Sample collection for liquid biopsy

Abstract

The present invention provides a method of the identification of a characteristic of cancer, comprising the steps of: (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent; and (ii) analysing the cell free nucleosomes to identify a cancer characteristic.

Description

[0001] SAMPLE COLLECTION FOR LIQUID BIOPSY

[0002] FIELD OF THE INVENTION

[0003] The invention relates to methods for processing body fluid samples for liquid biopsy. In particular, the invention also relates to sample preparation for the analysis of circulating chromatin fragments including cell free nucleosomes.

[0004] BACKGROUND OF THE INVENTION

[0005] Tissue biopsy involves the surgical removal and analysis of samples of tissue from the body and is a routine part of cancer medicine. Liquid biopsy is a term for the analysis of an actual or suspected cancer by means of a non-invasive body fluid (usually blood) test without the need for removal of solid tissue. Liquid biopsy is also used for the investigation of other diseases including the investigation of foetal diseases by analysis of maternal blood samples.

[0006] Currently most cancers are diagnosed symptomatically at a late stage when they are already metastatic with limited treatment options and poor patient outcomes. Liquid biopsy holds the promise to transform cancer treatment and save many lives by detecting ctDNA at early stage cancer by means of a blood test when treatment and outcomes for most patients are good. However, to date the promise of liquid biopsy to detect early stage cancer in a blood test has not been fulfilled and ctDNA tests are not used for routine cancer screening or diagnosis. Tests developed so far are complex, have long turnaround times (typically two weeks) and are provided at very high costs that are unaffordable to most healthcare systems.

[0007] When cancer (or other) cells die, the chromosomes are digested to small chromatin fragments. Most fragments are metabolised but some may be released into the blood. Liquid biopsy involves the analysis of these circulating cell free fragments of chromosomes in a blood sample to identify and analyse any such fragments that originate from diseased cells, particularly (but not only) cancer cells. However, the mechanisms underlying nucleosome release and metabolism in the blood are not entirely understood. In consequence, the preanalytics of blood samples collected for the measurement of circulating cell free nucleosomes are also not understood and current preanalytics are suboptimal.

[0008] Cell free DNA (cfDNA) circulates predominantly as nucleoprotein complexes that are chromatin fragments derived from cell chromosomes. The chromosome fragments most commonly analysed are small fragments of cfDNA that circulate predominantly (but not only) as cell free nucleosomes (cf-nucleosomes). Many liquid biopsy methods involve the analysis of cfDNA. These methods typically involve the extraction of cfDNA fragments from the sample (and hence also from the nucleosomes, or other nucleoprotein complexes) for analysis by DNA sequencing. In cancer, a proportion of the cfDNA fragments in circulation originate from the tumour and this is termed circulating tumour DNA (ctDNA). The DNA of cancer cells includes cancer associated mutations and the detection of mutated cfDNA fragments is an indicator of the presence of ctDNA and hence of a cancer in the subject tested.

[0009] There is a need in the art to provide improved methods for detecting biomarkers suitable for liquid biopsy, including optimal sample preparation and preanalytics to ensure the biomarker level measured is a true reflection of the level present in the circulation of the subject tested.

[0010] SUMMARY OF THE INVENTION

[0011] According to an aspect of the present invention there is provided a method of the identification of a characteristic of cancer, comprising the steps of:

[0012] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent; and

[0013] (ii) analysing the cell free nucleosomes to identify a cancer characteristic.

[0014] According to another aspect of the present invention there is provided a method for stabilising, storing and analysing cell free nucleosomes for a characteristic of cancer, comprising the steps of:

[0015] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;

[0016] (ii) separating plasma or serum from the blood sample; and

[0017] (iii) analysing the plasma or serum for cell free nucleosomes having a characteristic of cancer.

[0018] According to anotheraspect of the present invention there is provided a method for diagnosing or detecting a cancer, comprising the steps of:

[0019] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;

[0020] (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains stabilised cell free nucleosomes;

[0021] (iii) contacting the plasma sample or serum sample with a binding agent to detect or measure cell free nucleosomes; and (iv) using the cell free nucleosomes detected or measured to diagnose the subject with cancer.

[0022] According to another aspect of the present invention there is provided a method for determining the prognosis of a subject with a cancer, comprising the steps of:

[0023] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;

[0024] (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains stabilised cell free nucleosomes;

[0025] (iii) contacting the plasma sample or serum sample with a binding agent to detect or measure cell free nucleosomes; and

[0026] (iv) using the cell free nucleosomes detected or measured as indicative of the prognosis of said cancer.

[0027] According to another aspect of the present invention there is provided a method for monitoring the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer, comprising the steps of:

[0028] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;

[0029] (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains stabilised cell free nucleosomes;

[0030] (iii) contacting the plasma sample or serum sample with a binding agent to detect or measure cell free nucleosomes; and

[0031] (iv) comparing the cell free nucleosomes detected or measured with an earlier plasma sample or serum sample taken from said subject to determine the efficacy of said therapy.

[0032] According to anotheraspect of the present invention there is provided a method for quantifying cell free nucleosomes in a blood sample, comprising the steps of:

[0033] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;

[0034] (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains stabilised cell free nucleosomes;

[0035] (iii) contacting the plasma sample or serum sample with a nucleosome binding agent; and (iv) quantifying the concentration of cell free nucleosomes in the plasma sample or serum sample.

[0036] The concentration of cell free nucleosomes in the plasma sample or serum sample may be used to diagnose cancer in a subject, determine the prognosis of a subject with a cancer, or monitor the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer.

[0037] According to another aspect of the present invention there is provided a method for analysing DNA associated with cell free nucleosomes in a blood sample, comprising the steps of:

[0038] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;

[0039] (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains stabilised cell free nucleosomes;

[0040] (iii) analysing, detecting, measuring or quantifying the DNA fragments which are bound to or associated with the stabilised cell free nucleosomes.

[0041] Analysing, detecting, measuring or quantifying the DNA fragments may be used to diagnose cancer in a subject, determine the prognosis of a subject with a cancer, or monitor the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer.

[0042] In one embodiment the blood sample is contacted with the stabilising agent in a blood collection tube.

[0043] In one embodiment the stabilising agent is a cross-linking agent.

[0044] In one embodiment the cross-linking agent is selected from formaldehyde, paraformaldehyde, formalin, glutaraldehyde, Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE).

[0045] In one embodiment the cross-linking agent is a cross-linking agent releasing agent such as one selected from the group consisting of: diazolidinyl urea, imidazolidinyl urea, dimethoylol- 5,5dimethylhydantoin, dimethylol urea, 2-bromo-2.-nitropropane-1 ,3-diol, oxazolidines, sodium hydroxymethyl glycinate, 5-hydroxymethoxymethyl-1-1aza-3,7-dioxabicyclo [3.3.0]octane, 5-hydroxymethyl-1-1aza-3,7dioxabicyclo[3.3.0]octane, 5- hydroxypoly[methyleneoxy]methyl-1-1aza-3, 7dioxabicyclo[3.3.0]octane, quaternary adamantine, urotropine and any combination thereof.

[0046] In one embodiment of the method in step (i) the blood sample is also contacted with an anticoagulant, such as heparin, ethylenediamine tetraacetic acid (EDTA), citrate or oxalate.

[0047] In one embodiment the method, further includes the step of isolating the nucleosomes from the blood sample before analysis.

[0048] In one embodiment the method involves the steps, wherein (a) either or both of the isolating or analysing steps occurs up to 7 days after the blood sample is drawn from a subject, (b) either or both of the isolating or analysing steps occurs without freezing the blood sample; or both (a) and (b).

[0049] In one embodiment, the method comprises storing the blood sample, plasma sample or serum sample at room temperature for up to 7 days.

[0050] In one embodiment, the method comprises storing the blood sample, plasma sample or serum sample at about -80°C for up to two years.

[0051] In one embodiment, the method of analysing, detecting, measuring or quantifying comprises an immunoassay, immunochemical, mass spectroscopy, chromatographic, chromatin immunoprecipitation or biosensor method.

[0052] In one embodiment, the method of analysing, detecting, measuring or quantifying comprises an analysis of DNA fragments which are bound to or associated with the cell free nucleosomes. In one embodiment, the method comprises a step of extracting the DNA fragments which are bound to or associated with the stabilised cell free nucleosomes. In one embodiment, the method comprises sequencing the DNA fragments. In one embodiment, the method comprises amplifying the DNA fragments.

[0053] In one embodiment, the method of analysing, detecting, measuring or quantifying cell free nucleosomes comprises contacting the sample with a solid phase comprising a binding agent that detects cell free nucleosomes or a component thereof, and detecting binding to said binding agent. In one embodiment the blood is obtained from a human or an animal subject.

[0054] In one embodiment the subject is suspected of relapse to a cancer.

[0055] In one embodiment the method additionally comprises comparing the level of said cell free nucleosomes in said plasma sample or serum sample with one or more controls.

[0056] In one embodiment the control is a healthy subject or a subject with a non-cancer disease.

[0057] In one embodiment the level of cell free nucleosomes is elevated compared to a control. In particular, the level of cell free nucleosomes, e.g. in a blood, serum or plasma sample, obtained from a cancer patient is elevated compared to that of the control.

[0058] In one embodiment the cancer is a cancer of the bladder, breast, colon, cervix, oesophagus, kidney, large intestine, liver, lung, oral cavity, ovary, pancreas, prostate, rectum, skin or stomach or a vascular or haematological cancer.

[0059] In one embodiment cf-nucleosomes in a blood sample obtained from a pregnant subject are stabilised and analysed, detected, measured or quantified to determine the health of a foetus.

[0060] In one embodiment the cell free nucleosome is analysed by detecting or measuring a component of the nucleosome, such as a histone variant or histone isoform.

[0061] In one embodiment the cell free nucleosome is analysed by detecting or measuring an epigenetic feature of the cell free nucleosome.

[0062] In one embodiment the epigenetic feature of the cell free nucleosome is a histone isoform, such as a histone isoform of a core nucleosome, in particular a histone H3 isoform, such as H3.1.

[0063] In one embodiment the epigenetic feature of the cell free nucleosome is a histone post translational modification (PTM), such as a histone PTM of a core nucleosome, in particular a histone H3 PTM.

[0064] In one embodiment the histone PTM is selected from citrullination (such as H3 citrulline) or methylation (such as H3K27Me3, H3K36Me3, H3K9Me3). In one embodiment the binding agent comprises a detectable label. In one embodiment the binding agent is an anti-nucleosome antibody, an anti-DNA antibody, or an anti-histone antibody linked to a detectable label. In one embodiment the detectable label is an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP); a luminescent label, such as an acridinium ester derivative; a fluorescent label; or a radioactive label. In one embodiment the cell free nucleosomes are analysed, detected, measured or quantified using the detectable label.

[0065] In one embodiment the method further comprises the step of isolating the stabilised cell free nucleosomes from the plasma sample or serum sample before analysing, detecting, or measuring or quantifying cell free nucleosomes. In one embodiment the stabilised cell free nucleosomes are isolated by a nucleosome binding agent which binds to or is bound to a solid support such as a magnetic particle, a microplate, or a plate.

[0066] In one embodiment the method comprises contacting the plasma sample or serum sample with a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support, wherein the stabilised cell free nucleosomes are isolated from the plasma sample or serum sample using the second nucleosome binding agent and analysed, detected, measured or quantified using the detectable label.

[0067] In one embodiment the method does not comprise a step of contacting the blood sample or plasma sample or serum sample with a binding agent which binds to a transcription factor.

[0068] According to another aspect of the invention, there is provided the use of a stabilised nucleosome in a body fluid sample as a biomarker for cancer.

[0069] According to another aspect of the invention, there is provided the use of a cross-linked nucleosome in a body fluid sample as a biomarker for cancer.

[0070] According to another aspect of the invention, there is provided a kit comprising one or more reagents for carrying out the method as defined according to the present invention.

[0071] According to another aspect of the invention, there is provided use of a kit comprising: (i) one or more reagents to detect or measure the level of cell free nucleosomes or a component thereof, and (ii) a blood collection tube comprising a stabilising agent, to detect, monitor or diagnose cancer.

[0072] According to another aspect of the invention, there is provided a kit to detect, monitor or diagnose cancer in a subject, wherein said kit comprises (i) a first binding agent which specifically binds to an epigenetic feature of a cell free nucleosome and (ii) a second binding agent which specifically binds to cell free nucleosomes, for use with (iii) a blood collection tube comprising a stabilising agent.

[0073] According to another aspect of the invention, there is provided a blood collection tube comprising a stabilising agent for a cell free nucleosome for use in the method as defined according to the present invention.

[0074] According to another aspect of the invention, there is provided use of a blood collection tube comprising a stabilising agent for collecting a cell free nucleosome in a body fluid sample as a biomarker for cancer.

[0075] According to another aspect of the invention, there is provided a nucleosome binding agent for use in a diagnostic method, wherein the nucleosome binding agent is used in combination with a blood collection tube comprising a cross-linking agent.

[0076] According to another aspect of the invention, there is provided a nucleosome binding agent for use in a diagnostic method, wherein the method comprises obtaining a blood sample that contains cell free nucleosomes from a subject and: (i) contacting the blood sample with a cross-linking agent; (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains cross-linked cell free nucleosomes; and (iii) contacting the plasma sample or serum sample with the nucleosome binding agent to analyse, detect, measure or quantify the cross-linked cell free nucleosomes.

[0077] According to another aspect of the invention, there is provided a nucleosome binding agent for use in a method as defined according to the present invention.

[0078] According to another aspect of the invention, there is provided use of a nucleosome binding agent to analyse, detect, measure or quantify cross-linked cell free nucleosomes. According to another aspect of the invention, there is provided a kit for analysing, detecting, measuring or quantifying cell free nucleosomes in a blood sample, wherein the kit comprises a nucleosome binding agent and instructions for carrying out the method as defined according to the present invention.

[0079] According to another aspect of the invention, there is provided a combination of a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support, for use in a diagnostic method, wherein the first and second nucleosome binding agents are used in combination with a blood collection tube comprising a cross-linking agent.

[0080] According to another aspect of the invention, there is provided a combination of a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support, for use in a diagnostic method, wherein the method comprises obtaining a blood sample that contains cell free nucleosomes from a subject and: (i) contacting the blood sample with a cross-linking agent; (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains cross-linked cell free nucleosomes; (iii) and contacting the plasma sample or serum sample with the first and second nucleosome binding agents to isolate the cross-linked cell free nucleosomes from the plasma sample or serum sample and analyse, detect, measure or quantify the cross-linked cell free nucleosomes.

[0081] According to another aspect of the invention, there is provided a combination of a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support for use in a method as defined according to the present invention.

[0082] According to another aspect of the invention, there is provided use of a combination of a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support to isolate and analyse, detect, measure or quantify cross-linked cell free nucleosomes.

[0083] According to another aspect of the invention, there is provided a kit for analysing, detecting, measuring or quantifying cell free nucleosomes in a blood sample, wherein the kit comprises a first nucleosome binding agent comprising a detectable label, a second nucleosome binding agent which binds to or is bound to a solid support, and instructions for carrying out the method as defined according to the present invention.

[0084] According to another aspect of the invention, there is provided a cross-linking agent for use in a diagnostic method, wherein the cross-linking agent is used in combination with a nucleosome binding agent.

[0085] According to another aspect of the invention, there is provided a cross-linking agent for use in a diagnostic method, wherein the method comprises obtaining a blood sample that contains cell free nucleosomes from a subject and: (i) contacting the blood sample with the cross-linking agent; (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains cross-linked cell free nucleosomes; and (iii) contacting the plasma or serum sample with a nucleosome binding agent to analyse, detect, measure or quantify the cross-linked cell free nucleosomes.

[0086] According to another aspect of the invention, there is provided a cross-linking agent for use in a diagnostic method, wherein the cross-linking agent is used in combination with a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support.

[0087] According to another aspect of the invention, there is provided a cross-linking agent for use in a diagnostic method, wherein the method comprises obtaining a blood sample that contains cell free nucleosomes from a subject and: (i) contacting the blood sample with the cross-linking agent; (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains cross-linked cell free nucleosomes; and (iii) contacting the plasma sample or serum sample with a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support, to isolate the cross-linked cell free nucleosomes from the plasma sample or serum sample and analyse, detect, measure or quantify the cross-linked cell free nucleosomes.

[0088] According to another aspect of the invention, there is provided a cross-linking agent for use in a method as defined according to the present invention.

[0089] According to another aspect of the invention, there is provided use of a cross-linking agent to cross-link cell free nucleosomes prior to analysing, detecting, measuring or quantifying the cross-linked cell free nucleosomes with a nucleosome binding agent. According to another aspect of the invention, there is provided use of a cross-linking agent to cross-link cell free nucleosomes prior to isolating and analysing, detecting, measuring or quantifying the cross-linked cell free nucleosomes with a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support.

[0090] According to another aspect of the invention, there is provided a kit for quantifying cell free nucleosomes in a blood sample, wherein the kit comprises a cross-linking agent and instructions for carrying out the method as defined according to the present invention.

[0091] According to another aspect of the invention, there is provided a kit for quantifying cell free nucleosomes in a blood sample, wherein the kit comprises a nucleosome binding agent and a cross-linking agent, and optionally instructions for carrying out the method as defined according to the present invention.

[0092] According to another aspect of the invention, there is provided a kit for quantifying cell free nucleosomes in a blood sample, wherein the kit comprises a first nucleosome binding agent comprising a detectable label, a second nucleosome binding agent which binds to or is bound to a solid support, and a cross-linking agent, and optionally instructions for carrying out the method as defined according to the present invention.

[0093] According to another aspect of the invention, there is provided a plasma sample or serum sample comprising cross-linked cell free nucleosomes and a nucleosome binding agent comprising a detectable label.

[0094] According to another aspect of the invention, there is provided use of a crosslinking reagent or a crosslinking reagent releasing agent as an anticoagulant.

[0095] According to another aspect of the invention, there is provided use of formaldehyde or a formaldehyde releasing agent as an anticoagulant.

[0096] According to another aspect of the invention, there is provided use of a crosslinking reagent or a crosslinking reagent releasing agent as an inhibitor of NETosis. According to another aspect of the invention, there is provided use of formaldehyde or a formaldehyde releasing agent as an inhibitor of NETosis.

[0097] According to another aspect of the invention, there is provided use of a blood collection tube containing a crosslinking reagent or a crosslinking reagent releasing agent as an anticoagulant.

[0098] According to another aspect of the invention, there is provided use of a blood collection tube containing a formaldehyde or a formaldehyde releasing agent as an anticoagulant.

[0099] According to another aspect of the invention, there is provided use of a blood collection tube containing a crosslinking reagent or a crosslinking reagent releasing agent as an inhibitor of NETosis.

[0100] According to another aspect of the invention, there is provided use of a blood collection tube containing a formaldehyde or a formaldehyde releasing agent as an inhibitor of NETosis.

[0101] According to another aspect of the invention, there is provided a blood collection tube containing a crosslinking reagent or a crosslinking reagent releasing agent and no further anticoagulant.

[0102] According to another aspect of the invention, there is provided a blood collection tube containing a formaldehyde or a formaldehyde releasing agent and no further anticoagulant.

[0103] According to another aspect of the invention, there is provided a blood collection tube containing a crosslinking reagent or a crosslinking reagent releasing agent and no further additive.

[0104] According to another aspect of the invention, there is provided a blood collection tube containing a formaldehyde or a formaldehyde releasing agent and no further additive.

[0105] According to another aspect of the invention, there is provided a method for determining cell free nucleosome stability in a blood sample, comprising the steps of: (i) providing a first blood sample from a subject containing stabilised cell free nucleosomes and a second blood sample from the subject containing native cell free nucleosomes; (ii) separating plasma or serum from the blood, to provide a first plasma or serum sample containing stabilised cell free nucleosomes and a second plasma or serum sample containing native cell free nucleosomes; and (iii) analysing the DNA fragments which are bound to or associated with the stabilised cell free nucleosomes in the first plasma or serum sample and the DNA fragments which are bound to or associated with the native cell free nucleosomes in the second plasma or serum sample.

[0106] According to another aspect of the invention, there is provided use of a blood collection tube comprising a stabilising agent for collecting a body fluid sample and analysing, detecting, measuring or quantifying stabilised cell free nucleoproteins.

[0107] BRIEF DESCRIPTION OF THE FIGURES

[0108] Figure 1. Receiver Operating Characteristic (ROC) curve showing accuracy of detection of 99 cancer subjects assayed for stabilised H3.1 -nucleosomes. Cancer samples included from liquid and solid cancer patients. Control subjects were 73 healthy volunteers as described in detail in Example 1. Area under the Curve (AUC) = 0.878. 58% of cancers gave true positive results with no false positive results. Results were generated using an automated magnetic CLIA (chemiluminescent labelled immunoassay) system.

[0109] Figure 2. Levels of stabilised H3.1 -nucleosomes measured in plasma samples obtained from patients with a solid cancer diagnosis. The levels observed are stage dependent.

[0110] Figure 3. ROC curve showing accuracy of detection of subjects diagnosed with early stage cancer by assay for stabilised H3.1 -nucleosomes. Cancer samples included were from stage I and II solid cancer patients. Control subjects were healthy volunteers as described in detail in Example 1. (A) Stage I Cancer AUC = 0.800. (B) Stage II Cancer AUC = 0.789.

[0111] Figure 4. ROC showing accuracy of detection of subjects with later stage cancer by assay for stabilised H3.1 -nucleosomes. Cancer samples included were from Stage III and IV solid cancer patients. Control subjects were healthy volunteers as described in detail in Example 1. (A) Stage III Cancer AUC = 0.926. (B) Stage IV Cancer AUC = 0.950.

[0112] Figure 5. ROC curve showing accuracy of detection of subjects diagnosed with colorectal cancer by assay for stabilised H3.1 -nucleosomes. (A) Control subjects were healthy volunteers, versus Colorectal Cancer (CRC). AUC = 0.811 ; (B) Control subjects were diagnosed with an inflammatory disorder including 8 subjects with Crohn’s Disease or Colitis, versus CRC. AUC = 0.724.

[0113] Figure 6. ROC curve showing accuracy of detection of subjects diagnosed with 4 different solid cancer diseases by assay for stabilised H3.1 -nucleosomes. Control subjects were healthy volunteers as described in detail in Example 1 . (A) Breast cancer AUC = 0.855. (B) Lung cancer AUC = 0.976. (C) Hepatic cancer and Bile duct cancer AUC = 0.959. (D) Prostate cancer AUC = 0.829.

[0114] Figure 7. ROC curve showing accuracy of detection of subjects diagnosed with Acute Myeloid Leukaemia (AML) by assay for stabilised H3.1 -nucleosomes. Control subjects were healthy volunteers as described in detail in Example 1. AUC = 0.948.

[0115] Figure s. Levels of stabilised H3.1 -nucleosomes measured in plasma samples obtained from healthy subjects and patients with a gastroenterological inflammatory condition. The levels observed in 8 patients with gastroenterological inflammatory conditions (7 patients with Crohn’s Disease and 1 with Colitis) as well as 1 patient with Rheumatic OsteoArthritis and 1 with Polyarthritis, were not highly elevated above those observed for healthy subjects.

[0116] Figure 9. Box plot and Receiver Operating Characteristic (ROC) curve showing accuracy of detection of 229 cancer subjects assayed for stabilised H3.1 -nucleosomes. Cancer samples included from liquid and solid cancer patients. Control subjects were 105 healthy volunteers as described in detail in Example 2. (A) Box plot showing elevated stabilised H3.1 -nucleosomes levels for 21 cancer diseases for samples with levels. The graph shows samples with levels on a logarithmic scale to visualise the detection of lower levels as well as samples with higher levels up to 6000ng / ml. (B) ROC curve showing the True Positive Fraction (TPF) and False Positive Fraction (FPF) with AUC = 0.858. 49% of cancers gave a true positive result with zero false positive results.

