2. The results also indicated that this biomarker was particularly effective in identifying patients with lymphoma, as 84% ​​of NHL patients were distinguished from healthy subjects with 90% specificity (see Table 1).

[0158] The H3.1 concentrations in plasma samples from all AML, ALL, and NHL patients, derived from OD values ​​using standard curve interpolation, are shown in Table 1 compared to levels in healthy patients. Figure 1 H3.1 levels were significantly elevated in patients with blood cancer compared to healthy subjects.

[0159] The results showed that nucleosome levels, particularly those containing H3.1, could be used to detect blood cancers.

[0160] Example 2

[0161] The levels of H3.1-containing cell-free nucleosomes in the human plasma samples tested in Example 1 were further compared to the levels of H3.1 in plasma samples obtained from human patients with other forms of cancer, also collected as described in Example 1 .

[0162] The results are shown in Figure 2 Surprisingly, H3.1 levels were found to be useful in distinguishing subjects with blood cancers from patients with other forms of cancer. H3.1 levels were significantly elevated in patients with each type of blood cancer (ALL, AML, and NHL) compared to H3.1 levels in plasma samples from patients with bladder, bone, brain, esophageal, head and neck, skin, thyroid, tongue, uterine, cervical, and melanoma.

[0163] Example 3

[0164] The human plasma samples described in Example 1 were tested for levels of cell-free nucleosomes with post-translational modifications. The ELISA method was run in a similar manner to the ELISA method for H3.1 described in Example 1, except that the antibody directed against a post-translational histone modification selected from H3cit, H3K27Me3, and H4panAc was substituted for the antibody binding to histone H3.1. The results are summarized in Table 4. The results for all blood cancers and for each blood cancer type are also presented graphically (see Figure 3-5 ).

[0165] Table 4: Roc curves of PTM nucleosomes in plasma—all blood cancers

[0166]

[0167] All nucleosomal post-translational modifications tested were significantly elevated in patients with blood cancers compared to healthy controls. Even when blood cancers were differentiated by type (ALL, AML, NHL), levels of H3cit, H3K27Me3, and H4panAc cell-free nucleosomes were significantly elevated in plasma samples compared to healthy controls (see Figure 3 B. Figure 4 B and Figure 5 B) Post-translationally modified nucleosomes have the highest elevated levels in plasma samples obtained from ALL patients.

[0168] Example 4

[0169] The two best cell-free nucleosome markers (H3.1 and H3cit) were combined together or combined with the measurement of different interleukins. Cell-free nucleosome levels were measured as described previously, and plasma interleukin levels were measured using commercially available ELISA methods.

[0170] The assay results were modeled by logistic regression analysis to train a model or algorithm with the highest AUC for comparing human patients with blood cancers to normal human donors. The results are summarized in Table 5.

[0171] Table 5: Roc curves for combined biomarker panels in plasma – all blood cancers

[0172]

[0173] Cohort size: 54 cancer patients and 62 healthy subjects

[0174] As shown in Table 5, all models were able to distinguish more than 75% of blood cancer patients from healthy donors with 90% specificity. The results show that nucleosome levels can be combined with interleukin levels as an effective assay panel with associated algorithms for blood cancer detection.

[0175] Example 5

[0176] Only plasma samples from those human patients with NHL were modeled by logistic regression analysis as described in Example 4. The results are summarized in Table 6.

[0177] Table 6: Roc curves for combined biomarker panels in plasma – NHL cancer

[0178]

[0179] Cohort size: 25 NHL patients and 62 healthy subjects

[0180] As shown in Table 6, all models were able to distinguish 80% or more of NHL patients from healthy donors with a specificity of 90%. Specifically, the combination of H3.1 and IL-1β was able to distinguish 100% of NHL patients with a specificity of 80%. The results show that nucleosome and interleukin levels can be used as an effective assay panel with an associated algorithm for NHL detection.

[0181] Example 6

[0182] Plasma samples were collected from 73 dogs diagnosed with canine angiosarcoma, 127 dogs diagnosed with canine lymphoma, and 134 control dogs without cancer. The samples were analyzed for H3.1-containing nucleosomes by ELISA.

[0183] Healthy dogs were found to have a uniformly low concentration of circulating nucleosomes containing histone isoform H3.1, less than 67.4 ng / ml (mean 32 ng / ml, median 31 ng / ml).

[0184] Dogs diagnosed with lymphoma were found to have highly 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 graphically in Figure 6 A and Figure 6 In Figure 2, the AUC for detecting lymphoma was 87%. Using a cutoff of 67.4 ng / ml, the assay had a specificity of 100% and a sensitivity of 74%. Using a lower cutoff of 48.1 ng / ml, a sensitivity of 81% and a specificity of 90% were obtained.

[0185] Dogs diagnosed with angiosarcoma were found to have highly elevated levels of circulating nucleosomes containing the histone isoform H3.1 (mean 513 ng / ml, median 361 ng / ml). The dot plot and ROC curve of the results obtained for canine angiosarcoma are graphically presented in Figure 7 A and Figure 7In Figure B, the AUC for detecting angiosarcoma was 97.6%. Using a cutoff value of 67.4 ng / ml, the assay had a specificity of 100% and a sensitivity of 89%. Using a lower cutoff value of 48.1 ng / ml resulted in a sensitivity of 95% and a specificity of 90%.