[0117] Figure 10. ROC curve showing accuracy of detection of subjects diagnosed with cancer by assay for stabilised nucleosomes by ELISA. Samples containing stabilised nucleosomes obtained from 47 healthy volunteers and 119 subjects diagnosed with a variety of cancers, were assayed for H3.1 -nucleosomes (AUC=0.871), H3K36Me3-nucleosomes (AUC=0.879) and H3K27Me3-nucleosomes (AUC=0.860) by manual ELISA using a 96-well antibody coated microtitre plate and an alkaline phosphatase labelled antibody. Figure 11. ROC curve showing accuracy of detection of subjects diagnosed with cancer by assay for stabilised H3.1 -nucleosomes. Plasma samples containing stabilised nucleosomes obtained from 42 healthy volunteers and 111 subjects diagnosed with a variety of cancers, were assayed for stabilised H3.1 -nucleosomes, using an automated one-step magnetic chemiluminescent assay (AUC=0.860) and a two-step manual microtiter plate ELISA assay employing an alkaline phosphatase enzyme end-point (AUC=0.876).

[0118] Figure 12. ROC curve showing accuracy of detection of subjects diagnosed with cancer by assay for stabilised nucleosomes. Plasma samples containing stabilised nucleosomes obtained from 18 healthy volunteers and 21 subjects diagnosed with a variety of cancers, were assayed for stabilised H3.1 -nucleosomes, using an automated one-step magnetic chemiluminescent assay (AUC=0.800), a two-step manual microtiter plate ELISA assay employing an alkaline phosphatase enzyme end-point (AUC=0.886) and the Roche Cell Death Detection ELISA kit designed to detect all nucleosomes, or nucleosomes per se (AUC=0.807).

[0119] Figure 13. cf-DNA fragment size frequency profiles for cf-nucleosome-associated cfDNA isolated from Streck plasma samples by anti-H3.1 -nucleosome chromatin immunoprecipitation (ChIP) collected from four patients with CRC.

[0120] Figure 14. Levels of native and crosslinked H3.1-nucleosome levels measured in 8 patients with a cancer (•), 49 patients hospitalised with elevated levels of C-reactive protein (CRP) (A) but no cancer, 10 asymptomatic healthy volunteers (□).

[0121] Figure 15. 5 mL of whole blood from a healthy donor was added to a series of glass tubes containing increasing amounts of free formaldehyde to yield final (free) whole blood formaldehyde concentrations of zero to 0.5% v / v. The whole blood samples were left to stand at room temperature for 1 hour before processing by centrifugation. NETosis led to an approximately 7-fold increase in nucleosome level in normal serum (zero formaldehyde) over that observed for EDTA plasma. Addition of free formaldehyde prevented NETosis and / or prevented coagulation in whole blood leading to a fall in measured nucleosome level.

[0122] Figure 16. 5 mL of whole blood from a healthy donor was added to glass tubes containing the formaldehyde releasing agent imidazolidinyl urea (IDU) to yield a final whole blood IDU concentration of 2% m / v. The whole blood samples were left to stand at room temperature for 1 hour or 24 hours before processing by centrifugation. Addition of IDU prevented NETosis and / or prevented coagulation in whole blood. The result for whole blood processed at 24 hours and 1 hour were similar showing that the level measured was stable.

[0123] Figure 17. 5 mL of whole blood from a healthy donor was added to glass tubes containing the formaldehyde releasing agent diazolidinyl urea (DU) to yield a final whole blood DU concentration of 2% m / v. The whole blood samples were left to stand at room temperature for 1 hour or 24 hours before processing by centrifugation. Addition of DU prevented NETosis and / or prevented coagulation in whole blood. The result for whole blood processed at 24 hours and 1 hour were similar showing that the level measured was stable.

[0124] Figure 18. Results for the measurement of H3.1 -nucleosomes in plasma samples collected from 10 healthy subjects and 10 cancer patients crosslinked using urotropine as formaldehyde releaser by collection in Sarstedt S-Monovette cfDNA Exact blood collection tubes.

[0125] Figure 19. Cell free nucleosome levels in blood samples collected from 2 healthy donors into an EDTA blood collection tube (BCT), a Streck cfDNA BCT and into a glass or plastic tube containing sufficient aqueous IDU solution to provide a final IDU concentration of 0.125%, 0.25%, 0.5%, 1 % or 2% (m / v) when diluted by the added whole blood sample. Whole blood samples were left 1 hour (solid line) or 24 hours (dashed line) at room temperature after venipuncture before processing by centrifugation. H3.1 -nucleosome levels were measured in the supernatant collected after processing, (a) donor 1 blood sample collected in a glass tube; (b) donor 1 blood sample collected in a plastic tube; (c) donor 2 blood sample collected in a glass tube; (d) donor 2 blood sample collected in a plastic tube.

[0126] Figure 20. Recovery of native and crosslinked cfDNA from cf-nucleosomes isolated from EDTA plasma or Streck cfDNA BCT plasma respectively by ChIP, as a proportion of levels in whole plasma and the dependence of recovery on cf-nucleosome concentration, (a) in healthy subjects; (b) in cancer patients; (c) in patients with an inflammatory condition (with elevated CRP levels) but no cancer.

[0127] Figure 21. Recovery of native and crosslinked cfDNA from cf-nucleosomes isolated from EDTA plasma or Streck cfDNA BCT plasma respectively by ChIP, as a proportion of levels in whole plasma, in subjects with an elevated concentration of cf-nucleosomes. The results are shown for each patient group in ascending order of cf-nucleosome concentration and the actual concentrations are displayed numerically on the figure.

[0128] Figure 22. Correlation of cf-nucleosome levels measured by immunoassay in Streck plasma with (a) cfDNA levels measured by Qubit in EDTA plasma from healthy subjects and cancer patients, (b) cfDNA levels measured by Qubit in Streck plasma from healthy subjects and cancer patients, (c) cfDNA levels measured by Qubit in DNA extracts from ChIP isolated cf-nucleosomes in EDTA plasma samples obtained from healthy subjects and cancer patients, (d) cfDNA levels measured by Qubit in DNA extracts from ChIP isolated cf-nucleosomes in Streck plasma samples obtained from healthy subjects and cancer patients. The results show good agreement of Streck plasma cf-nucleosome levels with cfDNA levels both in plasma and in ChIP isolates.

[0129] Figure 23. Correlation of cf-nucleosome levels measured by immunoassay with cfDNA levels measured by Qubit as shown in Figure 22 with additional samples in each case (a), (b), (c) and (d) obtained from patients with an inflammatory condition. The results show good agreement of Streck plasma cf-nucleosome levels with cfDNA levels both in plasma and in ChIP isolates for healthy subjects and cancer patients but poor agreement for patients with an inflammatory condition.

[0130] Figure 24. Preanalytical effects of delayed plasma sample processing by up to 3 days for crosslinked cf-nucleosome measurements.

[0131] DETAILED DESCRIPTION OF THE INVENTION

[0132] Circulating nucleosomes are metabolised and have a limited half-life. The level of cf- nucleosomes present in a sample is a reflection of the rate of nucleosome release into the circulation and the rate of clearance from the circulation by nucleosome metabolism. In healthy individuals, the balance of release and clearance leads to a low level of circulating cf- nucleosomes. In subjects with a cancer disease, levels are higher due to increased nucleosome release as well as impaired clearance.

[0133] The mechanisms underlying nucleosome release and metabolism in the blood are not entirely understood. In consequence, the preanalytics of blood samples collected for the measurement of circulating cell free nucleosomes is also not entirely understood. However, circulating nucleosome metabolism involves digestion of nucleosome associated DNA by nucleases including DNase-1. Tightly bound DNA to the core of the nucleosome is protected from DNase digestion. However, DNA fragment ends that are less tightly bound to the nucleosome are susceptible to degradation. The length of DNA associated with a core nucleosome is 147 base pairs (bp). Nucleosome bound cfDNA fragments in the circulation comprise a variety of sizes from approximately 120-200bp. Sanchez et al (2018), demonstrated that the nucleosome associated cfDNA of cancer patients is more highly fragmented than that of healthy subjects including short fragments of less than 145bp down to 60bp in length. In addition, fragments bearing cancer associated mutations are most likely to be short. The smaller fragments represent nucleosomes with smaller DNA fragments than optimal for stability to degradation. Thus, not all circulating nucleosomes within a population are equally robust or resistant to degradation.

[0134] Some of these short cfDNA fragments may reflect nucleosomes with very short associated DNA fragments, as described above. Other short cfDNA fragments may reflect nucleosomes associated with >145bp of DNA, but where the DNA strand is broken or “nicked” leading to two or more smaller cfDNA fragments that derive from the same original nucleosome. The DNA of such nicked nucleosomes will be more accessible to nucleases such as DNase-1 , than intact 145bp DNA strands and, in consequence, such nucleosomes will be more susceptible to degradation.

[0135] Moreover, anti-coagulants such as EDTA or citrate used for plasma preparation function by binding to calcium (as well as magnesium and other bivalent) ions to remove these ions from solution which interrupts the coagulation cascade. However, the removal of magnesium and calcium ions further destabilises nucleosomes in plasma. Other anti-coagulants such as heparin are highly negatively charged and may interact with histones further destabilising nucleosomes in plasma.

[0136] Thus, for multiple reasons, not all nucleosomes are equally stable in plasma and the least stable may be cancer derived.

[0137] We have found that, in practice, it is better to use stabilised nucleosomes in a liquid biopsy setting. Moreover, we have found that stabilisation of nucleosomes in a sample, prior to analysis, gives rise to surprising clinical results in a low cost, high throughput, rapid (<1 hour), automated immunoassay liquid biopsy method, that provides discrimination between samples obtained from healthy subjects and subjects diagnosed with cancer, even in early stage cancer disease. We understand that we are the first to report the measurement of stabilised or cross-linked plasma or serum cell free nucleosomes.

[0138] In general terms, the invention relates to a method of cancer screening which includes the detecting and / or analysis of cell free (cf) nucleosomes within a blood sample. An inventive stabilisation (e.g. cross-linking) step acts to increase the amount of detectable and analysable cf nucleosomes. Whilst not wishing to be bound by any theory, it could be the case that such stabilisation (e.g. cross-linking) differentially stabilises (e.g. cross-links) cancer-derived nucleosomes compared to nucleosomes derived from a healthy population or even compared to nucleosomes derived from a sample derived from a patient with an autoimmune disease or inflammatory disorder. Therefore, the present invention allows for increased discrimination between cf nucleosomes derived from a cancer cell as opposed to cf nucleosomes derived from a healthy population and / or as opposed to cf nucleosomes derived from a population suffering from an autoimmune disease or an inflammatory disorder. In particular, the present invention enables the identification of cancers at an early stage providing obvious advantages in terms of treatment options.

[0139] In a study of 7 cancer types in 100 cancer and healthy subjects, the method of the present invention detected all cancer types studied, including more than 30% of stage I solid tumours at 100% specificity (Figure 1).

[0140] The present invention provides a method of the identification of a characteristic of cancer, comprising the steps of:

[0141] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent (e.g. a cross-linking agent); and

[0142] (ii) analysing the cell free nucleosomes to identify a cancer characteristic.

[0143] The present invention also provides a method for stabilising, storing and analysing cell free nucleosomes for a characteristic of cancer, comprising the steps of:

[0144] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent);

[0145] (ii) separating plasma or serum from the blood sample; and

[0146] (iii) analysing the plasma or serum for cell free nucleosomes having a characteristic of cancer. The characteristic of cancer may be a biomarker as described herein. In one embodiment, the characteristic of cancer is a cell free nucleosome (in particular, a stabilised cell free nucleosome once the stabilising agent has been applied to the sample). In a further embodiment, the characteristic of cancer is the level of cell free nucleosomes detected. In a further embodiment, the characteristic of cancer is the size profile of the DNA fragments associated with the stabilised nucleosomes. In a further embodiment, the characteristic of cancer is the presence or amount or proportion of short DNA fragments associated with the stabilised nucleosomes. For example, the short DNA fragments may have a length of 140 bp or less, 120 bp or less, 100 bp or less, 80 bp or less, or 60 bp or less.

[0147] The present invention also provides a method for diagnosing or detecting a cancer, comprising the steps of:

[0148] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent);

[0149] (ii) separating plasma or serum from the blood sample;

[0150] (iii) contacting the plasma sample or serum sample with a binding agent to detect or measure cell free nucleosomes; and

[0151] (iv) using the cell free nucleosomes detected to diagnose the subject with cancer.

[0152] The present invention further provides a method for determining the prognosis of a subject with a cancer, comprising the steps of:

[0153] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent);

[0154] (ii) separating plasma or serum from the blood sample;

[0155] (iii) contacting the plasma sample or serum sample with a binding agent to detect or measure cell free nucleosomes; and

[0156] (iv) using the cell free nucleosomes detected as indicative of the prognosis of said cancer.

[0157] The present invention additionally provides a method for monitoring the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer, comprising the steps of:

[0158] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent);

[0159] (ii) separating plasma or serum from the blood sample; (iii) contacting the plasma sample or serum sample with a binding agent to detect or measure cell free nucleosomes; and

[0160] (iv) comparing the cell free nucleosomes detected with an earlier plasma sample or serum sample taken from said subject to determine the efficacy of said therapy.

[0161] In one embodiment, the earlier plasma sample or serum sample taken from said subject is obtained from the subject prior to commencement of the therapy, or at an earlier stage of treatment using the therapy. In a further embodiment, the therapy is a treatment for cancer.

[0162] The sample(s) obtained from a subject to be tested by methods of the invention, and from which the cf nucleosomes may be detected, include any blood sample. The methods of the present invention allow for the efficient preservation of cf nucleosomes in a blood sample after blood draw for analysis.

[0163] In one embodiment, a whole blood sample containing cell free nucleosomes is contacted with a stabilising or cross-linking agent. In a further embodiment, a serum or plasma sample containing cell free nucleosomes derived from a whole blood sample, is contacted with a stabilising or cross-linking agent to stabilise the nucleosomes in the sample prior to analysis for nucleosomes.

[0164] This embodiment of the invention provides a method for diagnosing or detecting a cancer, comprising the steps of:

[0165] (i) contacting a blood plasma sample or serum sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent);

[0166] (ii) contacting the plasma sample or serum sample with a binding agent to detect or measure cell free nucleosomes; and

[0167] (iii) using the cell free nucleosomes detected to diagnose the subject with cancer.

[0168] In a further embodiment, the stabilising or cross-linking agent is added during, or concurrent with, the analysis, for example by inclusion as an additional assay reagent, or inclusion in an assay buffer or other assay reagent. In this embodiment the nucleosomes in the sample are stabilised concurrently with, or as part of, the nucleosome measurement or analytical process.

[0169] This embodiment of the invention provides a method for diagnosing or detecting a cancer, comprising the steps of: (i) contacting a blood plasma sample or serum sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent) and a binding agent to detect or measure cell free nucleosomes; and

[0170] (ii) using the cell free nucleosomes detected to diagnose the subject with cancer.

[0171] The tube as described herein (also referred to as a “blood collection tube”) may preferably include an anticoagulant agent and an active ingredient such as a fixative agent including but not limited to those active ingredients disclosed herein. The tube may also further include a nuclease inhibitor.

[0172] Any receptacle for collection of a body fluid sample may be used in place of a “blood collection tube” in the context of the present invention. A receptacle for collecting a body fluid sample is typically a sterile, sealed container, often a tube or vial, designed to hold the collected fluid without contamination or leakage. The present invention is not limited to the use of any particular receptacle and any suitable receptacle known to the skilled person may be used. In some embodiments, the receptacle is a blood collection tube or an evacuated blood collection tube. The receptacle may be made from any suitable material, such as plastic or glass. In some embodiments, the receptacle is a blood collection tube. In some embodiments, the receptacle is a glass receptacle. In some embodiments, the receptacle is a plastic receptacle.

[0173] As discussed herein, contacting a blood, plasma or serum sample with the stabilising agent allows the sample to be stored for a period of time prior to testing. More preferably, a blood, plasma or serum sample may be drawn at one location (e.g., a health care facility), contacted with the stabilising agent, and later transported to a different remote location (e.g., a laboratory) for the testing process.

[0174] In one embodiment, the body fluid sample obtained from a subject is a whole blood sample collected into a blood collection tube containing both a nucleosome stabiliser (e.g. a crosslinker) as well as a calcium ion sequestrator such as EDTA or sodium citrate.

[0175] In some preferred embodiments, the sample obtained from a subject may be any plasma sample, including a plasma sample produced using a calcium sequestrator such as EDTA plasma or citrate plasma, wherein the plasma sample is obtained by contacting a whole blood sample with a stabilising (e.g. cross-linking) agent and separating the plasma from the blood sample. In other embodiments, the sample obtained from a subject may be a serum sample, including a serum sample obtained by contacting a whole blood sample with a stabilising (e.g. crosslinking) agent and separating the serum from the blood sample.

[0176] The stabilising (e.g. cross-linking) agent may be contacted with whole blood in a stepwise process involving separately contacting a whole blood sample with a calcium ion chelating agent and then contacting the whole blood (or plasma or serum separated therefrom) with a stabilising (e.g. cross-linking agent). The stabilising (e.g. cross-linking) agent may be contacted with whole blood in a first step of a process involving: (1) contacting a whole blood sample with a stabilising (e.g. cross-linking) agent; (2) contacting the stabilised (e.g. crosslinked) sample with a calcium ion chelating agent; and (3) separating plasma or serum from the sample (e.g. isolating plasma from the sample).

[0177] Stabilising agents are well-known in the art and any suitable nucleosome stabiliser may be used. In preferred embodiments, the stabilising agent is a cross-linking agent. Cross-linking is a well-known technique in the art. The cross-linking agent may be an agent which gives rise to cross-linking through disulfide bridges. The most commonly used cross-linking reagent is formaldehyde which binds protein molecules to each other and to DNA. However, excess cross-linking may lead to changes in the structure of antibody binding epitopes in nucleosomes (and hence to loss of antibody binding) and even the cross-linking of nucleosomes to separate protein molecules or complexes. To prevent this, cross-linking is often quenched shortly after adding formaldehyde, for example by addition of excess glycine or tris(hydroxymethyl)aminomethane (TRIS), to stop further cross-linking.

[0178] Therefore, in one embodiment, the method comprises contacting the sample with a crosslinking agent and optionally adding a quenching agent to stop further cross-linking. The method may additionally comprise contacting the sample with a calcium ion chelating agent.

[0179] Excessive cross-linking can also be prevented by the use of a cross-linking releasing agent. These reagents disassociate to release small amounts of free cross-linker into solution in an equilibrium manner, maintaining a small concentration during cross-linking and minimising excess cross-linking.

[0180] In preferred embodiments, a whole blood sample obtained from a subject is collected in a blood collection tube containing formaldehyde or a formaldehyde releasing agent as a crosslinking agent and EDTA as a chelator of calcium ions to prevent coagulation and inhibit nuclease activity. In another embodiment the formaldehyde is added to whole blood following the collection of the whole blood sample, for example by adding the whole blood sample to a tube already containing formaldehyde. The tube is left for sufficient time for the cross-linking reaction to proceed. Optionally, the cross-linking reaction may be stopped by the addition of a quencher to prevent excess cross-linking of plasma components. The quencher is typically an amine compound such as glycine or TRIS that reacts with formaldehyde. The whole blood sample is then centrifuged and the plasma containing cross-linked nucleosomes, is isolated for analysis by methods of the invention.

[0181] For most stably bound DNA circulating chromatin fragments, cross-linking with formaldehyde in whole cultured cells or tissue samples is rapid and takes place in approximately 5 seconds (Poorey et al; 2013). We reasoned that, whilst 5 seconds may be required for diffusion and entry of formaldehyde into a cell, followed by entry into the nucleus, followed by cross-linking of chromatin, this time may be reduced in a whole blood context where the chromatin fragments are free in solution and immediately accessible to cross-linking. The cross-linking reagent used may be formaldehyde or be a formaldehyde releasing agent (also called a formaldehyde releaser, formaldehyde donor or formaldehyde releasing preservative). A formaldehyde releasing agent is a moiety that slowly releases formaldehyde. For example, a formaldehyde releasing agent may refer to a moiety that acts as a formaldehyde reservoir and maintains a low level of free formaldehyde in solution. Many formaldehyde releasing agents are known in the art and are commonly used as antimicrobial preservatives in the cosmetics industry, for example in skin care and hair care products where high levels of formaldehyde are avoided due to toxicity but low protective levels are maintained by release. Therefore, in one embodiment the cross-linking agent is a formaldehyde releasing agent.

[0182] In one embodiment of the invention, the cross-linking reagent may be added simultaneously with a calcium ion chelator. Blood collection tubes (BCT) containing both EDTA and a formaldehyde releasing agent are available commercially, for example the Cell-Free DNA BCT available from STRECK Inc. Whole blood added to such tubes is exposed simultaneously to EDTA and a cross-linking agent. As crosslinking is a rapid process, we reasoned that simultaneous addition rapidly stabilises nucleosome structures including against destabilising effects of magnesium and calcium sequestration by EDTA.

[0183] According to a further aspect of the invention, there is provided a blood collection tube comprising a stabilising agent (e.g. a crosslinking agent) for a cell free nucleosome for use in the method described herein. According to a further aspect of the invention, there is provided a use of a blood collection tube comprising a stabilising agent (e.g. a crosslinking agent) for collecting a cell free nucleosome in a body fluid sample as a biomarker for cancer.

[0184] According to a further aspect of the invention, there is provided a method for prevention of artefactually elevated measurements of cell free nucleosomes in solution in plasma, by prevention of the elution of cell surface bound cell free nucleosomes into free solution in plasma.

[0185] According to a further aspect of the invention, there is provided a method for stabilising the level of nucleosomes in a whole blood solution, wherein cell surface linked nucleosomes are cross linked to the cell surface to prevent their elution into free solution.

[0186] According to a further aspect of the invention, there is provided a method for prevention of the loss of nucleosome immunoreactivity or binding to antibodies (or other binders) leading to artefactually low measurements of cell free nucleosomes in solution in plasma, by stabilisation of the integrity, conformation, size or shape of nucleosomes in free solution in plasma or serum.

[0187] The cell free nucleosome may be a mononucleosome or oligonucleosome, or a mixture thereof.

[0188] Mononucleosomes and oligonucleosomes can be detected by Enzyme-Linked ImmunoSorbant 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 employ an anti-histone antibody (for example anti-H2B, anti-H3 or anti-H 1 , H2A, H2B, H3 and H4) as capture antibody and an anti-DNA or anti-H2A-H2B-DNA complex antibody as detection antibody.

[0189] Circulating nucleosomes are not a homogeneous group of protein-nucleic acid complexes. Rather, they are a heterogeneous group of chromatin fragments originating from the digestion of chromatin on cell death and include an immense variety of epigenetic structures including particular 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 an elevation in nucleosome levels will be associated with elevations in some circulating nucleosome subsets containing particular epigenetic signals including nucleosomes comprising particular histone isoforms (or variants), comprising particular post-translational histone modifications, comprising particular nucleotides or modified nucleotides and comprising particular protein adducts. Assays for these types of chromatin fragments are known in the art (for example, see WO 2005 / 019826, WO 2013 / 030579, WO 2013 / 030578, WO 2013 / 084002 which are herein incorporated by reference).

[0190] Methods of the invention enable the preparation of stabilised (e.g. cross-linked) cell free nucleosomes present in the sample, to provide a novel biomarker. The biomarker used in the uses and methods of the invention may be the level of (stabilised) cell free nucleosomes per se and / or the level or presence of an epigenetic feature of a (stabilised) cell free nucleosome. It will be understood that the terms “epigenetic signal structure” and “epigenetic feature” are used interchangeably herein. They refer to particular features of the nucleosome that may be detected. In one embodiment, the epigenetic feature of the nucleosome is selected from the group consisting of: a post-translational histone modification, a histone variant, a particular nucleotide and a protein adduct.

[0191] In one embodiment, the epigenetic feature of the nucleosome comprises one or more histone variants or isoforms. The epigenetic feature of the cell free nucleosome may be a histone isoform, such as a histone isoform of a core nucleosome, in particular a histone H3 isoform. The term “histone variant” and “histone isoform” may be used interchangeably herein. The structure of the nucleosome can also vary by the inclusion of alternative histone isoforms or variants which are different gene or splice products and have different amino acid sequences. Many histone isoforms are known in the art. Histone variants can be classed into a number of families which are subdivided into individual types. The nucleotide sequences of a large number of histone variants are known and 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), the GenBank (NIH genetic 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.

[0192] The nucleosomes measured in the present invention preferably contain the histone isoform H3.1. Therefore, in one embodiment, the histone isoform is H3.1. The structure of nucleosomes can vary by post translational modification (PTM) of histone proteins. PTM of histone proteins typically occurs on the tails of the 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 and 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). Therefore, in one embodiment, the epigenetic feature of the cell free nucleosome may be a histone post translational modification (PTM). The histone PTM may be present on a core nucleosome histone (e.g. H2A, H2B, H3 or H4), or a linker histone (e.g. H1 or H5). The histone PTM may be a histone PTM of a core nucleosome, e.g. H3, H2A, H2B or H4, in particular H3, H2A or H2B. In some embodiments, the histone PTM is a histone H3 PTM. Examples of such PTMs are described in WO 2005 / 019826 and WO 2017 / 068359.

[0193] For example, the post translational modification may include acetylation, methylation, which may be mono-, di-or tri-methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and / or isomerisation (see Ausio (2001) Biochem Cell Bio 79: 693). In one embodiment, the histone PTM is methylation of a lysine residue. In a further embodiment, the methylation is of a histone 3 lysine residue. In a yet further embodiment, the histone PTM is selected from H3K4Me, H3K4Me2, H3K9Me, H3K9Me3, H3K27Me3 or H3K36Me3. In a yet further embodiment, the histone PTM is selected from H3K27Me3 or H3K36Me3. In one embodiment, the histone PTM is acetylation of a lysine residue. In a further embodiment, the acetylation is of a histone 3 lysine residue. In a yet further embodiment, the histone PTM is selected from H3K9Ac, H3K14Ac, H3K18Ac or H3K27Ac. In another embodiment, the histone PTM is H4PanAc. In one embodiment, the histone PTM is phosphorylation of a serine residue. In a further embodiment, the phosphorylation is of an isoform X of histone 2A (H2AX) serine residue or phosphorylation of a histone 3 serine residue. In a yet further embodiment, the histone PTM is selected from pH2AX or H3S10Ph. In one embodiment, the histone PTM is selected from citrullination or ribosylation. In a further embodiment, the histone PTM is citrullinated H3 (H3cit) or citrullinated H4 (H4cit). In a further embodiment, the histone PTM is citrullination of a histone 3 arginine residue. In a yet further embodiment, the histone PTM is H3R8Cit. In one embodiment, the histone PTM is selected from the group consisting of: H3K4Me, H3K4Me2, H3K9Me, H3K9Me3, H3K27Me3, H3K36Me3, H3K9Ac, H3K14Ac, H3K18Ac, H3K27Ac, H4PanAc, pH2AX, H3S10Ph and H3R8Cit. In one embodiment the histone PTM is selected from the group consisting of: H3K27Me3, H3K36Me3 and H3K9Me3. A group or class of related histone post translational modifications (rather than a single modification) may also be detected. A typical example, without limitation, would involve a 2- site immunoassay employing one antibody or other selective binder directed to bind to nucleosomes and one antibody or other selective binder directed to bind the group of histone modifications in question. Examples of such antibodies directed to bind to a group of histone modifications would include, for illustrative purposes without limitation, anti-pan-acetylation antibodies (e.g. a Pan-acetyl H4 antibody [H4panAc]), anti-citrullination antibodies or antiubiquitin antibodies. In one embodiment, the histone PTM is selected from citrullination or ribosylation, in particular citrullination. In a further embodiment, the histone PTM is H3 citrulline (H3cit) or H4 citrulline (H4cit). In a yet further embodiment, the histone PTM is H3cit.

[0194] In one embodiment, the epigenetic feature of the nucleosome comprises one or more DNA modifications. In addition to the epigenetic signalling mediated by nucleosome histone isoform and PTM composition, nucleosomes also differ in their nucleotide and modified nucleotide composition. Global DNA hypomethylation is a hallmark of cancer cells and some nucleosomes may comprise 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.

[0195] In one embodiment, the epigenetic feature of the nucleosome comprises one or more proteinnucleosome adducts or complexes. A further type of circulating nucleosome subset is nucleosome protein adducts. It has been known for many years that chromatin comprises a large number of non-histone proteins bound to its constituent DNA and / or histones. These chromatin associated proteins are of a wide variety of types and have a variety of functions including transcription factors, transcription enhancement factors, transcription repression factors, histone modifying enzymes, DNA damage repair proteins and many more. These chromatin fragments including nucleosomes and other non-histone chromatin proteins or DNA and other non-histone chromatin proteins are described in the art.

[0196] In one embodiment, the protein adducted to the nucleosome (and which therefore may be used as a biomarker) is selected from: a transcription factor, a High Mobility Group Protein or chromatin modifying enzyme. References to “transcription factor” refer to proteins that bind to DNA and regulate gene expression by promoting ( / .e. activators) or suppressing ( / .e. repressors) transcription. Transcription factors contain one or more DNA-binding domains (DBDs), which attach to specific sequences of DNA adjacent to the genes that they regulate. All of the circulating nucleosomes and nucleosome moieties, types or subgroups described herein may be useful in the present invention.

[0197] In one embodiment, the epigenetic feature of the nucleosome does not comprise one or more protein-nucleosome adducts or complexes.

[0198] In some preferred embodiments, the method does not comprise a step of contacting the blood sample, plasma sample or serum sample with a binding agent which binds to a transcription factor.

[0199] In some preferred embodiments, the method does not comprise a step of contacting the blood sample, plasma sample or serum sample with a binding agent which binds to a protein other than a histone.

[0200] Another way the structure of nucleosomes may vary is by mutation. Therefore, in one embodiment, the epigenetic feature is a mutated histone. In a further embodiment, the mutation is in histone 3 (H3). In a yet further embodiment, the mutation in H3 is when lysine 27 is replaced by a methionine (H3K27M).

[0201] It will be understood that more than one epigenetic feature of cell free nucleosomes may be detected in methods and uses of the invention. Multiple biomarkers may be used as a combined biomarker. Therefore, in one embodiment, the use comprises more than one epigenetic feature of cell free nucleosomes as a combined biomarker. The epigenetic features may be the same type (e.g. PTMs, histone isoforms, nucleotides or protein adducts) or different types (e.g. a PTM in combination with a histone isoform). For example, a post- translational histone modification and a histone variant may be detected ( / .e. more than one type of epigenetic feature is detected). Alternatively, or additionally, more than one type of post-translational histone modification is detected, or more than one type of histone isoform is detected. In one aspect, the use comprises a post-translational histone modification and a histone isoform as a combined biomarker in a plasma sample or serum sample, for the diagnosis or detection of a cancer (such as a carcinoma). In one embodiment, the combined biomarker is H3.1 and H3cit. In an alternative embodiment, the combined biomarker is H3.1 and H3K27Me3. In an alternative embodiment, the combined biomarker is H3K27Me3 and H3K36Me3, optionally including H3.1. In an alternative embodiment the combined biomarker is H3K36Me3 and H3.1. In an alternative embodiment the combined biomarker is H3K9Me3 and H3.1. The term “biomarker” means a distinctive biological or biologically derived indicator of a process, event, or condition. Biomarkers can be used in methods of diagnosis, e.g. clinical screening, and prognosis assessment and in monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development. Biomarkers and uses thereof are valuable for identification of new drug treatments and for discovery of new targets for drug treatment.

[0202] The invention provides methods which can be used in the detection or diagnosis of patients with cancer. If a subject is determined to not have cancer, then the invention may still be used for the purposes of monitoring disease progression. For example, if the use comprises a sample from a subject determined not to have cancer, then the biomarker level measurements can be repeated at another time point to establish if the biomarker level has changed.

[0203] In one embodiment, the cancer is a cancer of the bladder, breast, colon, cervix, oesophagus, kidney, large intestine, liver, lung, oral cavity, ovary, pancreas, prostate, rectum, skin or stomach or a vascular or haematological cancer. In a further embodiment, the cancer is selected from blood (such as acute myeloid leukaemia (AML) or Non-Hodgkin’s Lymphoma (NHL)), breast, colorectal, liver (such as hepatic or bile duct cancer), lung, prostate, skin (such as melanoma) cancer. In one embodiment, the cancer is a haematopoietic cancer, such as a leukaemia, lymphoma or myeloma. In a particular embodiment, the cancer is a solid cancer, such as a carcinoma.

[0204] In one embodiment, the cancer is more specifically Acute lymphoblastic leukaemia (ALL), Acute myeloid leukaemia (AML), Advanced cancer, Advanced melanoma, Advanced prostate cancer, Anal cancer, Anaplastic large cell lymphoma (ALCL), Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma (AITL), Anorectal melanoma, Astrocytoma, Bile duct cancer (cholangiocarcinoma), Bladder cancer, Blood cancer, Bone cancer, Bowel cancer, Brain cancer, secondary, Brain tumours, Breast cancer, Breast cancer in men, Burkitt Lymphoma, Cancer of unknown primary (CUP), Cervical cancer, Chondrosarcoma, Chordoma, Chronic lymphocytic leukaemia (CLL), Chronic myeloid leukaemia (CML), Colon cancer, Craniopharyngioma, Cutaneous T-cell lymphoma (CTCL), Diffuse large B-cell lymphoma (DLBCL), Ductal carcinoma in situ (DCIS), Early (localised) prostate cancer, Ependymoma, Essential thrombocythaemia (ET), Ewing sarcoma, Eye cancer (ocular melanoma), Fallopian tube cancer, Follicular lymphoma (FL), Gallbladder cancer, Gastrointestinal stromal tumour (GIST), Germ cell ovarian cancer, Glioma, Haemangioblastoma, Head and neck cancer, Hodgkin lymphoma (HL), Inflammatory breast cancer, Kaposi's sarcoma, Kidney cancer, Laryngeal (larynx) cancer, Leiomyosarcoma, Leukaemia, Liver cancer, Locally advanced prostate cancer, Lung cancer, Lymph node cancer, secondary, Lymphoblastic lymphoma (LL), Lymphoma, MALT lymphoma, Mantle cell lymphoma, Medullary thyroid cancer, Medulloblastoma, Melanoma, Meningioma, Mesothelioma, Mouth cancer, Myelodysplasia (MDS), Myelofibrosis (MF), Myeloma, Nasal and sinus cancer, Nasopharyngeal cancer, Neuroendocrine tumours (NETs), Nodal marginal zone b-cell lymphoma (NMZI), Non-Hodgkin lymphoma (NHL), Non-small cell lung cancer (NSCLC), Oesophageal cancer, Oligodendroglioma, Oropharyngeal cancer, Osteosarcoma, Ovarian cancer, Paget's disease of the breast, Pancreatic cancer, Papillary and follicular thyroid cancer, Parathyroid cancer, Penile cancer, Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), Phyllodes tumours, Pineal region tumours, Pituitary gland tumours, Polycythaemia vera (PV), Primary CNS lymphoma, Primary mediastinal large B-cell lymphoma (PMBCL), Primary peritoneal cancer, Prostate cancer, Pseudomyxoma peritonei (PMP), Rare cancers, Rectal cancer, Recurrent melanoma, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Secondary bone cancer, Secondary breast cancer (metastatic breast cancer), Secondary cancer, Secondary liver cancer, Secondary lung cancer, Skin cancer, Small bowel cancer, Small cell lung cancer (SCLC), Small lymphocytic lymphoma (SLL), Soft tissue sarcoma, Spinal cord tumours, Splenic marginal zone lymphoma (SMZL), Stomach cancer, Testicular cancer, Throat cancer, Thymus cancer, Thyroid cancer, Tongue cancer, Tracheal cancer, Triple negative breast cancer, Upper urinary tract urothelial cancer (UTUC), Vaginal cancer, Vestibular schwannoma (acoustic neuroma) , Vulval cancer, Waldenstrom's macroglobulinaemia and Womb cancer.

[0205] In one embodiment, the methods described herein are for use in diagnosing, detection and / or management of subjects with early stage cancer. Cancer may be assigned as stage 0, stage I, stage II, stage III and stage IV. Stage definition varies with different cancer diseases and is known in the art. Typically, stage I is classified as when the cancer is small and confined locally to the tissue of origin. Stage II is classified as when the cancer has grown larger and beyond its origin into nearby tissues within the organ or to nearby lymph nodes. Stage III is classified as when the cancer has grown into nearby tissues beyond the organ of origin but has not spread to other more distant parts of the body. Stage IV is classified as when the cancer has spread to one or more distant parts of the body, such as the liver or lungs. Early stage cancer generally includes stages 0, I and II. Late stage cancer generally includes stages III and IV. In one embodiment, the cancer is a stage I (for example stage IA or stage IB), stage II (for example stage HA or stage 11 B), stage III (for example stage 11 IA, stage 111 B or stage 11 IC) or stage IV (for example stage IVA or stage IVB) cancer. The invention may find utility in detecting early stage cancers, in particular stages I and II. Therefore, in one embodiment the cancer is a stage I, II or III. In a further embodiment, the cancer is stage I or stage II. In an alternative embodiment, the cancer is stage II or stage III. The invention may also find utility in detecting late stage cancers, in particular stages III and IV. Therefore, in one embodiment the cancer is a stage III or IV. In a further embodiment, the cancer is stage IV.

[0206] In one embodiment, the condition is precancerous (also known as premalignant) wherein the condition could transform into a cancerous (malignant) condition. In a further embodiment the conditions could be of the skin such as actinic keratosis, Bowen’s disease or dyskeratosis. In one embodiment the conditions could be of the breast such as ductal carcinoma in situ, lobular carcinoma in situ, sclerosing adenosis or small duct papilloma. In one embodiment the conditions could be of gastrointestinal nature such as Barrett’s oesophagus, atrophic gastritis, colon polyp (including advanced adenomas), Plummer-Vinson syndrome, hereditary nonpolyposis colorectal cancer, anal dysplasia. In one embodiment the conditions could be of gynaecological nature such as cervical intraepithelial neoplasm (CIN), vaginal intraepithelial neoplasm (VAIN), Vulvar intraepithelial neoplasm, or lichen sclerosus.

[0207] Methods and uses described herein may be tested in body fluid samples, in particular blood, serum or plasma samples. The sample may be any biological fluid (or body fluid) sample taken from a subject including, without limitation, cerebrospinal fluid (CSF), whole blood, blood serum, plasma, menstrual blood, endometrial fluid, urine, saliva, or other bodily fluid (stool, tear fluid, synovial fluid, sputum), breath, e.g. as condensed breath, or an extract or purification therefrom, or dilution thereof. Biological samples also include specimens from a live subject, or taken post-mortem. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner. Preferably, blood samples, plasma samples or serum samples are used. Blood samples, plasma samples or serum samples may be collected in collection tubes containing one or more anticoagulants such as ethylenediamine tetraacetic acid (EDTA), heparin, or sodium citrate, in particular EDTA. In preferred embodiments, a plasma sample is used in the method of the invention. In other embodiments, a serum sample is used in the method of the invention.

[0208] Detecting and / or quantifying may be performed directly on a body fluid sample or on a purified or enriched nucleosome sample, or indirectly on an extract therefrom, or on a dilution thereof. Quantifying the amount of the biomarker present in a sample may include determining the concentration of the biomarker present in the sample. Uses and methods of detecting, monitoring and of diagnosis according to the invention described herein are useful to confirm the existence of a disease, to monitor development of the disease by assessing onset and progression, or to assess amelioration or regression of the disease. Uses and methods of detecting, monitoring and of diagnosis are also useful in methods for assessment of clinical screening, prognosis, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development.

[0209] The analysis, detection, measurement, or quantification may comprise an immunoassay, immunochemical, mass spectroscopy, chromatographic, chromatin immunoprecipitation or biosensor method. In particular, analysis, detection, measurement, or quantification may comprise a 2-site immunoassay method for nucleosome moieties. Such a method is preferred for the analysis, detection, measurement, or quantification of nucleosomes or nucleosome incorporated epigenetic features in situ employing two anti-nucleosome binding agents or an anti-nucleosome binding agent in combination with an anti-histone modification or anti-histone variant or anti-DNA or an anti-DNA modification or anti-adducted protein detection binding agent. Also, analysis, detection, measurement, or quantification may comprise a 2-site immunoassay employing a labelled anti-nucleosome detection binding agent in combination with an immobilised anti-histone modification or anti-histone variant or anti-DNA modification or anti-adducted protein binding agent. In one embodiment, the method does not employ an anti-adducted protein binding agent.

[0210] In one embodiment, the analysis, detection, measurement, or quantification comprises contacting the body fluid sample (e.g. plasma sample or serum sample) containing stabilised nucleosomes with an anti-histone antibody (for example anti-H2B, anti-H3 or anti-H1 , H2A, H2B, H3 and H4, i.e. an antibody that binds to a common histone or nucleosome epitope) and an anti-DNA or anti-H2A-H2B-DNA complex antibody. In particular, the anti-histone antibody may be a capture binding agent (e.g. bound to a solid phase) and the anti-DNA or anti-H2A- H2B-DNA complex antibody is the detection binding agent (e.g. comprising a detectable marker).

[0211] The inventors herein used a 2-site immunoassay for H3.1 -nucleosomes employing an immobilised anti-histone H3.1 antibody directed to bind to an epitope around amino acids SO- 33 of the histone H3.1 protein to capture clipped and non-clipped nucleosomes, together with a labelled anti-nucleosome antibody directed to bind to an epitope present in intact nucleosomes but not present on isolated (free) histone or DNA nucleosome components. This type of epitope may be referred to as a “conformational nucleosome epitope” herein because it requires the native three-dimensional configuration of the target nucleosome to be intact.

[0212] In one embodiment, the method of analysis, detection, measurement, or quantification comprises contacting the body fluid sample (e.g. plasma sample or serum sample) containing stabilised nucleosomes with a solid phase comprising a binding agent that detects cell free nucleosomes or a component thereof, and detecting binding to said binding agent.

[0213] In one embodiment, the method of analysis, detection, measurement, or quantification comprises: (i) contacting the body fluid sample (e.g. plasma sample or serum sample) containing stabilised nucleosomes with a first binding agent which binds to an epigenetic feature of a cell free nucleosome; (ii) contacting the sample or nucleosomes bound by the first binding agent in step (i) with a second binding agent which binds to cell free nucleosomes; and (iii) detecting or quantifying the binding of the second binding agent in the sample.

[0214] In another embodiment, the method of analysis, detection, measurement, or quantification comprises: (i) contacting the body fluid sample (e.g. plasma sample or serum sample) containing stabilised nucleosomes with a first binding agent which binds to cell free nucleosomes; (ii) contacting the sample or nucleosomes bound by the first binding agent in step (i) with a second binding agent which binds to an epigenetic feature of the cell free nucleosome; and (iii) detecting or quantifying the binding of the second binding agent in the sample.

[0215] Detecting or measuring the level of the biomarker(s) may be performed using one or more reagents, such as a suitable binding agent. For example, the one or more binding agents may comprise a ligand or binder specific for the desired biomarker, e.g. nucleosomes or component part thereof, an epigenetic feature of a nucleosome, a structural / shape mimic of the nucleosome or component part thereof.

[0216] It will be clear to those skilled in the art that the terms “binding agent”, “antibody”, “binder” or “ligand” as used herein are not limiting but are intended to include any binder capable of binding to particular molecules or entities and that any suitable binder can be used in the method of the invention. It will also be clear that the term “nucleosomes” is intended to include mononucleosomes, oligonucleosomes and polynucleosomes and any protein-DNA chromatin fragments that include a nucleosome. In one embodiment, the binding agent, such as the antibody, specifically binds to the target biomarker. The specificity of an antibody is the ability of the antibody to recognise a particular antigen as a unique molecular entity and distinguish it from another. An antibody that “specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and / or with greater affinity with a particular target antigen or epitope, than it does with alternative targets. An antibody “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances.

[0217] Methods of detecting biomarkers are known in the art. The reagents may comprise one or more ligands or binders, for example, naturally occurring or chemically synthesised compounds, capable of specific binding to the desired target. A ligand or binder may comprise a peptide, an antibody or a fragment thereof, or a synthetic ligand such as a plastic antibody, or an aptamer or oligonucleotide, capable of specific binding to the desired target. The antibody can be a monoclonal antibody or a fragment thereof. It will be understood that if an antibody fragment is used then it retains the ability to bind the biomarker so that the biomarker may be detected (in accordance with the present invention). A ligand / binder may be labelled with a detectable marker, such as a luminescent, fluorescent, enzyme or radioactive marker; alternatively or additionally a ligand according to the invention may be labelled with an affinity tag, e.g. a biotin, avidin, streptavidin or His (e.g. hexa-His) tag. Alternatively, ligand binding may be determined using a label-free technology for example that of ForteBio Inc.

[0218] The term “detecting” or “diagnosing” as used herein encompasses identification, confirmation, and / or characterisation of a disease state. Methods of detecting, monitoring and of diagnosis according to the invention are useful to confirm the existence of a disease, to monitor development of the disease by assessing onset and progression, or to assess amelioration or regression of the disease. Methods of detecting, monitoring and of diagnosis are also useful in methods for assessment of clinical screening, prognosis, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development.

[0219] In one embodiment, the method described herein is repeated on multiple occasions. This embodiment provides the advantage of allowing the detection results to be monitored over a time period. Such an arrangement will provide the benefit of monitoring or assessing the efficacy of treatment of a disease state. Such monitoring methods of the invention can be used to monitor onset, progression, stabilisation, amelioration, relapse and / or remission. In monitoring methods, test samples may be taken on two or more occasions. The method may further comprise comparing the level of the biomarker(s) present in the test sample with one or more control(s) and / or with one or more previous test sample(s) taken earlier from the same test subject, e.g. prior to commencement of therapy, and / or from the same test subject at an earlier stage of therapy. The method may comprise detecting a change in the nature or amount of the biomarker(s) in test samples taken on different occasions.

[0220] A change in the level of the biomarker in the test sample relative to the level in a previous test sample taken earlier from the same test subject may be indicative of a beneficial effect, e.g. stabilisation or improvement, of said therapy on the disorder or suspected disorder. Furthermore, once treatment has been completed, the method of the invention may be periodically repeated in order to monitor for the recurrence of a disease.

[0221] Methods for monitoring efficacy of a therapy can be used to monitor the therapeutic effectiveness of existing therapies and new therapies in human subjects and in non-human animals (e.g. in animal models). These monitoring methods can be incorporated into screens for new drug substances and combinations of substances.

[0222] In a further embodiment the monitoring of more rapid changes due to fast acting therapies may be conducted at shorter intervals of hours or days.

[0223] According to a further aspect, there is provided a kit comprising one or more reagents for carrying out the method as defined herein. According to a further aspect, there is provide the use of a kit comprising a blood collection tube comprising a stabilising agent and one or more reagents to detect or measure the level of cell free nucleosomes or a component thereof, to detect, monitor or diagnose cancer.