[0186] As we have found in human disease, the levels of circulating nucleosomes observed in dogs diagnosed with lymphoma or angiosarcoma were also much higher than those observed for the other canine tumors studied.

[0187] Most assays for human proteins are not transferable to other animals. However, the structure of the nucleosome is highly conserved across species and even across phyla. We have demonstrated that this means that assays for the human H3.1 nucleosome are transferable to other species, including dogs and horses. Furthermore, the results we observed for vascular and hematological cancers in canine subjects were very similar to those observed in human subjects. We conclude that the method of the present invention is a highly effective method for detecting vascular and hematological cancers in humans and animals.

[0188] Example 7

[0189] Serial plasma samples were obtained from two dogs being treated for angiosarcoma and two dogs being treated for lymphoma. All samples from the four dogs were collected and later analyzed. Figure 8 Nucleosomes containing the histone isoform H3.1 were analyzed after the last day of treatment, so this information does not inform clinical treatment decisions, which are made based on clinical evidence.

[0190] C-reactive protein (CRP) is a well-known biomarker of inflammation and this was also measured in the samples as a control to determine whether nucleosome levels reflect only inflammatory responses or provide additional information about the subject's status, prognosis, and treatment response.

[0191] Dog No. 1 Figure 8 Dog A) was diagnosed with angiosarcoma and treated with a 4-drug chemotherapy regimen called CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) from day 1 to day 129. Treatment was successful and on day 160, Dog 1 was determined to be in remission based on clinical evidence. The success of CHOP treatment was reflected in the downward trend in nucleosome levels over the course of treatment, and disease remission was predicted by near-normal nucleosome levels by day 122. The results demonstrate that nucleosome levels can be used as a prognostic indicator and can be used to guide treatment regimens and monitor disease recurrence in subjects in remission. CRP analysis should not be used for clinical purposes.

[0192] Dog No. 2 Figure 8Dog B was initially diagnosed with angiosarcoma in 2017 and treated with doxorubicin and immunomodulators. He was monitored regularly with whole-body CT scans every 2-3 months. A relapse was noted 2 years later, reflected in elevated circulating nucleosome levels. The relapse was successfully treated with doxorubicin, dacarbazine, and immunomodulators, as well as whole-lung radiation and stereotactic body radiation therapy (SBRT), and Dog 2 was determined to be in remission based on whole-body CT imaging. The success of the various treatments was reflected in near-normal nucleosome levels measured in January 2020. In February 2020, Dog 2 was diagnosed with a complete response based on imaging evidence, but interestingly, nucleosome levels began to rise in February, and imaging was not able to detect progressive disease until the next scan in April 2020. When progressive disease was noted in April, nucleosome levels were still elevated. Had nucleosome levels been known at the time of treatment, more intensive monitoring would have been instituted for this patient in February. The results suggest that nucleosome levels can be used to guide treatment regimens and monitor disease relapse in subjects in remission. CRP analysis is not for clinical use.

[0193] Dog No. 3 Figure 9 A) was initially diagnosed with lymphoma in 2018 and successfully treated, but has since passed remission and this is reflected in the measured nucleosome levels. As part of the CHOP chemotherapy regimen, he was treated with vincristine on day 1 for progressive disease (PD). Vincristine treatment resulted in an improvement in clinical status toward stable disease (SD), which was reflected in a decrease in nucleosome levels, as shown in Figure 3. Figure 9 Shown in the result among A. However, due to toxic reasons and the strong clinical reaction observed in lacking, CHOP treatment terminates. Started to add L-asparaginase (L-spar) treatment with lomustine (lomustine) on the 22nd day, then carried out further lomustine treatment at the 34th, 79th and 113th day. Determined by tumor measurement, clinical discovery improved to partial remission at the 34th and 79th day, then improved to complete response (CR) to treatment at the 113th and 145th day, now, No. 3 dog is in remission and does not use further chemotherapy. Nucleosome level rises between the 22nd-34th day, then continues to decline from the 34th day, predicts lomustine treatment success, after a while at the 113th day and 145th day clinical observation complete response. And, according to clinical discovery, nucleosome level falls into the scope observed for healthy dog ​​at the 145th day. CRP level is always within normal range and cannot be used for clinical purpose.