[0224] According to a further aspect of the invention, there is provided a kit to detect, monitor or diagnose cancer in a subject, wherein said kit comprises: (i) a blood collection tube comprising a stabilising agent, (ii) a first binding agent which specifically binds to an epigenetic feature of a cell free nucleosome (e.g. H3.1) and (iii) a second binding agent which specifically binds to cell free nucleosomes.

[0225] According to a further aspect of the invention, there is provided a kit to detect, monitor or diagnose cancer in a subject, wherein said kit comprises (i) a first binding agent which specifically binds to an epigenetic feature of a cell free nucleosome (e.g. H3.1) and (ii) a second binding agent which specifically binds to cell free nucleosomes, for use with (iii) a blood collection tube comprising a stabilising agent. It will be understood according to this aspect of the invention, that the blood collection tube can be provided separately from the kit. In particular, the kit may comprise instructions which instruct the user to use the kit (which detects cell free nucleosomes) with a blood collection tube comprising a stabilising agent.

[0226] Diagnostic or monitoring kits (or panels) are provided for performing methods of the invention. Such kits will suitably comprise one or more ligands or binders for detection and / or quantification of the biomarker according to the invention, and / or a biosensor, and / or an array as described herein, optionally together with instructions for use of the kit.

[0227] A further aspect of the invention is a kit for detecting the presence of a disease state, comprising a biosensor capable of detecting and / or quantifying one or more of the biomarkers as defined herein. As used herein, the term “biosensor” means anything capable of detecting the presence of the biomarker. Examples of biosensors are described herein. Biosensors may comprise a ligand binder or ligands, as described herein, capable of specific binding to the biomarker. Such biosensors are useful in detecting and / or quantifying a biomarker of the invention.

[0228] Suitably, biosensors for detection of one or more biomarkers combine biomolecular recognition with appropriate means to convert detection of the presence, or quantitation, of the biomarker in the sample into a signal. Biosensors can be adapted for “alternate site” diagnostic testing, e.g. in the ward, outpatients’ department, surgery, home, field and workplace. Biosensors to detect one or more biomarkers of the invention include acoustic, plasmon resonance, holographic, Bio-Layer Interferometry (BLI) and microengineered sensors. Imprinted recognition elements, thin film transistor technology, magnetic acoustic resonator devices and other novel acousto-electrical systems may be employed in biosensors for detection of the one or more biomarkers.

[0229] A further aspect of the invention is a lateral flow device for detecting the presence of a disease state, which is capable of detecting and / or quantifying one or more of the biomarkers as defined herein. As used herein, the term “lateral flow device” (which also may be known as a “lateral flow test”, “lateral flow immunochromatographic test” or “rapid test”) means a point of care or home use device intended to detect the presence of a target substance in a liquid sample. A common example of a lateral flow device is a home use test for human chorionic gonadotropin (hCG) to test for pregnancy. The lateral flow device of the present invention may be capable of detecting and / or quantifying cell free nucleosomes (e.g. stabilised cell free nucleosomes), for example using an anti-nucleosome antibody, an anti-DNA antibody, or an anti-histone antibody linked to a detectable label.

[0230] A further aspect of the invention is a microfluidic device for detecting the presence of a disease state, which is capable of detecting and / or quantifying one or more of the biomarkers as defined herein. As used herein, the term “microfluidic device” means a miniaturized instrument, often on a microchip, that manipulates and controls small amounts of fluids using channels with dimensions on the microscale. An example of a microfluidic device is the Element / available from Antech. The microfluidic device of the present invention may be capable of detecting and / or quantifying cell free nucleosomes (e.g. stabilised cell free nucleosomes), for example using an anti-nucleosome antibody, an anti-DNA antibody, or an anti-histone antibody linked to a detectable label.

[0231] Biomarkers for detecting the presence of a disease are essential targets for discovery of novel targets and drug molecules that retard or halt progression of the disorder. As the level of the biomarker is indicative of disorder and of drug response, the biomarker is useful for identification of novel therapeutic compounds in in vitro and / or in vivo assays. Biomarkers described herein can be employed in methods for screening for compounds that modulate the activity of the biomarker.

[0232] Thus, in a further aspect of the invention, there is provided the use of a binder or ligand, as described, which can be a peptide, antibody or fragment thereof or aptamer or oligonucleotide directed to a biomarker according to the invention; or the use of a biosensor, or an array, or a kit according to the invention, to identify a substance capable of promoting and / or of suppressing the generation of the biomarker.

[0233] The immunoassays described herein include any method employing one or more antibodies or other specific binders directed to bind to the biomarkers defined herein. Immunoassays include 2-site immunoassays or immunometric assays employing enzyme detection methods (for example ELISA), fluorescence labelled immunometric assays, time-resolved fluorescence labelled immunometric assays, chemiluminescent immunometric assays, immunoturbidimetric assays, particulate labelled immunometric assays and immunoradiometric assays as well as single-site immunoassays, reagent limited immunoassays, competitive immunoassay methods including labelled antigen and labelled antibody single antibody immunoassay methods with a variety of label types including radioactive, enzyme, fluorescent, time-resolved fluorescent and particulate labels. All of said immunoassay methods are well known in the art, see for example Salgame et al. (1997) and van Nieuwenhuijze et al. (2003).

[0234] 2-site immunoassays employed for methods of the invention may be one-step or two-step assays. In a one-step assay both antibodies or other specific binders and the sample are coincubated in a single reaction step. Alternatively, two-step assays may be employed in which one antibody or other specific binder and the sample are co-incubated in a first reaction step, following which the sample is removed and bound analyte is exposed to the second antibody or other specific binder in a separate reaction step.

[0235] Identifying, detecting and / or quantifying can be performed by any method suitable to identify the presence and / or amount of a cell free nucleosome in a biological sample from a subject or a purification or extract of a biological sample or a dilution thereof. In particular, quantifying may be performed by measuring the concentration of the target in the sample or samples. Biological samples that may be tested in a method of the invention include those as defined hereinbefore. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner. The present invention finds particular use in plasma samples or serum samples which may be obtained from the subject.

[0236] Identification, detection and / or quantification of biomarkers may be performed by detection of the biomarker or of a fragment thereof, e.g. a fragment with C-terminal truncation, or with N- terminal truncation. Fragments are suitably greater than 4 amino acids in length, for example 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. It is noted in particular that peptides of the same or related sequence to that of histone tails are particularly useful fragments of histone proteins.

[0237] For example, detecting and / or quantifying can be performed by one or more method(s) selected from the group consisting of: SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spec (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, LIPLC and other LC or LC MS-based techniques. Appropriate 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 could also be used. Methods involving detection and / or quantification of one or more biomarkers of the invention can be performed on bench-top instruments, or can be incorporated onto disposable, diagnostic or monitoring platforms that can be used in a non-laboratory environment, e.g. in the physician’s office or at the subject’s bedside. Suitable biosensors for performing methods of the invention include “credit” cards with optical or acoustic readers. Biosensors can be configured to allow the data collected to be electronically transmitted to the physician for interpretation and thus can form the basis for e-medicine.

[0238] The identification of biomarkers for a disease state permits integration of diagnostic procedures and therapeutic regimes. The biomarkers provide the means to indicate therapeutic response, failure to respond, unfavourable side-effect profile, degree of medication compliance and achievement of adequate serum drug levels. The biomarkers may be used to provide warning of adverse drug response. Biomarkers are useful in development of personalised therapies, as assessment of response can be used to fine-tune dosage, minimise the number of prescribed medications, reduce the delay in attaining effective therapy and avoid adverse drug reactions. Thus by monitoring a biomarker of the invention, subject care can be tailored precisely to match the needs determined by the disorder and the pharmacogenomic profile of the subject, the biomarker can thus be used to titrate the optimal dose, predict a positive therapeutic response and identify those subjects at high risk of severe side effects.

[0239] Biomarker-based tests provide a first line assessment of ‘new’ subjects, and provide objective measures for accurate and rapid diagnosis, not achievable using the current measures.

[0240] Biomarker monitoring methods, biosensors and kits are also vital as subject monitoring tools, to enable the physician to determine whether relapse is due to worsening of the disorder. If pharmacological treatment is assessed to be inadequate, then therapy can be reinstated or increased; a change in therapy can be given if appropriate. As the biomarkers are sensitive to the state of the disorder, they provide an indication of the impact of drug therapy.

[0241] References to “subject”, “individual” or “patient” are used interchangeably herein. The subject may be a human or an animal subject. In one embodiment, the subject is a human. In one embodiment, the subject is a (non-human) animal. In some embodiments the invention encompasses animal subjects (wild or domesticated). In some embodiments, the invention relates to veterinary uses including for livestock and companion animals such as cats, dogs, horses, donkeys, rats, rabbits, mice, guinea pigs, sheep, goats, pigs, deer, llamas, cows and cattle. In one embodiment, the subject is a non-human mammal, such as a dog, cat, mouse, rat or horse, in particular a dog.

[0242] The use, panels and methods described herein may be performed in vitro or ex vivo. The methods described herein are preferably performed in vitro. References to acts carried out on a body fluid sample “obtained” from a subject are intended to encompass acts carried out on a body fluid sample already obtained or “obtainable” from a subject and vice versa.

[0243] In one embodiment, the subject is suspected of relapse to a cancer. Minimal residual disease (MRD) is the name given to small numbers of cancer cells that remain in the person during treatment, or after treatment when the patient is in remission (i.e. patients with no symptoms or signs of disease). However, MRD is the major cause of relapse in cancer. Methods of the invention are therefore useful in monitoring patients who are suspected of relapse, particularly patients who are in remission from cancer.

[0244] The subject tested using the methods described herein may have no symptoms or may present with symptoms indicative of a cancer, for example change in bowel or bladder habits, a sore that does not heal, unusual bleeding or discharge, thickening lump in breasts or elsewhere, indigestion or difficulty in swallowing, change in wart or mole, nagging cough or hoarseness.

[0245] Detecting and / or quantifying may be compared to a cut-off level. Cut-off values can be predetermined by analysing results from multiple patients and controls, and determining a suitable value for classifying a subject as with or without the disease. For example, for diseases where the level of biomarker is higher in patients suffering from the disease, then if the level detected is higher than the cut-off, the patient is indicated to suffer from the disease. Alternatively, for diseases where the level of biomarker is lower in patients suffering from the disease, then if the level detected is lower than the cut-off, the patient is indicated to suffer from the disease. The advantages of using simple cut-off values include the ease with which clinicians are able to understand the test and the elimination of any need for software or other aids in the interpretation of the test results. Cut-off levels can be determined using methods in the art.

[0246] In one embodiment, the level of biomarker (i.e. stabilised cell free nucleosomes) is higher in patients suffering from cancer. The level of cell free nucleosomes measured in the plasma sample may be compared to a cut-off level. In one embodiment, the cut off level is greater than about 10ng / ml, 20ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml or 60 ng / ml, such as greater than about 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml, 100 ng / ml. In one embodiment, the cut off level is greater than about 31 ,7ng / ml.

[0247] Detecting and / or quantifying may also be compared to a control. It will be clear to those skilled in the art that the control subjects may be selected on a variety of basis which may include, for example, subjects known to be free of the disease or may be subjects with a different disease (for example, for the investigation of differential diagnosis). The “control” may comprise a healthy subject, a non-diseased subject and / or a subject without a cancer. Comparison with a control is well known in the field of diagnostics.

[0248] Therefore, in one embodiment, the method additionally comprises comparing the level of said cell free nucleosomes in said blood sample, plasma sample or serum sample with one or more controls. For example, the method may comprise comparing the level of cell free nucleosomes present in a blood sample, plasma sample or serum sample obtained from the subject with the level of cell free nucleosomes present in a blood sample, plasma sample or serum sample obtained from a normal subject. The control may be a healthy subject. Alternatively, the control may be a diseased subject, such as a subject with an inflammatory disorder.

[0249] Alternatively, the control is a subject with a different type of cancer or a cancer which affects a different organ in the body or a vascular or haematological cancer.

[0250] In one embodiment, the level of cell free nucleosomes is elevated compared to the control.

[0251] It will be understood that it is not necessary to measure control levels for comparative purposes on every occasion. For example, for healthy / non-diseased controls, once the ‘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 multiple control subjects without a particular cancer and testing for the level of biomarker. Results (i.e. biomarker levels) for subjects suspected to have a cancer can then be examined to see if they fall within, or outside of, the respective normal range. Use of a ‘normal range’ is standard practice for the detection of disease.

[0252] The elevated concentration of cell free nucleosomes may be used to diagnose cancer in a subject, determine the prognosis of a subject with a cancer, or monitor the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer. In one embodiment, the subject is diagnosed with cancer if the concentration of cell free nucleosomes in the blood sample or plasma sample or serum sample is elevated compared to a normal range. In one embodiment, the subject is diagnosed with cancer if the concentration of cell free nucleosomes in the blood sample or plasma sample or serum sample is elevated compared to samples from a normal subject (e.g. a healthy subject).

[0253] In one embodiment, the subject is diagnosed with cancer if the concentration of cell free nucleosomes (e.g. cell free H3.1 nucleosomes) in the plasma sample is about 10 ng / ml or higher, 20 ng / ml or higher, 30 ng / ml or higher, about 40 ng / ml or higher, about 50 ng / ml or higher, about 60 ng / ml or higher, about 65 ng / ml or higher, about 70 ng / ml or higher, about 75 ng / ml or higher, about 80 ng / ml or higher, about 85 ng / ml or higher, about 90 ng / ml or higher, about 95 ng / ml or higher, or about 100 ng / ml or higher.

[0254] In one embodiment, the subject is diagnosed with cancer if the concentration of cell free nucleosomes (e.g. cell free H3.1 nucleosomes) in the serum sample is about 10 ng / ml or higher, 20 ng / ml or higher, 30 ng / ml or higher, about 40 ng / ml or higher, about 50 ng / ml or higher, about 60 ng / ml or higher, about 65 ng / ml or higher, about 70 ng / ml or higher, about 75 ng / ml or higher, about 80 ng / ml or higher, about 85 ng / ml or higher, about 90 ng / ml or higher, about 95 ng / ml or higher, or about 100 ng / ml or higher.

[0255] In one embodiment, the subject is diagnosed as being at high risk of having a cancer if the concentration of cell free nucleosomes in the blood sample, plasma sample or serum sample is elevated compared to a normal range. This embodiment is analogous to cancer screening methods such as faecal immunochemical testing (FIT) in which the finding of blood in the stool is not diagnostic for cancer but identifies a subject as being at high risk for CRC. Similarly, scanning cancer screening methods such as mammography or low dose computed tomography identify subjects with lumps or nodules and are not diagnostic for cancer, but identify a subject as being at high risk for breast and lung cancer respectively. In one embodiment, the subject is diagnosed with being at high risk of having a cancer if the concentration of cell free nucleosomes in the blood sample, plasma sample or serum sample is elevated compared to samples from a normal subject (e.g. a healthy subject).

[0256] In one embodiment, the method additionally comprises determining at least one clinical parameter for the patient. This parameter can be used in the interpretation of results. Clinical parameters may include any relevant clinical information for example, without limitation, gender, weight, Body Mass Index (BMI), smoking status and dietary habits. Therefore, in one embodiment, the clinical parameter is selected from the group consisting of: smoking status, family history of lung cancer, age, sex and body mass index (BMI). In a further embodiment, the clinical parameter is selected from the group consisting of: smoking status and family history of cancer.

[0257] It will be clear to those skilled in the art, that any combination of the biomarkers disclosed herein may be used in panels and algorithms for the detection of cancer and that further markers may be added to a panel including these markers.

[0258] In one embodiment, the method of the invention is performed to identify a subject at high risk of having a cancer and therefore in need of further testing (i.e. further cancer investigations). The further testing may involve one or more of: biopsy (such as bone marrow biopsy or lymph node biopsy), cytogenetic testing, immunophenotyping, CT scanning, LDCT scanning, PET scanning, X-ray (in particular chest X-ray) and / or lumbar puncture.

[0259] Methods and biomarkers described herein may be used to identify if a patient is in need of a biopsy. Therefore, according to a further aspect of the invention there is provided a method of identifying a patient in need of a biopsy comprising obtaining a plasma or serum sample from said patient, detecting the level of (stabilised) cell free nucleosomes in the plasma or serum sample, and using the results obtained to identify whether the patient is in need of a biopsy.

[0260] According to a further aspect, there is provided a method of treating a cancer in a subject, which comprises the following steps:

[0261] (i) obtaining a blood sample which contains cell free nucleosomes from the subject;

[0262] (ii) contacting the nucleosomes or the blood sample with a stabilising agent;

[0263] (iii) detecting or measuring the level of cell free nucleosomes or a component thereof;

[0264] (iv) using the level measured in step (iii) as indicative of the presence of said cancer in the subject; and

[0265] (v) treating surgically or administering a therapeutic agent if the subject is determined to have said cancer in step (iv).

[0266] According to a further aspect, there is provided a method of treating a cancer in a subject in need thereof, which comprises the step of treating surgically or administering a therapeutic agent to a subject identified as having differing levels of stabilised cell free nucleosomes in a blood sample, plasma sample or serum sample obtained from said subject, when compared to the level of stabilised cell free nucleosomes in a sample obtained from a control subject.

[0267] In one embodiment, the treatment is selected from one or more of: surgery, chemotherapy, immunotherapy, hormone therapy, biological therapy and radiotherapy.

[0268] The methods may comprise:

[0269] (i) contacting the cell free nucleosomes in a blood sample obtained from the subject, with a stabilising agent;

[0270] (ii) measuring the level of cell free nucleosomes (optionally in combination with the level of one or more additional biomarkers) in the sample;

[0271] (iii) identifying the subject as suffering from a cancer based on a higher level of cell free nucleosomes compared to a control; and

[0272] (iv) administering a treatment to the subject.

[0273] One aspect of the teachings herein contemplates a method for the analysis of stabilised cf nucleosomes of foetal origin circulating in maternal whole blood. The method may be performed on a single sample or on a multitude of samples (e.g., in a multi-well plate). The method includes contacting the maternal whole blood sample with a stabilising (e.g. crosslinking) agent optionally in the form of a stabilising reagent as discussed above. The stabilising reagent may further include a DNase inhibitor to maintain the structural integrity of the cf nucleosome. After contacting the maternal blood sample with the stabilising agent, the sample may be centrifuged to separate the plasma and the supernatant is discarded.

[0274] Another aspect of the teachings herein contemplates a method for the analysis of stabilised cf nucleosomes of foetal origin circulating in maternal plasma. The method may be performed on a single sample or on a multitude of samples (e.g., in a multi-well plate). The method includes contacting the maternal plasma sample with a stabilising (e.g. cross-linking) agent optionally in the form of a stabilising reagent as discussed above. The stabilising reagent may further include a DNase inhibitor to maintain the structural integrity of the cf nucleosome.

[0275] By contacting a blood sample, plasma sample or serum sample with the stabilising agent, the blood, plasma or serum sample does not necessarily require immediate processing and may be stored for a prolonged period, such as up to about 7 days or up to about 3 days at room temperature. Thus the inventions herein contemplate one or more steps of storing and / or otherwise waiting a relatively lengthy period from the time of blood draw and / or contacting until the time of screening, testing or other analysis.

[0276] Samples may be stored prior to analysis. Storage may occur on ice or at any temperature between -80°C and 30°C. For example, a sample (e.g. a plasma or serum sample) may be stored at about -80°C. For example, a sample may be stored under refrigeration (e.g. about 2-8°C). A sample may also be stored at room temperature (e.g. about 20-22°C) and thus free of freeze / thaw cycles upon analysis.

[0277] In one embodiment, the method of the invention comprises storing the blood sample at room temperature after contacting it with the stabilising agent (e.g. before separating plasma or serum from the blood sample). In one embodiment, the blood sample is stored at room temperature for up to about 7 days, up to about 6 days, up to about 5 days, up to about 4 days, or up to about 3 days. In one embodiment, the blood sample is stored at room temperature for 1 day or more, 2 days or more, or 3 days or more.

[0278] In one embodiment, the method of the invention comprises storing the blood sample under refrigeration after contacting it with the stabilising agent (e.g. before separating plasma or serum from the blood sample). In one embodiment, the blood sample is stored under refrigeration for up to about 42 days, up to about 28 days, up to about 14 days, or up to about 7 days. In one embodiment, the blood sample is stored under refrigeration for 3 days or more, 5 days or more, or 7 days or more.

[0279] In one embodiment, the method of the invention comprises storing the plasma or serum sample at room temperature after separating plasma from the blood sample (e.g. before contacting the plasma or serum sample with a nucleosome binding agent). In one embodiment, the plasma or serum sample is stored at room temperature for up to about 7 days, up to about 6 days, up to about 5 days, up to about 4 days, or up to about 3 days. In one embodiment, the plasma or serum sample is stored at room temperature for 1 day or more, 2 days or more, or 3 days or more.

[0280] In one embodiment, the method of the invention comprises storing the plasma or serum sample under refrigeration after separating plasma from the blood sample (e.g. before contacting the plasma or serum sample with a nucleosome binding agent). In one embodiment, the plasma or serum sample is stored under refrigeration for up to about 42 days, up to about 28 days, up to about 14 days, or up to about 7 days. In one embodiment, the plasma or serum sample is stored under refrigeration for 3 days or more, 5 days or more, or 7 days or more.

[0281] In one embodiment, the method of the invention comprises storing the plasma or serum sample at -80°C after separating plasma or serum from the blood sample (e.g. before contacting the plasma or serum sample with a nucleosome binding agent). In one embodiment, plasma or serum sample is stored at -80°C for up to about two years, or up to about one year. In one embodiment, the plasma or serum sample is stored at -80°C for 1 week or more, 2 weeks or more, or one month or more.

[0282] Centrifugation may be performed at a suitable rate. For example, centrifugation may be done at about 300g to about 20,000g. Centrifugation may occur at about 1 ,000 to 16,000g. Centrifugation may be performed at about room temperature or cooler. For example, it may be performed at about 1-20°C, or still more specifically at about 4-9°C.

[0283] NUCLEOSOME STABILISATION

[0284] The present inventors have shown that the effect of stabilisation of cf-nucleosomes present in body fluid samples, particularly in whole blood samples, differs in samples obtained from subjects or patients with different disease statuses.

[0285] The present inventors investigated the stability of crosslinked and native (not crosslinked) cf- nucleosomes in whole blood. Cf-nucleosomes were isolated from samples by ChIP and the amount of DNA retained by the antibody bound cf-nucleosomes was measured by Qubit. Without being bound by theory, mononucleosomes are nucleoprotein complexes containing both histone protein and DNA. Retention of the DNA by isolated antibody bound cf- mononucleosomes indicates that they retained both their susceptibility to histone antibody binding and their DNA component. A high cfDNA recovery indicates a high mononucleosome stability. Conversely, a low cfDNA recovery indicates a low mononucleosome stability.

[0286] Matched plasma samples were collected in EDTA and Streck BCTs from 6 healthy subjects, 6 patients hospitalised with an inflammatory condition and 7 patients diagnosed with a cancer. Two of the 6 healthy subjects were found to have crosslinked cf-nucleosome levels above the cutoff used in EXAMPLE 2 (about 31.7 ng / ml). These were the first observed false positive results obtained for the measurement of crosslinked plasma nucleosomes. This result would reduce the observed specificity of the method from 100% (reported in Examples 1-4) to approximately 98%. The present inventors observed that the cfDNA recovery from samples collected from healthy subjects in EDTA BCTs containing native cf-nucleosomes was around 30-50% in samples with low nucleosome levels in the normal range. cfDNA recovery was reduced in samples with increasing nucleosome level above the normal range (see Figure 20(a)). The results were similar for native and crosslinked nucleosomes indicating a similar stability for native and crosslinked nucleosomes in healthy blood samples.