[0194] Dog No. 4 Figure 9Dog B) was diagnosed with stage Vb intra-abdominal hypercalcemic lymphoma. He was started on the CHOP chemotherapy regimen of vincristine and doxorubicin from day 1 to day 93, and his response to treatment was monitored based on circulating calcium levels. The veterinarian recognized clinically that there was some response to vincristine, but no response to doxorubicin. Treatment was then switched from CHOP to lomustine plus L-spar on days 108-164, to which he also did not respond. Because a response to vincristine had been noted, treatment with COP (CHOP without doxorubicin) with vincristine was initiated on day 164 (a blood sample could not be obtained on day 167). Dog No. 4 responded to COP therapy and remains responsive to treatment at the time of this writing. The measured nucleosome levels reflected the observed clinical findings ( Figure 9 B). At most time points after treatment with vincristine, nucleosome levels decreased, reflecting a response to this therapy, while nucleosome levels increased after each dose of doxorubicin, reflecting a lack of response to this therapy. Dog 4 was also found to have a partial response to cyclophosphamide clinically, which was also reflected in a decrease in nucleosome concentrations. However, nucleosome levels remained above normal, indicating a continued need for further treatment. Nucleosome measurements clearly correlate with clinical findings and can be used to monitor disease remission and guide treatment selection. For example, nucleosome levels predicted a lack of response to doxorubicin therapy, and doxorubicin therapy could have been stopped earlier based on information from nucleosome levels. CRP analysis did not show any significant changes and is not useful for clinical purposes.

Claims

1. Use of a binding agent capable of binding to H3.1 histone isoforms of cell-free nucleosomes as a biomarker in a plasma sample in the preparation of a kit for diagnosing or detecting vascular cancer or hematological cancer.

2. The use as defined in claim 1, wherein the cell-free nucleosomes are mononucleosomes or oligonucleosomes.

3. The use as defined in claim 1 or 2, wherein the blood cancer is selected from leukemia, lymphoma or myeloma.

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

5. The use as defined in claim 3, wherein the lymphoma is non-Hodgkin's lymphoma (NHL).

6. The use as defined in claim 1 or 2, wherein the vascular cancer is angiosarcoma.

7. The use as defined in claim 1 or 2, wherein the vascular cancer is hemangiosarcoma.

8. Use of a binding agent capable of binding to an H3.1 histone isoform of cell-free nucleosomes in the preparation of a kit for use in a method for diagnosing or detecting vascular cancer or hematological cancer, comprising the following steps: (i) contacting a plasma sample obtained from a subject with the binding agent to detect or measure cell-free nucleosomes containing histone isoform H3.1; as well as (ii) using the detected level of cell-free nucleosomes containing histone isoform H3.1 to diagnose said subject as having said vascular cancer or hematological cancer.

9. Use of a binding agent capable of binding to an H3.1 histone isoform of cell-free nucleosomes in the preparation of a kit for use in a method for determining the prognosis of a subject suffering from a vascular cancer or a hematological cancer, comprising the steps of: (i) contacting a plasma sample obtained from the subject with the binding agent to detect or measure cell-free nucleosomes containing histone isoform H3.1; as well as (ii) using the detected level of cell-free nucleosomes containing histone isoform H3.1 as a prognostic indicator for said vascular or hematological cancer.

10. Use of a binding agent capable of binding to an H3.1 histone isoform on cell-free nucleosomes in the preparation of a kit for use in a method for monitoring the efficacy of a therapy in a subject having, suspected of having, or susceptible to a vascular or hematological cancer, comprising the steps of: (i) contacting a plasma sample obtained from the subject with the binding agent to detect or measure cell-free nucleosomes containing histone isoform H3.1; as well as (ii) comparing the detected levels of cell-free nucleosomes containing histone isoform H3.1 to earlier plasma samples taken from the subject to determine the efficacy of the therapy.

11. The use as defined in any one of claims 8 to 10, wherein the detecting or measuring comprises an immunochemical method, a mass spectrometry method, a chromatography method or a biosensor method.

12. The use as defined in any one of claims 8 to 10, wherein the detecting or measuring comprises an immunoassay or a chromatin immunoprecipitation method.

13. Use as defined in any one of claims 8 to 10, wherein the detecting or measuring comprises a 2-site immunoassay employing a combination of a labeled anti-nucleosome detection binding agent and an immobilized anti-histone H3.1 binding agent.

14. Use as defined in any one of claims 8 to 10, wherein the subject is a human or animal subject.

15. Use as defined in any one of claims 8 to 10, wherein the subject is suspected of having a recurrence of a vascular or hematological cancer.

16. The use as defined in any one of claims 8 to 10, wherein the subject has a high level of leukocytosis or a high white blood cell count.

17. Use as defined in any one of claims 8 to 10, wherein the method further comprises comparing the level of the cell-free nucleosomes containing histone isoform H3.1 in the plasma sample with one or more controls.

18. Use as defined in claim 17, wherein the control is a healthy subject.

19. Use as defined in claim 17, wherein the control is a subject having a cancer that is not a vascular cancer or a hematological cancer.

20. The use as defined in claim 17, wherein the control is a subject with a high level of leukocytosis.

21. The use as defined in claim 17, wherein the level of cell-free nucleosomes containing histone isoform H3.1 is increased compared to the control.

22. Use as defined in any one of claims 8 to 10, wherein the level of cell-free nucleosomes containing histone isoform H3.1 is detected or measured as one of the panel measurements.

23. Use as defined in claim 22, wherein the panel comprises one or more interleukins.

24. Use as defined in claim 23, wherein the one or more interleukins are selected from the group consisting of IL-6, IL-10 and IL-1 β.

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