[0287] DNA recovery of native cf-nucleosomes in blood samples collected from cancer patients in EDTA BCTs gave recovery results similar to those of native cf-nucleosomes from healthy subjects. The DNA recovery was around 30-50% in samples with nucleosome levels in the normal range with a reduction in recovery in samples with higher cf-nucleosome concentrations. Crosslinking of cf-nucleosomes in cancer blood samples by collection in Streck BCTs had little effect on cancer samples with low cf-nucleosome levels in the normal range. However, crosslinking greatly increased cfDNA recovery from ChIP isolated cf- nucleosomes in cancer samples with elevated cf-nucleosome levels (see Figure 20(b)). These results indicate that cf-nucleosomes present at elevated levels in blood samples from cancer patients are stabilised by crosslinking.

[0288] DNA recovery of native cf-nucleosomes in blood samples collected from patients hospitalised with an inflammatory condition in EDTA BCTs differed from that observed for healthy subjects or cancer patients and was above 27% at all nucleosome concentrations tested. In contrast to the effect in cancer patients, crosslinking of cf-nucleosomes resulted in a reduction in cfDNA recovery (see Figure 20(c)). This surprising finding indicates, counterintuitively, that the use of a stabilising or preservative reagent destabilises cf-nucleosomes in samples from patients with an inflammatory condition.

[0289] The results indicate that cf-nucleosomes present in the circulation of healthy subjects, cancer patients and patients with an inflammatory condition all differ in structure.

[0290] Without being bound by theory, cf-nucleosomes present in the circulation of healthy subjects may consist primarily of intact mononucleosomes. “Healthy” native cf-mononucleosomes are relatively stable and their stability is not greatly increased by intra-nucleosomal crosslinking. In cancer patients, cf-nucleosomes may comprise a significant proportion of nicked or clipped (i.e. not intact) mononucleosomes. Native non-intact “cancer” cf-nucleosomes may be less stable than cf-nucleosomes in healthy subjects and the stability (and hence DNA retention) of cancer cf-nucleosomes is markedly increased by intra-nucleosomal crosslinking. In patients with an inflammatory condition, cf-nucleosomes may comprise a significant proportion of white blood cell derived extracellular trap oligonucleosomes or polynucleosomes. The reason for a reduction of cfDNA on crosslinking may be that, in addition to intra-nucleosomal crosslinking, the oligonucleosomes and polynucleosomes characteristic of inflammatory derived cf- nucleosomes, may undergo inter-nucleosomal crosslinking. Inter-nucleosomal crosslinking may result in the coalescence and clumping of inflammatory polynucleosome strings leading to loss from solution and / or reduced antibody binding.

[0291] Figure 21 shows DNA recovery from native (EDTA) and crosslinked (Streck) cf-nucleosomes in plasma samples with elevated cf-nucleosome levels. The recovery of cfDNA in samples from cancer patients is higher for crosslinked cf-nucleosomes in Streck plasma. In contrast, the recovery of cfDNA in samples from patients with inflammatory conditions with elevated CRP levels is higher from native cf-nucleosomes in EDTA plasma. This difference may be used to identify an elevated level of nucleosomes observed for a patient as being caused by a cancer condition or by an inflammatory condition.

[0292] The recovery of cfDNA from the cf-nucleosomes of the two samples from healthy subjects that gave rise to false positive results for cancer by measurement of crosslinked cf-nucleosomes, was not increased by crosslinking as would be expected for a cancer patient. This difference may be used to identify an elevated level of nucleosomes observed for a patient as being associated with a cancer condition or as a false positive cancer result.

[0293] The present invention provides a method for determining cell free nucleosome stability in a body fluid sample, comprising the steps of:

[0294] (i) providing a first body fluid sample from a subject containing stabilised cell free nucleosomes and a second body fluid sample from the subject containing native cell free nucleosomes;

[0295] (ii) separating a cell-free supernatant liquid from the body fluid samples, to provide a first supernatant sample containing stabilised cell free nucleosomes and a second supernatant sample containing native cell free nucleosomes; and

[0296] (iii) analysing the DNA fragments which are bound to or associated with the stabilised cell free nucleosomes in the first supernatant sample and the DNA fragments which are bound to or associated with the native cell free nucleosomes in the second supernatant sample. The body fluid samples may be any described herein. In some embodiments, the first body fluid sample and the second body fluid sample are blood samples. In some embodiments, the first blood sample and the second blood sample are whole blood samples. In some embodiments, the cell-free supernatant liquid is plasma or serum, the first supernatant sample is a first plasma or serum sample, and the second supernatant sample is a second plasma or serum sample.

[0297] The cell free nucleosomes may be stabilised by any method described herein, for example by contacting the body fluid with a stabilising agent. In preferred embodiments, the stabilising agent is a cross-linking agent or the stabilising agent releases a cross-linking agent. In one embodiment, the cross-linking agent is formaldehyde or paraformaldehyde. In a particular embodiment, the cross-linking agent is formaldehyde. In one particular embodiment, the stabilising agent is imidazolidinyl urea.

[0298] The term “native cell free nucleosomes” may refer to cell free nucleosomes which have not been stabilised, for example cell free nucleosomes which have not been cross-linked. The second body fluid sample may be provided by any suitable method, for example by collecting the sample in an EDTA blood collection tube.

[0299] The DNA fragments may be analysed by any method described herein. In some embodiments, the step of analysing the DNA fragments comprises: (i) isolating the cell free nucleosomes from the supernatant sample; (ii) optionally, extracting and / or purifying DNA fragments which are bound to or associated with the isolated cell free nucleosomes; and (iii) analysing the DNA fragments (e.g. detecting, measuring, or quantifying the DNA fragments or sequencing the DNA fragments).

[0300] The cell free nucleosomes may be isolated by any suitable method described herein or known in the art. In one embodiment, the supernatant sample is contacted with a nucleosome binding agent which binds to or is bound to a solid support, such as a magnetic particle, a microplate, or a plate. In one embodiment, the supernatant sample is contacted with magnetic particles coated with a nucleosome binding agent and isolated using the magnetic particles. In one embodiment, the DNA fragments which are bound to or associated with the (isolated) stabilised cell free nucleosomes are extracted and / or purified. The DNA fragments may be extracted and / or purified by any suitable method described herein or known in the art. In some embodiments, the method comprises quantifying the DNA fragments which are bound to or associated with the stabilised cell free nucleosomes in the first supernatant sample and the DNA fragments which are bound to or associated with the native cell free nucleosomes in the second supernatant sample. There are many methods known in the art to quantify DNA, for example ultra-violet spectroscopy measurements, electrophoretic methods, and methods involving coloured or fluorescent DNA binding or intercalating dyes. In some embodiments, the DNA fragments which are bound to or associated with the stabilised cell free nucleosomes are quantified by a fluorometer using a fluorescent DNA intercalating dye (e.g. by a Qubit fluorometer).

[0301] In some embodiments, the step of analysing the DNA fragments is used to determine the relative stability of the stabilised and native cell free nucleosomes in body fluid samples (e.g. blood samples) from a subject. The relative stability may be used as a biomarker for health and disease, for example to diagnose cancer in a subject, determine the prognosis of a subject with a cancer, or monitor the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer. The relative stability may be used to diagnose an inflammatory condition or disease in a subject, determine the prognosis of a subject with an inflammatory condition or disease, or monitor the efficacy of a therapy in a subject having, suspected of having, or being predisposed to an inflammatory condition or disease.

[0302] In some embodiments, the difference in DNA fragments quantified from the first sample and the second sample is used as a biomarker for health or disease. In some embodiments, the difference in DNA fragments quantified from the first sample and the second sample is used as a biomarker for cancer. In some embodiments, the difference in DNA fragments quantified from the first sample and the second sample is used as a biomarker for an inflammatory condition or disease. For example, the subject may be diagnosed as having cancer if the concentration of DNA fragments in the first sample is two times or greater, three times or greater, four times or greater, or five times or greater than the second sample. For example, the subject may be considered to have an inflammatory condition or disease if the concentration of DNA fragments in the second sample is two times or greater than the first sample. For example, the subject may be considered healthy if the concentration of DNA fragments in the first sample and the second sample are to significantly different, e.g. about ±50%, about ±20% or about ±10%. According to another aspect of the present invention there is provided a method for the identification of cell free nucleosomes in a blood or other body fluid sample as healthy, inflammatory or cancerous in nature, comprising the steps of:

[0303] (i) determining the recovery of nucleosome associated cfDNAfrom native cell free nucleosomes isolated from a blood or other body fluid sample;

[0304] (ii) determining the recovery of nucleosome associated cfDNAfrom stabilised (e.g. cross-linked) cell free nucleosomes isolated from a blood or other body fluid sample; and

[0305] (iii) using the measured recoveries to characterize the cell free nucleosomes present in the samples as healthy, inflammatory or cancerous in nature.

[0306] According to another aspect of the present invention there is provided a method for the identification of cell free nucleosomes in a blood or other body fluid sample as inflammatory or cancerous in nature, comprising the steps of:

[0307] (i) determining the recovery of nucleosome associated cfDNAfrom native cell free nucleosomes isolated from a blood or other body fluid sample;

[0308] (ii) determining the recovery of nucleosome associated cfDNAfrom stabilised (e.g. cross-linked) cell free nucleosomes isolated from a blood or other body fluid sample; and

[0309] (iii) using the measured recoveries to characterize the cell free nucleosomes present in the samples as inflammatory or cancerous in nature.

[0310] According to anotheraspect of the present invention there is provided a method for diagnosing or detecting a cancer in a subject, determining the prognosis of a subject with a cancer, or for monitoring the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer, the method comprising the steps of:

[0311] (i) obtaining two whole blood samples from a subject and contacting one of the blood samples with a stabilising agent, and optionally separating plasma or serum from the two blood samples;

[0312] (ii) analysing (e.g. quantifying) the cfDNA present in the native and stabilised blood, plasma or serum samples obtained in step (i);

[0313] (iii) contacting the native and stabilised blood, plasma or serum samples obtained in step (i) with a nucleosome binding agent and isolating cell free nucleosomes;

[0314] (iv) analysing (e.g. quantifying) the cfDNA present in the native and stabilised blood, plasma or serum cell free nucleosomes isolated in step (iii); and (v) using the native and stabilised cfDNA measurements or analyses to indicate the presence of a cancer in the subject, the prognosis of a cancer in the subject, or to monitor the efficacy of a therapy in the subject.

[0315] In another aspect of the invention there is provided the stability of cell free nucleosomes in a body fluid sample (e.g. whole blood) as a biomarker for health or disease.

[0316] In another aspect of the invention there is provided the stability of cell free nucleosomes in a body fluid sample (e.g. whole blood) as a biomarker for cancer.

[0317] In another aspect of the invention there is provided the stability of cell free nucleosomes in a body fluid sample (e.g. whole blood) as a biomarker for an inflammatory condition or disease.

[0318] In another aspect of the invention there is provided the relative stability of native and stabilized (e.g. crosslinked) cell free nucleosomes in a body fluid sample (e.g. whole blood) as a biomarker for health or disease.

[0319] In another aspect of the invention there is provided the relative stability of native and stabilized (e.g. crosslinked) cell free nucleosomes in a body fluid sample (e.g. whole blood) as a biomarker for cancer.

[0320] In another aspect of the invention there is provided the relative stability of native and stabilized (e.g. crosslinked) cell free nucleosomes in a body fluid sample (e.g. whole blood) as a biomarker for an inflammatory condition or disease.

[0321] As used herein, an “inflammatory condition or disease” may refer to a condition or disease in which the immune system attacks the body's own tissues, resulting in inflammation. The present invention is not limited to any specific inflammatory condition or disease. In some embodiments, the inflammatory condition or disease is a systemic inflammatory condition including sepsis or SIRS (systemic inflammatory response syndrome). In some embodiments, the inflammatory condition is caused by an infection including influenza, COVID, pneumonia, or a urinary tract infection. In some embodiments, the inflammatory condition or disease is a pulmonary inflammatory disease, such as ARDS (acute respiratory distress syndrome). In some embodiments, the inflammatory condition or disease is selected from Crohn’s disease, colitis, rheumatoid arthritis, or polyarthritis. In some embodiments, the inflammatory condition or disease is a gastroenterological inflammatory disease, such as Crohn’s disease or colitis. In some embodiments, the inflammatory condition or disease is arthritis, such as rheumatoid arthritis or polyarthritis.

[0322] The method for determining cell free nucleosome stability in a body fluid sample may be used alone, for example to diagnose cancer or an inflammatory condition or disease, or may be used in combination with any of the other methods of the present invention described herein, for example to detect false positive results. For example, a method for quantifying cell free nucleosomes in a blood sample as described herein may be used in combination with a method for determining cell free nucleosome stability described herein.

[0323] STABILISING AGENTS

[0324] The present invention provides a stabilising agent (e.g. a cross-linking agent) for use in a diagnostic method, wherein the stabilising agent is used in combination with one or more nucleosome binding agents. The stabilising agent may be comprised in a blood collection tube.

[0325] The process of improved cf nucleosome detection begins by contacting the sample with a stabilising agent (which may also be referred to as a “protective agent” or “preservative agent”) containing an active ingredient to maintain the integrity of the nucleosome within the sample. Preferred stabilising agents are cross-linking agents. Such cross-linking agents may include but are not limited to formaldehyde, paraformaldehyde, formalin, glutaraldehyde, urotropine, Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE). In one embodiment the cross-linking agent is formaldehyde or paraformaldehyde. In a particular embodiment the cross-linking agent is formaldehyde.

[0326] In one embodiment the concentration of the stabilising agent prior to dilution by the sample due to the contacting step is between about 0.1 g / ml and about 0.8 g / ml, or between about 0.4 g / ml and about 0.8 g / ml.

[0327] In one embodiment, the concentration of the stabilising agent is less than about 20 mg / ml after dilution in the blood sample.

[0328] In one embodiment the process involves contacting a blood sample with a stabilising agent which releases a cross-linking agent, particularly a formaldehyde releasing agent. Stabilising agents that may be used include, but are not limited to, diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5dimethylhydantoin, dimethylol urea, 2-bromo-2.-nitropropane-1 ,3-diol, oxazolidines, sodium hydroxymethyl glycinate, 5-hydroxymethoxymethyl-1-1aza-3,7- dioxabicyclo[3.3.0]octane, 5-hydroxymethyl-1-1aza-3,7dioxabicyclo[3.3.0]octane, 5- hydroxypoly[methyleneoxy]methyl-1-1aza-3, 7dioxabicyclo [3.3.0]octane, quaternary adamantine, 2-aminoacetic acid or any combination thereof. Preferred stabilising agents, particularly where the cross-linking agent is formaldehyde, are selected from the group consisting of diazolidinyl urea (DU), imidazolidinyl urea (I DU), methenamine, urotropine (hexamethylenetetramine) and any combination thereof. In one particular embodiment the stabilising agent is imidazolidinyl urea.

[0329] Formaldehyde releasing agents have the advantage of maintaining a low equilibrium concentration of free formaldehyde in solution for cross-linking which can be continuously replenished. The formaldehyde releasing agents act as a reservoir for free formaldehyde by disassociation when free formaldehyde is depleted through cross-linking. For example, a 0.6% solution of imidazolidinyl urea will act as reservoir for an equilibrium free formaldehyde concentration of 0.012% at pH6. Similarly, a 0.5% solution of diazolidinyl urea will act as reservoir for an equilibrium free formaldehyde concentration of 0.036% (Emeis et al, 2007).

[0330] The level of free formaldehyde use for cross-linking proteins, nucleosomes and nucleoproteins in cell culture varies widely from approximately 0.1% to 3%. In one embodiment, similar levels may be used for the present invention. In one embodiment, the level of free formaldehyde required for effective cross-linking of cell-free nucleosomes in solution is less than required in cell culture. In one embodiment the level of free formaldehyde used in the sample is in the range 0.01 to 0.05%. In further embodiments, the level of free formaldehyde used in the sample are <0.1 %, or <0.05% or even less than 0.01%. The levels of releasing agent, as well as the conditions, used may be adjusted to produce a suitable level of free formaldehyde in the sample. Similarly, the stabilising agent or releasing agent may be provided as a concentrate to produce a suitable free formaldehyde level after dilution with the sample. For example, the stabilising agent or releasing agent may be provided as a 5x, 10x, 20x, 100x or greater than a 100x concentrate.

[0331] It is common practice in cross-linking in cell culture, to use a quencher to remove excess formaldehyde (or other cross-linker). Quencher moieties, such as glycine, lysine, urea or other quencher (without limitation) may similarly be added to the stabilising reagent for use in the present invention. EDTA used as an anticoagulant also inhibits nuclease enzymes which further helps to prevent nucleosome degradation. Other nuclease inhibitors are known in the art and may also be added to the stabilising reagent. The stabilising reagent to be added to the sample may consist essentially of the formaldehyde releasing agent. It may be at least about 10%, 50%, or even 80% by volume of the formaldehyde releasing agent. For instance, the amount of formaldehyde releasing agent within the stabilising reagent used may be generally about 100 to about 800 grams per litre. The amount of formaldehyde releasing agent within the stabilising reagent may be at least about 25 grams per litre or even 50 grams per litre. The amount of formaldehyde releasing agent within the stabilising reagent may be less than about 1000 grams per litre. For example, the stabilising reagent may comprise about 0.05 to about 0.4 grams per ml of a formaldehyde releasing agent (e.g., IDU). The stabilising reagent may be diluted by addition to the sample. For example, 0.2 ml of the stabilising reagent may be added to approximately 10ml of whole blood or other sample.

[0332] The stabilising reagent may include a nuclease inhibitor in a suitable amount (e.g. about 10% to 50% by weight) to prevent DNase activity from decreasing the quality and amount of nucleosomes present in a sample as compared with a sample that does not include a nuclease inhibitor. Nuclease inhibitors that may be used include, but are not limited to diethyl pyrocarbonate, ethanol, aurintricarboxylic acid (ATA), formamide, vanadyl-ribonucleoside complexes, macaloid, ethylenediamine tetraacetic acid (EDTA), heparin, hydroxylamine- oxygen-cupric ion, bentonite, ammonium sulphate, dithiothreitol (DTT), beta-mercaptoethanol, cysteine, dithioerythritol, tris (2-carboxyethyl) phosphene hydrochloride, or a divalent cation such as Mg2+, Mn2+, Zn2+, Fe2+, Ca2+, Cu2+ or any combination thereof. Further, the stabilising reagent may be substantially free of guanidinium salts, sodium dodecyl sulphate (SDS), or any combination thereof.

[0333] The chemistry of cross-linking by formaldehyde is well known in the art. Following reaction with formaldehyde, the cross-linked moieties are connected by a CH2 group with mass 14 Daltons that replaces a hydrogen atom in each of the linked moieties (2 Daltons) leading to an observed mass increase of 12 Daltons which can be detected by mass spectrometry.

[0334] Cell-Free DNA blood collection tubes (BCT) available from STRECK Inc contain a formaldehyde releasing agent. Mass spectrometry has been used to demonstrate that proteins, including histone components of nucleosomes, are cross-linked in whole blood collected in Streck Cell-Free DNA BCT (EP 2814981 B1 , Roth et al', 2023 and Dhondt et al', 2023). S-Monovette cfDNA Exact blood collection tubes available from Sarstedt (US2021310045 A1) contain the formaldehyde releasing agent hexamethylenetetramine. A hexamethylenetetramine stabilising reagent may be produced by adding 18% (wt / vol) hexamethylenetetramine to a 0.5M citrate buffered solution pH 4.2. The stabilising reagent may be used by addition to whole blood to a final level of 10% (eg; 1 ml stabiliser added to 9ml whole blood).

[0335] Streck or Sarstedt cfDNA tubes may therefore be useful for implementing methods of the present invention.

[0336] The initial contacting of the blood sample may be for a time sufficient to inhibit one or both of nucleosome degradation, nuclease activity, or any combination thereof. Contacting may occur for at least about 10 seconds, more preferably at least about 1 minute, still more preferably at least about 2 minutes. Contacting can occur for longer periods of time. For example, contacting may be commenced substantially contemporaneously from the time of blood draw (e.g., immediately on blood draw or within less than about 10 minutes of the blood draw) and it may last until nucleosomes are isolated, and / or tested. The contacting step may also be employed to provide a sample with a longer shelf life. Thus, it is possible that a lapse of time of at least about 2 hours, more preferably at least about 6 hours, at least about 24 hours, at least about 7 days or even at least about 14 days can elapse between the time of blood draw (which may be substantially contemporaneous with the contacting step), and the time of any testing or screening of the sample, and / or isolation of the nucleosomes.

[0337] The stabilising reagent may comprise an active agent in solution. Suitable solvents include water, saline, dimethylsulfoxide, alcohol and mixtures thereof. The stabilising reagent may comprise diazolidinyl urea (DU) and / or imidazolidinyl urea (IDU) in a buffered salt solution. The stabilising reagent may further comprise EDTA and 2-aminoacetic acid. Alternatively, the stabilising reagent may contain only a fixative (e.g., an active ingredient) and may be free of any additional additives.

[0338] The amount of any active stabilising ingredient within the stabilising reagent may generally be about 10% to about 90% by weight. The active ingredient or fixative may comprise about 70% to about 90% by weight of the stabilising reagent. The stabilising reagent may further contain an anticoagulant such as about 5% to about 20% by weight EDTA. The stabilising reagent may contain about 10% by weight EDTA. The stabilising reagent may include from about 1% to about 40% by weight of nuclease inhibitor. The amount of active ingredient or cross-linking agent (e.g. the formaldehyde releaser) relative to the amount of EDTA may be about 1 to about 10 parts (more preferably about 2 to about 8 parts) by weight of fixative to about 1 part by weight EDTA. The amount of stabilising reagent within a tube prior to blood draw may be about 0.05 to about 1 .0 ml and more preferably about 0.1 to about 0.3 ml.

[0339] The combination of an active ingredient or cross-linking agent (e.g. the formaldehyde releaser) and anticoagulant within the stabilising reagent results in improved ability to maintain the amount and quality of cf nucleosomes within a blood sample.

[0340] The following illustrates how a blood collection method, in accordance with the present teachings, can preserve cf nucleosomes. Blood samples may be drawn from patients into blood collection tubes containing a nucleosome stabilising agent as taught herein and kept at ambient temperature. For example, the stabilising agent may be in the form of a protective or cross-linking agent and may include about 500 g / L IDU and optionally about 81 g / L Tripotassium EDTA, and about 47 g / l glycine. The stabilising reagent of the present teachings may be placed within a tube so that the tube contains about 0.2-0.5 ml (e.g. about 0.20 ml) of the stabilising reagent. The tube containing the stabilising reagent may receive about 10 ml of patient blood. The patient blood may be drawn directly into the tube containing the stabilising reagent. It is believed that results shown will vary by about ± 25% of that described across a range of about 300 to about 700 g / L IDU (with similar results expected for other formaldehyde releasers described herein) and from about 60 to about 100 g / L Tripotassium EDTA, and about 20 to about 60 g / L glycine. The stabilising reagent may include roughly about 6 parts by weight IDU per about 1 part by weight EDTA, and roughly about 10 parts by weight IDU per about 1 part glycine. The stabilising reagent may include about 80% by volume of IDU, 12.8% by volume Tripotassium EDTA, and 7.25 by volume glycine.

[0341] In one embodiment the cross-linker releasing agent is hexamethylenetetramine (urotropine). The use of this formaldehyde releasing agent to stabilise white blood cells in whole blood is described in US2021310045, which is herein incorporated by reference. An example formulation of a hexamethylenetetramine-based stabilising reagent is 18% (w / v) hexamethylenetetramine in 0.5 M sodium citrate buffer pH 4.2. The stabilising reagent may be used by addition to whole blood at a 1 :10 dilution (e.g. 0.5ml stabilising reagent added to 4.5ml whole blood). The stabilising reagent may be located within a specialized device. The device may be a sample collection container, for example a blood collection tube or an evacuated blood collection tube. Examples, without limitation, of commercially available tubes in accordance with the present teachings containing EDTA as anticoagulant and a formaldehyde releasing agent stabiliser are sold under the name Cell-Free DNA BCT by Streck, Inc., Omaha, Nebraska and S-Monovette® cfDNA Exact by Sarstedt AG of Germany.

[0342] Formaldehyde crosslinking inhibits enzyme activity. In a further aspect of the invention there is provided the use of formaldehyde or a formaldehyde releasing agent as a nuclease or protease inhibitor in a blood collection tube. The coagulation cascade is a serial enzyme process which is inhibited at each step by crosslinking. In a further aspect of the invention there is provided the use of formaldehyde or a formaldehyde releasing agent as an anticoagulant in a blood collection tube. In a further aspect of the invention there is provided a blood collection tube that contains a nucleosome stabilising agent with no requirement for a (further) anticoagulant agent.

[0343] Formaldehyde crosslinking crosslinks all chromatin present in a blood collection tube. NETosis occurs in whole blood collected in serum (but not plasma) blood collection tubes and this rapidly adds contaminant cell derived nucleosomes (and DNA) to serum. In a further aspect of the invention there is provided a blood collection tube that contains a crosslinking agent or a crosslinking releasing agent that both stabilises cell free nucleosomes and prevents contamination by NETosis.

[0344] In one embodiment the blood collection tube contains an amount or quantity of formaldehyde (or formaldehyde solution) to enable a final formaldehyde level of approximately 0.1% in the whole blood collected (for example, a 10ml blood collection tube containing 0.1 ml of a 10% formaldehyde solution would enable a final formaldehyde level of approximately 0.1 % in the blood sample collected).

[0345] In one embodiment the blood collection tube contains a formaldehyde releasing agent such that the final formaldehyde releasing agent level produced after dilution in the blood sample collected is approximately 0.1 %-5%. This provides a low level of free formaldehyde in the blood sample that is continuously replenished, when consumed by crosslinking, from the reservoir present in the releasing agent. In preferred embodiments the formaldehyde releasing agent is DU, I DU or hexamethylenetetramine (urotropine). In one embodiment the blood collection tube contains formaldehyde or a formaldehyde releasing agent or other nucleosome stabiliser and no additional anticoagulant(s) or enzyme inhibitors. In this embodiment, the separated sample provided following centrifugation of whole blood may be serum or plasma depending on whether or not the stabiliser used also acts as an anticoagulant (for example by crosslinking coagulation cascade factors and thereby preventing their function). If the stabiliser selected for use also acts as an anti-coagulant, the separated sample will be plasma. If the stabiliser selected for use is not also an anti-coagulant, the separated sample will be serum. Normal serum collection tubes (without a stabiliser reagent) are normally considered unsuitable for collection of blood samples for the analysis of nucleosomes or DNA, because clotting may induce the production of neutrophil extracellular traps (NETs) by white blood cells. NETs material consists of polynucleosomes and DNA and so interferes in nucleosome and DNA analysis. However, the extracellular release of NETs requires intracellular chromatin unwinding and cell lysis which are prevented by crosslinking, enabling the use of formaldehyde crosslinked serum for circulating nucleosome or DNA analysis. As crosslinking is known to lead to whole or partial enzyme inactivation, the use of a crosslinking agent will also act as a nuclease and protease inhibitor.

[0346] EDTA blood collection tubes are designed to provide a final concentration of approximately 5mg / ml in the blood sample collected. The actual concentration present in a blood sample will vary with the volume of blood added to the tube, for example between approximately 3- 8mg / ml.

[0347] In one embodiment the blood collection tube contains formaldehyde or a formaldehyde releasing agent or other nucleosome stabiliser and sufficient EDTA to enable a final level of approximately 3-8mg / ml in the whole blood collected.

[0348] According to a further aspect of the invention, there is provided the use of a stabilised nucleosome in a body fluid sample as a biomarker for cancer. According to another aspect of the invention, there is provided the use of a cross-linked nucleosome in a body fluid sample as a biomarker for cancer.

[0349] The stabilised (e.g. cross-linked) nucleosomes may be used according to the methods of the invention as described herein.

[0350] BLOOD COLLECTION TUBES According to a further aspect of the invention, there is provided the use of a receptacle for the collection of a body fluid sample and analysing, detecting, measuring or quantifying stabilised (e.g. crosslinked) cell free nucleoproteins, wherein the receptacle contains a stabilising agent (e.g. a crosslinking agent or a crosslinker releasing agent).

[0351] In a preferred embodiment the receptacle is a blood collection tube or an evacuated blood collection tube. In a preferred embodiment the stabilising agent is IDU. In one embodiment the receptacle is a glass receptacle. In one embodiment the receptacle is a plastic receptacle. In one embodiment the receptacle contains IDU and water only. In one embodiment the IDU concentration provided in the receptacle is sufficient to provide a final concentration after dilution with the sample of about 1 % or higher. In further embodiments the receptacle contains an anticoagulant and / or other or further solvents, and / or enzyme inhibitors and / or detergents as well as a stabilising agent (e.g. a crosslinking agent or crosslinker releasing agent).

[0352] In a preferred embodiment the stabilised (e.g. crosslinked) cell free nucleoproteins to be analysed are stabilised cell free nucleosomes (e.g. crosslinked cell free nucleosomes). The stabilised (e.g. crosslinked) cell free nucleoproteins may be analysed, detected, measured or quantified by any method described herein. In one embodiment the stabilised (e.g. crosslinked) cell free nucleoproteins are analysed, detected, measured or quantified by an immunochemical method. In a preferred embodiment the stabilised (e.g. crosslinked) cell free nucleoproteins are analysed, detected, measured or quantified by an immunoassay method. In one embodiment the stabilised (e.g. crosslinked) nucleoproteins are analysed, detected, measured or quantified by a mass spectrometry or proteomic method. In one embodiment the stabilised (e.g. crosslinked nucleoproteins) are analysed, detected, measured or quantified by a method comprising extraction and measurement or analysis or sequencing of ChIP isolated crosslinked nucleosome associated cfDNA.

[0353] In some embodiments, the methods of the present invention further comprise a step of preparing a receptacle (e.g. a blood collection tube) including a stabilising agent (e.g. a crosslinking agent or a crosslinker releasing agent). In some embodiments, the methods of the present invention further comprise a step of adding or drawing blood into the receptacle (e.g. blood collection tube). In some embodiments, the methods of the present invention further comprise a step of centrifuging the blood and isolating the supernatant (e.g. serum or plasma) component of the blood. In one embodiment there is provided a method for analysing, detecting, measuring, or quantifying stabilised (e.g. crosslinked) cell free nucleosomes in a blood sample comprising the steps of;

[0354] (i) preparing a receptacle (e.g. a blood collection tube) including a stabilising agent (e.g. a crosslinking agent or a crosslinker releasing agent);

[0355] (ii) adding or drawing blood into the receptacle (e.g. blood collection tube);

[0356] (iii) optionally centrifuging the blood and isolating the supernatant (e.g. serum or plasma) component of the blood; and

[0357] (iv) analysing, detecting, measuring, or quantifying stabilised (e.g. crosslinked) cell free nucleosome in the blood or isolated supernatant (e.g. serum or plasma).

[0358] SEPARATION

[0359] We observed a high sensitivity and specificity for cancer detection with an immunoassay for stabilised (e.g. cross-linked) nucleosomes. To our knowledge, this is the first report of the measurement of stabilised (e.g. cross-linked) plasma cf-nucleosomes.

[0360] Nucleoproteins have evolved for the packing and epigenetic regulation of DNA in the nucleus but are labile and may be degraded, altered in conformation or wholly or partially dissociated outside of the nucleus.

[0361] The effects of crosslinking of endogenous plasma cf-nucleosomes have not been investigated, although, the effects of crosslinking cellular chromatin and nucleosomes have been studied. Studies of crosslinked cellular chromatin indicate a number of potential effects that may lead to an increase or decrease in measured cellular nucleosomes extracted from the nucleus of cells. A decrease in measured levels may arise from interference in the chemistry of the assay, either directly or through alteration of nucleosome conformation at antibody binding sites. Crosslinking of nucleosomes, particularly oligonucleosomes, may lead to precipitation of cellular chromatin or nucleosomes.

[0362] Whole blood samples collected for cell free DNA analysis are liable to contamination with cellular DNA contained inside the nucleus of white blood cells (i.e. DNA that was not cell free at venipuncture), by leakage out of the cells if cell membrane lysis occurs. A number of specialized cfDNA blood collection tubes to preserve cfDNA in whole blood for several days prior to processing at a central laboratory are commercially available. The principle employed is the stabilisation of white blood cells to prevent lysis and leakage of cellular DNA and consequent artifactual contamination of the plasma sample with cellular DNA. We, on the other hand, have investigated the effects of cross linking of cell free proteins, including histones, and cf-nucleosomes in blood. Cell free nucleosomes in free solution in blood may occur as intact cf-nucleosomes, or as degraded cf-nucleosomes. Examples of degraded cf-nucleosomes include clipped cf-nucleosomes (nucleosomes in which some or all histone tails have been removed) and nicked cf-nucleosomes (nucleosomes in which the associated DNA has single and / or double-stranded breaks) or both. However, not all cf- nucleosomes in blood are in free solution but cf-nucleosomes may also occur attached to red and white blood cells by association with the exterior surface membrane (i.e. nucleosomes that were cell free at venipuncture and not inside a cell). Cell free nucleosomes may be associated with membranes by electrostatic attraction. As a result of our investigations, we have provided a method for the enrichment or partitioning of a particular population (i.e. subpopulation) of cf-nucleoproteins according to the source of the nucleoproteins. In other words, we are able to enrich or partition a population of cf-nucleoproteins on the basis of their chemical properties or structure. This method can be achieved irrespective of the amino acid sequence or the sequence of the attached DNA.

[0363] We have identified that circulating nucleoproteins found in free solution in the plasma are distinct from the nucleoproteins attached to a cell membrane (usually as long chains of oligonucleosomes). In practice, circulating cancer derived cf-nucleoproteins may occur preferentially in free solution, whereas cf-nucleoproteins attached to a cell membrane may be preferentially derived from healthy cells. Without wishing to be bound by any theory, cf- nucleoproteins which are stabilised on cell membranes can be fractionated from the stabilised cf-nucleoproteins in circulation or vice versa as crosslinking of cell surface bound cf- nucleosomes covalently attaches them to cell membranes. For example, immunoprecipitation can be used to pull down a particular population of cf-nucleoproteins with a binding agent based on, for example, their epigenetic structure or structural integrity. The remaining populations of nucleoproteins can be separated by techniques such as centrifugation or washing. As nicked and clipped cf-nucleosomes are reported to be more common in the circulation of cancer patients and the stabilised cf-nucleoproteins from circulation are left in the plasma, the present invention provides a means of enriching for a population of cancer- associated cf-nucleoproteins.

[0364] NUCLEOSOME BINDING AGENTS

[0365] The present invention provides one or more nucleosome binding agents for use in a diagnostic method, wherein the one or more nucleosome binding agents are used in combination with a stabilising agent (e.g. a cross-linking agent). The stabilising agent may be comprised in a blood collection tube.

[0366] A nucleosome binding agent may refer to an agent which specifically binds to a nucleosome, including mononucleosomes, oligonucleosomes and polynucleosomes and any protein-DNA chromatin fragments that include a nucleosome. A nucleosome binding agent may comprise an antibody or a fragment thereof, a peptide, an aptamer or an oligonucleotide which is capable of specific binding to a nucleosome. The term “a nucleosome binding agent” used herein may be used interchangeably with “one or more nucleosome binding agents”.

[0367] In preferred embodiments, a nucleosome binding agent comprises an antibody or a fragment thereof. Suitable antibody fragments include an antigen-binding fragment (Fab), a fragment antibody (F(ab’)2), a single chain antibody (scFv), or a single-domain antibody (sdAb). In one embodiment, a nucleosome binding agent comprises a full length antibody. The antibody or fragment thereof may specifically bind to any part of a nucleosome, including the histone DNA- complex, histones, or DNA. In one embodiment, a nucleosome binding agent comprises an anti-nucleosome antibody or fragment thereof, an anti-DNA antibody or fragment thereof, or an anti-histone antibody or fragment thereof. Such antibodies are commercially available and can be readily generated by the skilled person using methods known in the art.

[0368] In one embodiment, the nucleosome binding agent is an anti-nucleosome antibody or fragment thereof. An anti-nucleosome antibody or fragment thereof may specifically bind to histone-DNA complexes. In one embodiment, the anti-nucleosome antibody or fragment thereof is an anti-H2A-H2B-DNA complex antibody or fragment thereof.

[0369] In one embodiment, the nucleosome binding agent is an anti-histone antibody or fragment thereof. An anti-histone antibody or fragment thereof may specifically bind to histones, including histone variants, histone isoforms, and histones with one or more post-translational modifications (histone PTM). Histones H2A, H2B, H3 and H4 are known as core nucleosome histones, while histones H1 / H5 are known as linker histones. In one embodiment, the anti- histone antibody or fragment thereof is an anti-core nucleosome histone antibody or fragment thereof. Histone variants and histone isoforms are described herein. In one embodiment, the anti-histone antibody or fragment thereof is an anti-H3.1 antibody or fragment thereof. Histone PTMs are described herein. In one embodiment, the anti-histone antibody or fragment thereof is an anti-histone PTM antibody or fragment thereof, such as a histone PTM of a core nucleosome, in particular a histone H3 PTM. In one embodiment, the anti-histone antibody or fragment thereof is an anti-H3 citrulline antibody or fragment thereof, an anti-H3K27Me3 antibody or fragment thereof, an anti-H3K36Me3 antibody or fragment thereof, or an anti- H3K9Me3 antibody or fragment thereof.

[0370] In one embodiment, the nucleosome binding agent is an anti-nucleosome antibody or fragment thereof that specifically binds to both clipped and non-clipped nucleosomes. In one embodiment, the nucleosome binding agent is an anti-histone antibody of fragment thereof that specifically binds to both clipped and non-clipped histones. In one embodiment, the inventors used an immunoassay for H3.1 -nucleosomes employing an immobilised anti-histone H3.1 antibody directed to bind to an epitope around amino acids 30-33 of the histone H3.1 protein to capture clipped and non-clipped nucleosomes, optionally together with a labelled anti-nucleosome antibody directed to bind to an epitope present in intact nucleosomes but not present on isolated (free) histone or DNA nucleosome components.

[0371] In one embodiment, the antibody specifically binds to an epitope present in histone H3.1 (an “anti-histone H3.1 antibody”). The amino acid sequence of histone H3.1 is known in the art and is described at UniProt Accession No. P68431. In mammals, histone H3 variants include: H3.1 , H3.2, H3.3, H3t and Centromeric Protein A (CENP-A). Human H3.1 and H3.2 are 99% identical (differing by only one amino acid), whereas histone H3t is 96% identical to H3.1 (differing at four amino acid positions) and histone H3.3 is 96% identical to H3.1 (differing at five amino acid positions). CENP-A shares only 46% identity with H3.1. In one embodiment, the antibody selectively binds histone H3.1 , H3.2 and H3t. It will be understood in this embodiment, the antibody does not bind to other histone H3 isoforms, such as histone H3.3 and CENP-A.

[0372] Nucleosomes are subject to clipping in which the histone tail is physically and irreversibly removed by regulated proteolysis, or clipping. On histone H3, clipping is reported to occur around amino acid position 21 (Yi and Kim (2018) BMB Reports, 51 (5): 211-218). Therefore, in one embodiment, the antibody specifically binds to an epitope located higher than amino acid position 21. This enables capture of both clipped and non-clipped histones / nucleosomes.

[0373] The amino acid sequence of histone H3.1 at positions 27-36 is KSAPATGGVK (SEQ ID NO: 1). The amino acid sequence at positions 29-35 does not include any commonly post- translationally modified amino acids (for example lysine, serine or arginine). Therefore, antibodies directed to bind to this epitope (i.e. amino acid positions 29-35) are unaffected, or minimally affected, by the post-translational modification status of the nucleosome, and will bind to all or most nucleosomes containing histone H3.1 , regardless of PTM structure. In one embodiment, the anti-histone H3.1 antibody specifically binds to an epitope comprising amino acids 30-33 (PATG, SEQ ID NO: 2) of histone H3.1. In another embodiment, the anti-histone H3.1 antibody specifically binds to an epitope comprising amino acids 28-32 (SAPAT, SEQ ID NO: 3) of histone H3.1. In particular embodiments, the epitope is in or around amino acid 31 of histone H3 which is an alanine in H3.1 , but a serine in H3.3. Binding in these regions ensures that both intact and clipped histones / nucleosomes are captured by the antibody regardless of their PTM status. This maximises the capture of H3.1-nucleosomes.

[0374] In one embodiment, the anti-nucleosome antibody specifically binds to an epitope present in intact nucleosomes. The antibody does not bind (or binds weakly) to free histone octamer complexes, free histones (i.e. without DNA), free DNA or free histones. Again, the antibody may be relatively unaffected by the histone PTM composition of the nucleosomes to be bound. This type of epitope may be referred to as a “conformational nucleosome epitope” herein because it requires the native three-dimensional configuration of the target nucleosome to be intact.

[0375] Therefore, in one embodiment of the invention the antibody is directed to bind to a conformational nucleosome epitope present in intact nucleosomes containing a histone octamer core complexed with DNA.

[0376] In one embodiment, the nucleosome binding agent is an anti-DNA antibody or fragment thereof. An anti-DNA antibody or fragment thereof may specifically bind to the DNA of histone- DNA complexes. In one embodiment, the anti-DNA antibody or fragment thereof is an anti- double-stranded (ds)DNA antibody or fragment thereof.

[0377] In one embodiment, the nucleosome binding agent comprises a detectable label. A nucleosome binding agent comprising a detectable label may be used to analyse, detect, measure or quantify the stabilised cell free nucleosomes. In one embodiment, the nucleosome binding agent may be an antibody or a fragment thereof linked to a detectable label. In one embodiment, the nucleosome binding agent is an anti-nucleosome antibody or fragment thereof, an anti-DNA antibody or fragment thereof, or an anti-histone antibody or fragment thereof linked to a detectable label.

[0378] Detectable labels or detectable markers are used in immunoassays to generate a measurable signal that indicates the binding of an antibody or other specific binder to an antigen. Labels can be detected in a variety of ways, including colour change, luminescence, fluorescence, or radiation emission (see e.g. Darwish, LA., 2006. International journal of biomedical science: UBS, 2(3), p.217). Any suitable detectable label may be used, for example an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP); a luminescent label, such as an acridinium ester derivative; a fluorescent label; or a radioactive label. In one embodiment, the detectable label is an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). In one embodiment, the detectable label is a luminescent label, such as an acridinium ester derivative.

[0379] In one embodiment, the nucleosome binding agent is an anti-nucleosome antibody linked to a detectable label (e.g. an acridinium ester derivative or a horseradish peroxidase).

[0380] In one embodiment, the nucleosome binding agent is an anti-DNA antibody linked to a detectable label (e.g. a horseradish peroxidase).

[0381] In one embodiment, the nucleosome binding agent is an anti-histone antibody linked to a detectable label.

[0382] In one embodiment, the nucleosome binding agent comprises a nucleosome binding agent which binds to or is bound to a solid support. A nucleosome binding agent which binds to or is bound to a solid support may be used to isolate the stabilised cell free nucleosomes for analysing, detecting, measuring or quantifying. In one embodiment, the nucleosome binding agent may comprise an antibody or a fragment thereof and binds to or is bound to a solid support. In one embodiment, the nucleosome binding agent comprises an anti-nucleosome antibody or fragment thereof, an anti-DNA antibody or fragment thereof, or an anti-histone antibody or fragment thereof and binds to or is bound to a solid support. The solid support or solid phase may be any which is suitable for immobilising the nucleosome binding agent. For example, a magnetic particle, a microplate, or a plate.

[0383] In one embodiment, the nucleosome binding agent binds to or is capable of binding to a solid support. For example, a specific functional group may be introduced into the nucleosome binding agent (e.g. biotin) to enable binding to a solid support (e.g. streptavidin) (see e.g. Kusnezow, W. and Hoheisel, J.D., 2003. Journal of molecular recognition, 16(4), pp.165-176). In one embodiment, the nucleosome binding agent is a biotinylated antibody or fragment thereof. In one embodiment, the nucleosome binding agent is a biotinylated anti-nucleosome antibody or fragment thereof, a biotinylated anti-DNA antibody or fragment thereof, or a biotinylated anti-histone antibody or fragment thereof.

[0384] In one embodiment, the nucleosome binding agent is a biotinylated anti-histone antibody.

[0385] In one embodiment, the nucleosome binding agent is bound to a solid support. For example, the nucleosome binding agent may be in the form of a magnetic particle, microplate or plate coated with an antibody or fragment thereof (see e.g. Galkin, O.Y., et al., 2018. Regulatory Mechanisms in Biosystems, 9(1), pp.47-55 and Kusnezow, W. and Hoheisel, J.D., 2003. Journal of molecular recognition, 16(4), pp.165-176).

[0386] In one embodiment, the nucleosome binding agent is a magnetic particle coated with an antinucleosome antibody or fragment thereof, a magnetic particle coated with an anti-DNA antibody or fragment thereof, or a magnetic particle coated with an anti-histone antibody or fragment thereof.

[0387] In one embodiment, the nucleosome binding agent is a magnetic particle coated with an anti- histone antibody (e.g. an anti-H3.1 antibody, an anti-H3K27Me3, or anti-H3K36Me3 antibody).

[0388] In one embodiment, the nucleosome binding agent is a magnetic particle coated with an antinucleosome antibody or fragment thereof, a magnetic particle coated with an anti-DNA antibody or fragment thereof, or a magnetic particle coated with an anti-histone antibody or fragment thereof.

[0389] In one embodiment, the nucleosome binding agent is a microplate or plate coated with the anti-histone antibody (e.g. an anti-H3.1 antibody).

[0390] In some embodiments, two or more different nucleosome binding agents are used in combination. For example, a first nucleosome binding agent comprising a detectable label may be used in combination with a second nucleosome binding agent which binds to or is bound to a solid support. For example, the methods of the present invention may comprise contacting the plasma or serum sample with a first nucleosome binding agent comprising a detectable label and / or a second nucleosome binding agent which binds to or is bound to a solid support. In one embodiment, the methods of the present invention comprise contacting the plasma or serum sample with a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support.

[0391] The combination of first and second nucleosome binding agents may be used to isolate the stabilised cell free nucleosomes from the plasma or serum sample and analyse, detect, measure or quantify the stabilised cell free nucleosomes. The first nucleosome binding agent comprising a detectable label may be used to analyse, detect, measure or quantify the stabilised cell free nucleosomes. The second nucleosome binding agent which binds to or is bound to a solid support may be used to isolate the stabilised cell free nucleosomes from the plasma or serum sample.

[0392] In preferred embodiments, the first and second nucleosome binding agents each comprise an antibody or a fragment thereof. In more preferred embodiments, the first and second nucleosome binding agents each comprise an antibody. In one embodiment, the first and second nucleosome binding agents bind different parts of a nucleosome. In one embodiment, the first nucleosome binding agent is an anti-nucleosome antibody or an anti-DNA antibody linked to a detectable label and the second nucleosome binding agent is an anti-histone antibody which binds to or is bound to a solid support.

[0393] In one embodiment, the first nucleosome binding agent is an anti-nucleosome antibody linked to a detectable label (e.g. an acridinium ester derivative) and the second nucleosome binding agent is an anti-histone antibody (e.g. an anti-H3.1 antibody, an anti-H3K27Me3, or anti- H3K36Me3 antibody) which binds to or is bound to a solid support (e.g. a magnetic particle coated with the anti-histone antibody).

[0394] In one embodiment, the first nucleosome binding agent is an anti-nucleosome antibody linked to a detectable label (e.g. a horseradish peroxidase) and the second nucleosome binding agent is an anti-histone antibody (e.g. an anti-H3.1 antibody) which binds to or is bound to a solid support (e.g. a microplate or plate coated with the anti-histone antibody).

[0395] In one embodiment, the first nucleosome binding agent is an anti-DNA antibody linked to a detectable label (e.g. a horseradish peroxidase) and the second nucleosome binding agent is an anti-histone antibody which binds to or is bound to a solid support (e.g. a biotinylated anti- histone antibody). ANTICOAGULATION

[0396] The two main sample types derived from whole blood for analysis of circulating biomarkers are serum and plasma. Serum is not normally used for cfDNA analysis because the coagulation of blood in the tube post venipuncture is associated with the release of neutrophil extracellular traps (NETs) that comprise nucleosomes and DNA in a process termed NETosis. NETosis artifactually contaminates serum with cellular nucleosomes and DNA, impairing or preventing the analysis of cell free nucleosomes or DNA. However, serum can be used in the present invention.

[0397] Plasma is usually a convenient sample and EDTA plasma is the sample matrix of choice for the analysis of circulating cfDNA in most laboratories. EDTA plasma may be used without further additives as sample matrix for cfDNA. However, the cells present in a blood sample begin to perish within a few hours of venepuncture and start to release cellular genomic DNA. As a result, the original cfDNA portion in the plasma is contaminated by released cellular DNA and cfDNA analysis is impaired or not possible. For this reason, many laboratories use plasma prepared from whole blood collected in specialized cfDNA blood collection tubes (BCTs) designed to stabilise blood cells. These cfDNA BCTs comprise a tube containing a stabiliser in addition to an anticoagulant (typically EDTA). The whole blood of a subject is added to the tube for later processing, isolation of plasma and cfDNA analysis. CfDNA BCTs are available commercially including, for example, those available from Streck (cfDNA BCT), Roche Diagnostics (Cell-Free DNA Collection Tube), Qiagen (PAXgene Blood ccfDNA Tube), Magen Biotech (Cell-Free DNA Blood Collection Tube) and Sarstedt (S-Monovette® cfDNA Exact).

[0398] The anticoagulant commonly used is EDTA but other anticoagulants, for example citrate or heparin, may be used. A variety of stabilisers may be used including detergents and crosslinking agents. Detergents may stabilise cell surface membranes preventing cell rupture or lysis and preventing contamination of cfDNA by cellular genomic DNA derived from the mitochondria or nuclei of ruptured blood cells. Crosslinking agents stabilise cell membranes by crosslinking and also crosslink cell constituents more widely including chromatin. The crosslinker commonly used is formaldehyde. Typically, formaldehyde is not added to the tube directly, but in the form of a formaldehyde releasing agent such as those described above.

[0399] Membrane stabilisation enables whole blood to be stored for up to 2 weeks without contamination enabling the collection of blood at centres convenient to patients for transport to a distant central laboratory for processing by centrifugation and analysis of cfDNA. To further stabilise whole blood, inhibitors of protease and nuclease enzymes are also typically added to the BCT (in addition to the cell membrane stabiliser).

[0400] Typical cfDNA BCTs comprise an anticoagulant (usually EDTA), a membrane stabiliser and one or more enzyme inhibitors.

[0401] However, as we have demonstrated here, production of plasma through use of anticoagulants based on chelation of bivalent calcium ions (for example citrates, acid citrate dextrose, citrate phosphate dextrose, oxalates, EDTA, EGTA) is suboptimal because it may destabilise some nucleoproteins and lead to redistribution of the equilibrium between cell surface bound and free solution nucleosomes. Other anticoagulants, such as heparin (a family of highly sulphated polysaccharide chains) bind to nucleosomes through electrochemical binding of negatively charged heparin sulphate residues with positive histone structures, causing interference in nucleosome measurements. These anticoagulants also are not used in samples for cfDNA analysis as they interfere in cfDNA extraction from plasma.

[0402] There is a clear need for a plasma sample matrix which contains an anticoagulant that does not chelate bivalent metal ions or bind to nucleosomes or nucleoproteins.

[0403] Therefore, in a further aspect of the invention, there is provided an anticoagulation additive for the preparation of plasma which does not chelate bivalent metal ions and does not include negatively charged sulphate groups.

[0404] The mechanism of coagulation of blood is complex and described by a multi-step coagulation cascade involving 12 principle coagulation factors. The cascade involves a series of steps, in which each step is triggered by the preceding one, resulting eventually in the release of fibrin from fibrinogen by thrombin and the formation of a fibrin based clot. Anticoagulants interrupt the cascade at one or more steps and inhibit clot formation. Calcium ions are a coagulation factor responsible for complete activation of several other coagulation factors and their sequestration (eg; by EDTA) inhibits fibrin clot formation. Antithrombin III is a protein that inhibits coagulation through inactivation of thrombin. Heparin binding to antithrombin III greatly increases the inhibition of coagulation.

[0405] It is clear that interruption of the coagulation cascade prevents fibrin clot formation. The principle underlying this aspect of the current invention is the interruption of the coagulation cascade by use of a cross linker. The addition of a crosslinker to a whole blood sample crosslinks protein components of the sample. Crosslinking of all the numerous enzyme and other coagulation factors present in a whole blood sample interrupts the coagulation cascade at one or more steps and prevents fibrin clot formation.

[0406] The use of a crosslinker as an anticoagulant enables the preparation of a plasma sample containing crosslinked nucleosomes or other plasma components without the need for any further anticoagulant moiety.

[0407] In preferred embodiments of the invention the anticoagulant additive is a crosslinker. In preferred embodiments the anticoagulant additive is formaldehyde or a formaldehyde releasing agent.

[0408] It will be understood that the function of other enzymes, such as nucleases and proteases is also inhibited by crosslinking. Moreover, crosslinking agents are effective antibacterial, antiviral and antimicrobial preservatives.

[0409] Therefore, in a further aspect of the invention there is provided a crosslinking agent added to, or used as a component of, a blood collection tube which has multiple functions including acting simultaneously as an anticoagulant, an inhibitor of enzyme activity, an antimicrobial preservative and a stabiliser of nucleosomes, nucleoproteins and other proteins.

[0410] In one embodiment of the invention there is provided an additive to be added to a blood collection tube or to a whole blood sample, wherein said additive comprises a crosslinker and no further anticoagulant is added or present in the sample or tube.

[0411] In preferred embodiments the crosslinker in the additive is formaldehyde or a formaldehyde releasing agent.

[0412] In another embodiment of the invention there is provided a blood collection tube containing an additive comprising a crosslinker and no further anticoagulant.

[0413] In preferred embodiments the crosslinker in the additive is formaldehyde or a formaldehyde releasing agent.

[0414] In another aspect of the invention there is provided a receptacle (e.g. a blood collection tube) containing a stabilising agent (e.g. a crosslinking agent or a crosslinker releasing agent), wherein the stabilising agent does not act as an anticoagulant and no other anticoagulant is used. In this aspect, the sample preparation will produce a serum sample that is suitable for use in methods of the invention because all cells in the sample, including all intracellular chromatin and nucleoproteins, are fixed and no cellular chromatin is released into the liquid serum.

[0415] ANALYSIS OF DNA FRAGMENTS

[0416] In some embodiments of the present invention, the DNA fragments which are bound to or associated with the cell free nucleosomes are analysed. The stabilising agent (e.g. crosslinking agent) may be used to stabilise histone-DNA complexes prior to analysing, detecting, measuring or quantifying nucleosome associated DNA fragments.

[0417] The present invention provides a method for analysing DNA fragments which are bound to or associated with the cell free nucleosomes in a blood sample, comprising the steps of:

[0418] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent; and

[0419] (ii) analysing the DNA fragments which are bound to or associated with the stabilised cell free nucleosomes.

[0420] The present invention also provides a method for analysing DNA fragments which are bound to or associated with the cell free nucleosomes in a blood sample, comprising the steps of:

[0421] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;

[0422] (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains stabilised cell free nucleosomes; and

[0423] (iii) analysing the DNA fragments which are bound to or associated with the stabilised cell free nucleosomes.

[0424] Analysing, detecting, measuring or quantifying the DNA fragments may be used to diagnose cancer in a subject, determine the prognosis of a subject with a cancer, or monitor the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer. As described above, Sanchez et al (2018), demonstrated that the nucleosome associated cfDNA of cancer patients is more highly fragmented than that of healthy subjects including short DNA fragments of less than 145bp down to 60bp in length. The present invention also provides a method of the identification of a characteristic of cancer, comprising the steps of:

[0425] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent); and

[0426] (ii) analysing DNA fragments which are bound to or associated with the cell free nucleosomes to identify a cancer characteristic.

[0427] The present invention also provides a method for stabilising, storing and analysing DNA fragments which are bound to or associated with the cell free nucleosomes for a characteristic of cancer, comprising the steps of:

[0428] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent);

[0429] (ii) separating plasma or serum from the blood sample; and

[0430] (iii) analysing the plasma or serum for DNA fragments having a characteristic of cancer which are bound to or associated with the cell free nucleosomes.

[0431] The present invention also provides a method for diagnosing or detecting a cancer, comprising the steps of:

[0432] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent);

[0433] (ii) separating plasma or serum from the blood sample;

[0434] (iii) analysing the DNA fragments which are bound to or associated with the cell free nucleosomes; and

[0435] (iv) using the DNA fragments analysed to diagnose the subject with cancer.

[0436] The present invention further provides a method for determining the prognosis of a subject with a cancer, comprising the steps of:

[0437] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent);

[0438] (ii) separating plasma or serum from the blood sample;

[0439] (iii) analysing the DNA fragments which are bound to or associated with the cell free nucleosomes; and

[0440] (iv) using the DNA fragments analysed as indicative of the prognosis of said cancer. The present invention additionally provides a method for monitoring the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer, comprising the steps of:

[0441] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent);

[0442] (ii) separating plasma or serum from the blood sample;

[0443] (iii) analysing the DNA fragments which are bound to or associated with the cell free nucleosomes; and

[0444] (iv) comparing the DNA fragments analysed with an earlier plasma sample or serum sample taken from said subject to determine the efficacy of said therapy.

[0445] The present invention additionally provides a method for diagnosing or detecting a cancer, comprising the steps of:

[0446] (i) contacting a blood plasma or serum sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent);

[0447] (ii) analysing the DNA fragments which are bound to or associated with the cell free nucleosomes; and

[0448] (iii) using the DNA fragments analysed to diagnose the subject with cancer.

[0449] The present invention additionally provides a method for diagnosing or detecting a cancer, comprising the steps of:

[0450] (i) contacting a blood plasma or serum sample that contains cell free nucleosomes with a stabilising agent (e.g. a crosslinking agent) and analysing the DNA fragments which are bound to or associated with the cell free nucleosomes; and

[0451] (ii) using the DNA fragments analysed to diagnose the subject with cancer.

[0452] According to a further aspect, there is provided a method of treating a cancer in a subject, which comprises the following steps:

[0453] (i) obtaining a blood sample which contains cell free nucleosomes from the subject;

[0454] (ii) contacting the nucleosomes or the blood sample with a stabilising agent;

[0455] (iii) analysing the DNA fragments which are bound to or associated with the cell free nucleosomes;

[0456] (iv) using the DNA fragments analysed as indicative of the presence of said cancer in the subject; and

[0457] (v) treating surgically or administering a therapeutic agent if the subject is determined to have said cancer in step (iv), optionally the treatment is selected from one or more of: surgery, chemotherapy, immunotherapy, hormone therapy, biological therapy and radiotherapy.

[0458] In one embodiment, the step of analysing the DNA fragments comprises: (i) isolating the stabilised cell free nucleosomes from the plasma or serum sample; (ii) optionally, extracting and / or purifying DNA fragments which are bound to or associated with the isolated stabilised cell free nucleosomes; and (iii) analysing the DNA fragments (e.g. detecting, measure, or quantifying the DNA fragments or sequencing the DNA fragments).

[0459] The stabilised cell free nucleosomes may be isolated by any suitable method described herein or known in the art. In one embodiment, the sample (e.g. plasma sample or serum sample) is contacted with a nucleosome binding agent which binds to or is bound to a solid support, such as a magnetic particle, a microplate, or a plate. In one embodiment, the sample (e.g. plasma sample or serum sample) is contacted with magnetic particles coated with a nucleosome binding agent and isolated using the magnetic particles.

[0460] In one embodiment, the DNA fragments which are bound to or associated with the (isolated) stabilised cell free nucleosomes are extracted and / or purified. The DNA fragments may be extracted and / or purified by any suitable method described herein or known in the art. For example, the (isolated) stabilised cell free nucleosomes may be treated with a protease (e.g. Proteinase K) to digest proteins and / or an RNase (e.g. RNase A) to degrade RNA. For example, the DNA fragments may be purified using a DNA purification kit or equivalent method (e.g. phenol-chloroform extraction). In one embodiment, the DNA fragments are treated to reverse cross-linking (e.g. by heating). Suitable conditions to reverse cross-linking are known in the art. In one embodiment, the (isolated) stabilised cell free nucleosomes are heat-treated, digested with Proteinase K, optionally treated with RNase A, and optionally purified using a DNA purification kit or equivalent method.

[0461] The DNA analysis may include any suitable method to detect, measure, or quantify the DNA fragments or analyse the sequences of the DNA fragments. In one embodiment, the step of analysing the DNA fragments comprises detecting, measuring, or quantifying the DNA fragments. In one embodiment, the step of analysing the DNA fragments comprises sequencing of the DNA fragments.

[0462] In one embodiment, the step of analysing the DNA fragments comprises: (i) isolating the stabilised cell free nucleosomes from the plasma or serum sample; (ii) optionally, extracting and / or purifying DNA fragments which are bound to or associated with the isolated stabilised cell free nucleosomes; and (iii) detecting, measuring, or quantifying the DNA fragments.

[0463] In another embodiment, the step of analysing the DNA fragments comprises: (i) isolating the stabilised cell free nucleosomes from the plasma or serum sample; (ii) optionally, extracting and / or purifying DNA fragments which are bound to or associated with the isolated stabilised cell free nucleosomes; and (iii) sequencing the DNA fragments.

[0464] There are many methods known in the art to detect, measure, or quantify DNA. Any DNA analysis method may be employed for methods of the current invention including, without limitation, ultra-violet spectroscopy measurements, electrophoretic methods, methods involving coloured or fluorescent DNA binding or intercalating dyes (for example SYBR Green), next generation sequencing methods or polymerase chain reaction (PGR) methods.

[0465] There are many methods known in the art to analyse or identify a DNA sequence and any DNA analysis method may be employed for methods of the current invention including, without limitation, next generation sequencing methods, isothermal DNA amplification, cold PGR (coamplification at lower denaturation temperature-PCR), MAP (MIDI-Activated Pyrophosphorolysis), PARE (personalised analysis of rearranged ends), DNA hybridization methods (including gene chip methods and in situ hybridization methods). In addition, the gene sequence may also be analysed for epigenetically altered DNA sequences by epigenetic DNA sequencing analysis (e.g. for sequences containing 5-methylcytosine using bisulphite conversion of unmodified cytosine to uracil).

[0466] In one embodiment, the DNA fragments are analysed using DNA sequencing, for example a sequencing method selected from Next Generation Sequencing (targeted or whole genome) and methylated DNA sequencing analysis, BEAMing, PGR including digital PGR and cold PGR (co-amplification at lower denaturation temperature-PCR), isothermal amplification, hybridization, MIDI-Activated Pyrophosphorolysis (MAP) or Personalised Analysis of Rearranged Ends (PARE).

[0467] In one embodiment, the step of analysing the DNA fragments comprises amplifying the DNA fragments, for example by PCR. In one embodiment, the DNA fragments are detected, measured, or quantified by PCR (e.g. qPCR). In one embodiment, the DNA fragments are amplified by PCR prior to sequencing (e.g. for sequencing library preparation). Methods for sequencing library preparation are well-known in the art and any library preparation method may be used for methods of the invention. Typical sequencing library preparation methods involve DNA amplification and the use of adapter DNA molecules linked to each end of the DNA fragments to be analysed. Library preparation methods may be considered as single-stranded or double-stranded DNA library preparation methods. In one embodiment a double-stranded DNA library preparation method is used. In one embodiment a single-stranded DNA library preparation method is used. Single-stranded DNA library preparation methods have an advantage in better recovery of short <100bp DNA fragments.

[0468] Short cfDNA is a characteristic of plasma samples from cancer patients. A large proportion of cf-DNA is highly fragmented in cancer patients with a large proportion of ctDNA fragments being less than 100 bp in length, but low levels of short cf-DNA are reported for the plasma of healthy subjects. The present invention can be used to isolate and analyse small nicked- nucleosome derived <100bp cfDNA in a ChIP pull-down (such as a H3.1-ChlP pull-down) from cancer plasma or serum. Such a method allows for the enrichment of ctDNA. For example, cfDNA extracted from ChIP isolated crosslinked cf-nucleosomes may be sequenced. The sequences of nicked nucleosome derived short cfDNA fragments are enriched for ctDNA sequences.

[0469] Detecting small (<100bp) fragments using the methods of the present invention can be used to detect tumour-derived cf-DNA and therefore used in a method to analyse or detect cancer. cfDNA in samples from healthy subjects includes few fragments shorter than 100bp. cfDNA in samples from cancer patients contains much more short <100bp cfDNA. The cfDNA fragment size profile we obtained for CRC samples in Figure 13 shows that these short cfDNA fragments are nucleosome derived, indicating that nicked nucleosomes are prevalent in cancer samples. In one embodiment, the characteristic of cancer for use in methods of the invention is the cfDNA fragment size profile. In one embodiment, the characteristic of cancer for use in methods of the invention is the amount or proportion of short cfDNA fragments present in a sample. In one embodiment, the characteristic of cancer for use in methods of the invention is the amount or proportion of nicked nucleosomes present in a sample.

[0470] In one embodiment there is provided the use of a nicked cell free nucleosome as a biomarker for cancer. It will be understood that a nicked nucleosome as described herein is a histone associated DNA fragment that is shorter than 140bp in length, such as shorter than 130bp, shorter than 120bp, shorter than 110bp, shorter than 100bp, shorter than 90bp, shorter than 80bp, shorter than 70bp, shorter than 60bp or shorter than 50bp in length. In one embodiment the cell free nucleosome is a crosslinked nicked cell free nucleosome.

[0471] It will be understood that the embodiments described herein may be applied to all aspects of the invention, i.e. the embodiment described for the uses may equally apply to the claimed methods and so forth.

[0472] The invention will now be illustrated with reference to the following non-limiting examples.

[0473] EXAMPLES

[0474] EXAMPLE 1

[0475] To investigate the diagnostic potential of the measurement of stabilised nucleosomes in oncology, we collected blood plasma samples from 73 healthy subjects, 10 subjects diagnosed with an inflammatory disorder (8 subjects diagnosed with Crohn’s Disease or Colitis and 2 subjects diagnosed with rheumatoid arthritis or polyarthritis) and 99 patients diagnosed with cancer, of which 33 were colorectal cancer cases, 10 were breast cancer, 11 were lung cancer, 10 were liver cancer (comprising 5 hepatic cancer and 5 bile duct cancer cases), 10 were prostate cancer, 3 were melanoma, 2 were Non-Hodgkin’s Lymphoma and 20 were acute myeloid leukaemia (AML) cases.

[0476] Whole blood was collected into EDTA plasma blood collection tubes (BCT) containing formaldehyde as a cross-linking agent. For this purpose we used blood collection tubes containing a formaldehyde-releasing agent (Streck Cell Free DNA BCT tubes). Blood tubes were processed according to the manufacturer’s instructions. Separated plasma was transferred to 1 ml cryotubes and frozen until assayed.

[0477] Samples were assayed for the level of nucleosomes containing histone isoform H3.1 (H3.1 -nucleosomes) using an automated one-step chemiluminescent immunoassay (CLIA) system. Briefly, calibrant or sample (50pl) was incubated with an acridinium ester labelled antinucleosome antibody (50pl) and assay buffer (1 OOpI) for 1800 seconds at 37°C. Magnetic beads coated with an anti-histone H3.1 -nucleosome antibody (20pl) were added and the mixture was incubated for a further 900 seconds. The magnetic beads were then isolated, washed 3 times and magnetic bound acridinium ester was determined by luminescence output over 7000 milliseconds in RLU. In this assay the plasma sample, the solid-phase nucleosome capture antibody and the acridinium ester labelled anti-nucleosome antibody were all co-incubated in a single solution. This assay design may be referred to as a single incubation step assay. The results are shown in Tables 1-3 and Figures 1-8.

[0478] Table 1 : Solid cancer detection by H3.1 -nucleosome level by disease type, where specificity is determined using healthy subjects as control. Table 2: Solid cancer detection by H3.1 -nucleosome level by disease stage, where specificity is determined using healthy subjects as control.

[0479] Table 3: Colorectal cancer detection by H3.1-nucleosome level, where specificity is determined using subjects with gastroenterological inflammatory disease as control. The results demonstrate highly sensitive detection of cancer using an automated, simple, rapid (<1 hour), low cost immunoassay that compares favourably with sophisticated cancer detection modalities based on next generation sequencing of circulating tumour DNA (ctDNA). For example, the Galleri test, which takes 2 weeks to complete and is expensive, has an overall sensitivity for cancer detection of 51.5% at a specificity of 99.5%, with sensitivity of 16.8% at stage I, 40.4% at stage II, 77.0% at stage III and 90.1% at stage IV (Klein et al. 2021).

[0480] In addition, we investigated whether use of the stabilised nucleosome assay would lead to false positive results in samples obtained from patients with inflammatory diseases. We selected colorectal cancer and the gastroenterological inflammatory diseases of Crohn’s Disease and Colitis for this purpose. Tables 1 and 3 and Figures 5 and 8 show that stabilised nucleosome levels observed were similar in inflammatory disease subjects to those observed for healthy subjects.

[0481] EXAMPLE 2

[0482] We collected and analysed additional samples from healthy volunteers and cancer patients to obtain similar results to that described in Example 1 , but based on larger numbers of patients and 21 different cancer diseases. Whole blood samples were collected from 344 subjects in blood tubes containing a formaldehyde-releasing agent (Streck Cell Free DNA BCT tubes). The tubes were processed to produce plasma according to the manufacturer’s instructions and separated plasma was frozen until assayed. The subjects included 105 healthy volunteers and 229 subjects diagnosed with a variety of cancers including pharyngeal, AML, bladder, breast, cervix, colorectal, endometrial, bile duct, oesophagus, brain, kidney, liver, lung, melanoma, myeloma, NHL, ovarian, pancreatic, prostate, stomach, and thyroid cancer.

[0483] The 344 plasma samples were assayed for stabilised H3.1 -nucleosomes as described in Example 1. The results (Figure 9 and Table 4) show that all the 21 cancers were detectable at 100% specificity. Figure 9(A) shows that all cancers included cases that were above the highest healthy result (shown by dotted line). Figure 9(B) shows that 49% of all cancers were positive with no false positives among 105 healthy subjects (i.e. observed specificity of 100%). Table 4 shows the quartile results for healthy subjects, subjects with Crohn’s, Colitis or arthritis and subjects with 21 different cancers. The highest healthy stabilised H3.1 -nucleosomes result (32ng / ml) was used as cut-off to determine true positive results at 100% specificity (i.e. with zero false positive results for healthy subjects). The Crohn’s, Colitis and arthritis results were all below 32ng / ml (classified as true negatives for cancer). The quartile results for cancer diseases above 32ng / ml (classified as true positives) are shaded grey in Table 4. This shows that the observed sensitivity was above 25% for all 21 cancer diseases, was above 50% for 9 of the 21 cancers and above 75% for 4 of the 21 cancer diseases with zero false positive results.

[0484] Importantly, we observed high levels of stabilised H3.1 -nucleosomes in solid cancer diseases. For comparison we have observed that H3.1 -nucleosome levels measured for patients diagnosed with solid cancers, in EDTA plasma samples without crosslinking (i.e. for native H3.1 -nucleosomes) are lower than the levels observed with crosslinking in Figure 9(A). For example, levels observed for native H3.1 -nucleosomes in EDTA plasma for colorectal cancer are very rarely elevated above 150ng / ml. In contrast, 10 of 63 samples analysed for crosslinked stabilised nucleosomes were above 150ng / ml using the same assay system as shown Figure 9(A). In addition, the native (not cross-linked) H3.1 -nucleosome level measured in 135 healthy subjects was up to 173ng / ml with a 95th percentile cutoff set at 65ng / ml. The normal range measured for crosslinked H3.1 -nucleosomes in 100 subjects was up to 31.7ng / ml and the result at the 95th percentile was 24.1 ng / ml. Use of a stabilising (e.g. crosslinking) agent therefore produced both higher results in samples from cancer patients as well as lower results in samples from healthy subjects compared to the results measured for native H3.1- nucleosomes. This dual effect resulted in the detection of early stage cancer as well as an increase in the accuracy of detection of all stages of cancer compared to native nucleosome measurements which are elevated only at late stage disease and are not used clinically for cancer detection in humans.

[0485] Without being bound by theory, it may be that this dual effect relates to (i) prevention of elution of cell surface bound nucleosomes into free solution in EDTA plasma by crosslinking (leading to a fall in measured levels) and (ii) stabilisation of nucleosomes in free solution in EDTA plasma by crosslinking (leading to an increase in measured levels).

[0486] Although EDTA plasma is the standard sample matrix used for cf-nucleosome analysis, the electrochemistry of EDTA and its effects on cf-nucleosomes in solution in plasma has not been investigated. The physical chemistry of the nucleosome core particle (NCP) and the properties of cf-nucleosomes in the blood stream are incompletely understood. However, three NCP properties are of particular interest in the context of plasma cf-nucleosome measurements. Firstly, the properties of the NCP in solution are strongly influenced by electrochemical interactions with bivalent Ca2+and Mg2+cations due to the strong net negative NCP charge in excess of -100 which is stabilised by cations. Secondly, as well as being dependent on ambient Ca2+and Mg2+levels, the conformation and stability of the solvated NCP in aqueous solution is also dependent on NCP structure including whether or not the DNA fragment associated with the nucleosome is intact (ie; whether the nucleosome is a nicked nucleosome), the presence or absence of histone tails (ie; whether the nucleosome is a clipped nucleosome), and the presence or absence of linker DNA. Thirdly, cf-nucleosomes do not occur only free in solution, but also as cell-surface bound cell free nucleosomes (csb-cf-nucleosomes) and the equilibrium between the cell bound and free solution compartments is disturbed by alterations of the ambient Ca2+and Mg2+ion concentrations, including through bivalent cation sequestration by EDTA.

[0487] The NCP comprises a DNA fragment of approximately 145 base pairs in length bound to a histone octamer (145bp cf-nucleosome). Histone-DNA binding occurs primarily through the DNA sugar phosphate backbone where this faces inwards towards the octamer surface in the helical turn of the DNA. Histone proteins bind to DNA through electrostatic interactions of amino groups with DNA phosphate residues, but also through salt bridges, hydrogen bonds and nonpolar contacts.

[0488] The DNA component of the NCP has a negative charge of approximately -290. The histone octamer contains large numbers of lysine and arginine residues and is positively charged. The globular region of the histone octamer is in contact with the DNA and has a net charge of +58. The histone tails have a net charge of +98 and can interact with DNA, as well as facilitating inter-nucleosome interactions and chromatin folding. The negative charge of the DNA is therefore only partially attenuated by association with histone proteins and the NCP is surrounded by a strong negative electrostatic field. The surface charge density is not uniform but is strongest surrounding the coiled surface DNA and weaker around the histone core surface. The negative charge is stabilised by K+, Na+, Ca2+and Mg2+ions. The size, shape and stability of nucleosomes, as well as inter-nucleosomal interactions, are dependent on ionic strength and bivalent cation concentration.

[0489] Ca2+and Mg2+ions are involved in the packing of nucleosomes into higher order structures and are essential to the integrity of chromatin in vivo. Sequestration of bivalent cations from 30nm chromatin fibres by addition of 0.3mM EDTA, leads to unwinding of chromatin to produce strings of nucleosomes connected by linker DNA. (Vengerov and Popenko, 1977). We conclude that this likely reflects a reduction in ionic neutralization of the core nucleosome charge, an increase in intra-chromatosome binding of histone tails to linker DNA and a concomitant loss of inter-nucleosomal binding. The NCP may have specific Ca2+and Mg2+ion receptors (Yang and Hayes, 2011).

[0490] Intact 145bp cf-nucleosomes bind to Hela cell membranes through non-specific electrostatic interactions between positively charged groups on histone tails, as well as bivalent cation salt bridges, with negatively charged groups on cell membrane glycosaminoglycans. Removal of the histone tails through clipping by proteases, prevents these interactions and leads to weak binding of clipped nucleosomes to cell membranes. Addition of linker DNA to an intact NCP involves an increase in negative charge, leading to increased intra-chromatosomal binding of histone tails to linker DNA rather than cell membrane glycosaminoglycans and, in consequence, 185bp cf-nucleosomes bind weakly to cell surfaces (Wang et al, 2021).

[0491] Bryzgunova et al demonstrated that cfDNA fragments bind to the surfaces of red and white blood cells. The proportion of total cfDNA that is free in solution varies greatly between individuals from <5% to >80%. In most healthy subjects the majority of the total cfDNA present is cell-bound with only a small proportion (mean 13%) occurring free in solution. A similar variation occurs in cancer patients but the mean proportion in free solution is typically higher (41 % for prostate cancer). Leukocyte and erythrocyte bound cfDNA comprises long DNA fragments of 10, 000-25, OOObp as well as smaller fragments of 250-500bp. The binding of cfDNA to cell surfaces involves bivalent cations and exposure of cells to 5 mM EDTA in phosphate buffered saline leads to bivalent cation sequestration and elution of some csb-cf- nucleosomes into free solution. The proportion of csb-cf-nucleosomes eluted in 5 mM EDTA varies greatly up to around 40% (Bryzgunova et al, 2015). As the concentration of EDTA used as anticoagulant in plasma is similar (approximately 3-5 mM) we hypothesised that some csb- cf-nucleosomes may similarly be eluted into free solution in EDTA plasma.

[0492] Plasma cf-nucleosomes bear a multitude of protein related epigenetic signals including different histone isoforms and post translational histone modifications (PTM). Most histone PTMs are located on the histone tails. Removal of histone tails through the action of proteases, often called clipping, is well described and may act as an epigenetic eraser. The +98 charged histone tails contribute to NCP stability and their removal in clipped nucleosomes greatly increases the NCP surface negative charge, weakens the binding of DNA to the histone octamer and increases the accessibility of DNA to cleavage by hydroxyl radicals or nuclease activity leading to nucleosomes with nicked DNA. However, loss of histone tail stabilisation of histone-DNA interactions within the NCP may be at least partially compensated by Ca2+and Mg2+ion stabilisation and clipped nucleosomes are stable in solution in the presence of bivalent cations but unstable if they are removed (Yang and Hayes, 2011).

[0493] Circulating cf-nucleosomes comprise a heterogeneous mixture of nucleoproteins that have a variety of origins and occur in various states of metabolism. There are multiple potential effects of EDTA sequestration of bivalent cations in whole blood on cf-nucleosomes, including elution of some csb-cf-nucleosomes into free solution as well as changes in conformation and a reduction in the stability of clipped or nicked cf-nucleosomes. In this model, the overall effect of EDTA on the measured cf-nucleosome level in a sample will be determined by a combination of factors including, the proportion of total cf-nucleosomes in free solution, nucleosome structure and degree of metabolism, including the degree and the propensity of csb-cf-nucleosomes to elution by EDTA.

[0494] We hypothesised that crosslinking might both stabilise cf-nucleosomes in EDTA plasma and chemically link csb-cf-nucleosomes to the cell surfaces on which they are located.

[0495] Table 4: Measured stabilised H3.1 -nucleosome levels (nq / ml) by quartile (Lowest value, 1stquartile value, median value, 3rdquartile value and highest value) for 21 cancer diseases compared to results for healthy volunteers and patients with colonic inflammatory disorders.

[0496] EXAMPLE 3

[0497] In a similar experiment, plasma samples containing stabilised nucleosomes obtained from 47 healthy volunteers and 119 subjects diagnosed with a variety of cancers, were assayed for H3.1 -nucleosomes as well as stabilised nucleosomes containing histone H3 modified by trimethylation at lysine 27 (H3K27Me3-nucleosomes), using a similar automated immunoassay system to that described in Example 1 but employing magnetic beads coated with an antibody directed to bind to H3K27Me3 and the same acridinium ester labelled anti-nucleosome antibody. The results are shown in Figure 10 and confirm that this assay for nucleosomes containing a specific histone modification is a suitable assay for a method of the invention providing a similar AUC in a ROC curve to the previously described automated H3.1- nucleosome assay. The disease stage dependency for the sensitivity of solid cancer detection at 100% specificity is shown in Table 5.

[0498] EXAMPLE 4

[0499] In a further experiment, plasma samples containing stabilised nucleosomes obtained from 47 healthy volunteers and 119 subjects diagnosed with a variety of cancers, were assayed for stabilised H3.1 -nucleosomes as well as stabilised nucleosomes containing histone H3 modified by tri-methylation at lysine 36 (H3K36Me3), using a similar automated immunoassay system to that described in Example 1 but employing magnetic beads coated with an antibody directed to bind to H3K36Me3 and the same acridinium ester labelled anti-nucleosome antibody. The results are shown in Figure 10 and confirm that this assay for nucleosomes containing a specific histone modification is a suitable assay for a method of the invention providing a similar AUC in a ROC curve to the previously described automated H3.1- nucleosome assay. The disease stage dependency for the sensitivity of solid cancer detection at 100% specificity is shown in Table 5. Table 5: Sensitivity for solid cancer disease detection at 100% specificity (zero FPF) for stabilised H3.1 -nucleosome, H3K27Me3-nucleosome and H3K36Me3-nucleosome levels (nq / ml).

[0500] EXAMPLE 5

[0501] In a further experiment we investigated whether a nucleosome assay, designed to detect all nucleosomes or nucleosomes per se, for an entirely different purpose would be useful as an assay system for use as a method of the invention. For this purpose we selected the manual Roche Cell Death Detection ELISA for the qualitative and quantitative in vitro determination of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) after induced cell death. The Roche Cell Death Detection ELISA does not output concentrations as it has no calibrator and results are obtained as absorbance units. However, this is adequate to rank the samples by nucleosome level and hence obtain a ROC curve.

[0502] Plasma samples containing stabilised nucleosomes obtained from 18 healthy volunteers and 21 subjects diagnosed with a variety of cancers were assayed for stabilised H3.1- nucleosomes, using the same one-step automated and two-step manual ELISA immunoassay systems for stabilised H3.1 -nucleosomes described in Example 5. In addition the samples were ranked for their nucleosome level using the Roche Cell Death Detection ELISA according to the manufacturer’s instructions. The results are shown in Figure 12 and confirm that the Roche Cell Death Detection ELISA is a suitable assay for a method of the invention providing a similar AUC in a ROC curve to the previously described assay for H3.1 -nucleosomes.

[0503] Examples 3 to 6 demonstrate that assays for all nucleosomes, nucleosomes per se, nucleosomes containing particular histone isoforms (e.g. histone isoform H3.1) and modified nucleosomes (e.g. containing histone modifications H3K27Me3 or H3K36Me3) are all useful in methods of the invention. In addition these examples demonstrate that the nucleosome assays employed may be automated or manual, one-step or two-step, employ any endpoint (e.g. enzyme or chemiluminescent), and may or may not employ a calibrant. Together these examples demonstrate that any measurement for all nucleosomes or particular nucleosome types may be used for the measurement of stabilised nucleosomes and hence be useful as a method of the invention.

[0504] EXAMPLE 6

[0505] In a further experiment, plasma samples containing stabilised nucleosomes obtained from 42 healthy volunteers and 111 subjects diagnosed with a variety of cancers, were assayed for stabilised H3.1 -nucleosomes, using the same one-step automated immunoassay system to that described in Example 1 as well as a two-step manual ELISA assay for stabilised H3.1- nucleosomes employing a 96-well microtiter plate coated with an antibody directed to bind to histone H3.1 and an alkaline phosphatase enzyme labelled anti-nucleosome antibody. In this two-step assay format, the sample is reacted firstly with the solid phase antibody coated to a microtiter well plastic surface. The sample is then discarded and the microtiter well-bound nucleosomes are thoroughly washed to remove any remaining sample or unbound nucleosomes. The enzyme labelled antibody is then reacted with the washed microtiter bound nucleosomes in a separate second reaction step. The results are shown in Figure 11 and confirm that the two-step manual assay for H3.1 -nucleosomes is a suitable assay for a method of the invention providing a similar AUC in a ROC curve to the previously described automated one-step CLIA assay for H3.1 -nucleosomes.

[0506] EXAMPLE 7

[0507] Intact plasma cf-mononucleosomes comprise a histone core and approximately 130-200bp DNA. Nicked nucleosomes in which the cf-nucleosome associated DNA fragment includes one or more single or double-stranded cuts or nicks, would be expected to yield shorter DNA fragments. To ascertain whether any short fragments occur in crosslinked cf-nucleosomes from cancer patients, and whether these fragments are bound by the solid phase antibody employed in the H3.1 -nucleosome immunoassay, we investigated the fragment size frequency profile for cfDNA associated with crosslinked H3.1-nucleosomes bound by the antibody. cf-nucleosomes were isolated by ChIP from Streck plasma samples collected from four patients with CRC using the magnetic solid phase antibody employed in the H3.1 -nucleosome immunoassay. DNA extracted from the antibody bound crosslinked cf-nucleosomes was sequenced and fragment size frequency profiles produced. Consistent with previous findings (Thierry, 2023), the profiles confirm the presence of a significant, but variable, level of nicked crosslinked cf-nucleosome derived short cfDNA fragments in the four CRC patient samples and also that crosslinked nicked cf-nucleosomes are bound by the magnetic antibody used in the H3.1-nucleosome assay (Figure 13). DNA fragment size profiles obtained for the ChIP isolates were similar to profiles for cfDNA extracted directly from the same whole Streck plasma samples, confirming that most plasma cfDNA is nucleosome-protected and indicating a high efficiency of immunoprecipitation.

[0508] EXAMPLE 8

[0509] The effect of crosslinking cf-nucleosomes in whole blood samples prior to sample processing on immunoassay results was investigated by comparing results obtained for EDTA plasma (native) and Streck plasma (crosslinked) samples collected from 10 healthy subjects, 8 CRC patients and 49 hospitalised patients with elevated levels of CRP (>5mg / L). We observed that 3 out of 8 CRC patients have higher H3.1 results when measured crosslinked in whole blood than native in EDTA, but 0 of 10 healthy volunteers or 39 hospitalised patients with elevated inflammatory markers (FIGURE 14).

[0510] EXAMPLE 9

[0511] Whole blood was collected from a healthy volunteer into an EDTA plasma BCT and 6 glass tubes containing varying amounts of aqueous formaldehyde such that addition of 5mls whole blood would lead to a final whole blood formaldehyde concentration of zero, 0.015%, 0.03%, 0.1 %, 0.2% and 0.5%. As these glass tubes contained no EDTA or other known anticoagulant they would be assumed by those skilled in the art to be serum tubes. The EDTA and glass tubes containing whole blood were left for 1 hour at room temperature. The tubes were then processed by centrifugation and the supernatant liquid was removed from the sedimented blood cells. The supernatant liquid was assayed for H3.1 -nucleosomes. The results (Figure 15) demonstrate that the level of nucleosomes measured in a glass tube containing no formaldehyde (ie; normal serum) was 7-fold higher than the result obtained in EDTA plasma. This effect is well described in the literature and occurs because NETosis occurs in whole blood collected in serum tubes post venipuncture. However, addition of formaldehyde prevented either or both of NETosis and coagulation.

[0512] EXAMPLE 10

[0513] Whole blood was collected from a healthy volunteer into an EDTA plasma BCT, a Streck cell free DNA BCT and 2 glass tubes containing IDU such that addition of 5ml whole blood would lead to a final whole blood IDU concentration of 2% m / v. As these glass tubes contained no EDTA or other known anticoagulant they would be assumed by those skilled in the art to be serum tubes. The EDTA, Streck and glass tubes containing whole blood were left for 1 hour or 24 hours at room temperature. The tubes were then processed by centrifugation and the supernatant liquid was removed from the sedimented blood cells. The supernatant liquid was assayed for H3.1-nucleosomes. The results (Figure 16) demonstrate that addition of 2% IDU to whole blood prevented either or both of NETosis and coagulation. Moreover the level was stable over 24 hours indicating that the nucleosome level was stabilised in whole blood. The stabilisation is caused by formaldehyde fixation of blood cells present in the sample preventing leakage of intracellular chromatin into the liquid extracellular phase of the blood, as well as by stabilisation of extracellular cf-nucleosomes through intra-nucleosome crosslinking.

[0514] EXAMPLE 11

[0515] Whole blood was collected from a healthy volunteer into 2 glass tubes containing DU such that addition of 5ml whole blood would lead to a final whole blood DU concentration of 2% m / v. As these glass tubes contained no EDTA or other known anticoagulant they would be assumed by those skilled in the art to be serum tubes. The glass tubes containing whole blood were left for 1 hour or 24 hours at room temperature. The tubes were then processed by centrifugation and the supernatant liquid was removed from the sedimented blood cells. The supernatant liquid was assayed for H3.1 -nucleosomes. The results (Figure 17) demonstrate that addition of 2% DU to whole blood prevented either or both of NETosis and coagulation. Moreover the level was stable over 24 hours indicating that the nucleosome level was stabilised in whole blood. As in EXAMPLE 10, this stabilisation is caused by formaldehyde fixation of blood cells present in the sample as well as by stabilisation of extracellular cf-nucleosomes through intra- nucleosome crosslinking.

[0516] EXAMPLE 12

[0517] Chromatin extracted from Hela cells in culture is digested with micrococcal nuclease under a variety of conditions for 1 hour: a) in buffer b) in buffer containing formaldehyde c) in buffer containing imidazolidinyl urea d) in buffer containing diazolidinyl urea

[0518] The treated chromatin solutions are investigated for size profile by Bioanalyzer. The digested chromatin in buffer is observed to comprise predominantly nucleosome sized (100-200bp) length DNA fragments. The formaldehyde, imidazolidinyl urea and diazolidinyl urea treated extracts are observed to comprise more large DNA fragments >1000bp in length. The result demonstrates that use of a crosslinker inhibits nuclease enzyme activity. EXAMPLE 13

[0519] Whole blood samples are collected in duplicate from each of 20 healthy volunteers and 20 patients diagnosed with NSCLC in glass or plastic tubes containing aqueous imidazolidinyl urea or diazolidinyl urea and no other additive. The whole blood samples are processed by centrifugation at 1600 x g for 10 minutes at room temperature and the supernatant liquid is separated and stored at -80°C until assayed for H3.1 -nucleosomes. The H3.1 -nucleosome assay results for the NSCLC patients are observed to be elevated above those observed for the healthy volunteers.

[0520] EXAMPLE 14

[0521] Whole blood samples were collected from each of 10 healthy volunteers and 10 cancer patients in Sarstedt collection tubes (S-Monovette® cfDNA Exact). The whole blood samples were processed by centrifugation and the supernatant liquid was separated. The H3.1- nucleosome assay results for the cancer patients were elevated above those observed for healthy volunteers (Figure 18). S-Monovette® cfDNA Exact blood collection tubes contain the formaldehyde releasing agent urotropine.

[0522] EXAMPLE 15

[0523] 2g IDU was dissolved in 10ml water to produce a 20% IDU solution. Increasing volumes (32pL, 62.5pL, 125pL, 250pL and 500pL) of 20% IDU were added to a series of glass and plastic tubes. 5mL whole blood was collected from 2 healthy doners in the glass and plastic tubes so that the blood collected contained 0.125%, 0.25%, 0.5%, 1% and 2% IDU. The tubes were left at room temperature for 1 hour or 24 hours before centrifugation and the upper liquid layer was transferred to a cryotube and stored refrigerated until assayed. A whole blood sample was also collected from each volunteer in an EDTA BCT and a Streck BCT and processed according to manufacturers’ instructions. All samples were analysed for H3.1 -nucleosome concentration. The measured H3.1-nucleosome level decreased with the IDU concentration present in whole blood at levels of up to approximately 0.5% and became less dependent, or independent, of IDU concentration at higher levels. Furthermore, whilst the measured H3.1- nucleosome levels were not stable in samples left 24 hours before centrifugation at low whole blood IDU concentrations, they were stable at IDU concentrations of approximately 1 % or higher. The results indicate that whole blood IDU concentrations of approximately 1 % or higher are suitable for methods of the invention, such as 1%, 2%, 3%, 4% or higher as shown in Figure 19. The results were similar when whole blood samples were collected in plastic or glass tubes demonstrating the production of simple crosslinking blood collection tubes containing water and a formaldehyde releasing agent for blood nucleosome measurements. Without being bound by theory, the results shown in Figures 16, 17 and 19 indicate that formaldehyde release by IDU or DU in whole blood results in fixation of blood cells. Fixing of cells preserves cellular structures, effectively locking structures in place, whilst also arresting cellular activity. Fixed cells are generally considered to be dead. The fixation by IDU includes fixation of intracellular chromatin by crosslinking, locking its structure in place inside the cell whilst also preserving the structure of the cell thereby preventing passive nucleosome loss to the extracellular fluid of the blood. As fixed cells are effectively dead and inactive, they do not actively release nucleosomes or NETs through active mechanisms including NETosis.

[0524] EXAMPLE 16

[0525] Evacuated blood collection tubes (vacutainers) containing IDU are produced by adding IDU to a glass or plastic tube and producing a vacuum within the tube by methods well known by manufacturers in the art.

[0526] EXAMPLE 17

[0527] We hypothesised that cancer derived, inflammatory derived and healthy cf-nucleosomes are mixtures of different structures. Without being bound by theory, healthy cf-nucleosomes are likely to include predominantly intact mono-nucleosomes, cancer cf-nucleosomes are likely to include a significant proportion of nicked and / or clipped mononucleosomes and inflammatory cf-nucleosomes are likely to include a significant proportion of oligonucleosomes or polynucleosomes. We further hypothesised that native nicked and / or clipped native cf-nucleosome structures may be less stable than intact native cf-nucleosomes in EDTA plasma and may, in consequence, be more susceptible to stabilisation by crosslinking.

[0528] A cf-nucleosome immunoassay typically includes a step involving nucleosome binding to a solid phase anti-nucleosome antibody (i.e. chromatin immunoprecipitation or ChIP). We investigated cf-nucleosome stability as determined by the core histone-DNA integrity of nucleosomes bound to a solid phase anti-nucleosome antibody. We measured the proportion of cf-nucleosomes that retain the associated cfDNA after ChIP. To determine whether healthy, cancer derived or inflammation derived cf-nucleosomes are affected differently by crosslinking, we tested the recovery of plasma cfDNA from crosslinked or native cf- nucleosomes isolated by binding to magnetic anti-H3.1 -nucleosome antibody (magnetic ChIP as occurs in H3.1 -nucleosome immunoassays). Matched whole blood samples were collected in EDTA BCTs and Streck Cell-Free DNA BCTs from 6 healthy subjects, 6 patients diagnosed with a cancer and 7 patients with an inflammatory condition with an elevated CRP level (but no cancer). Two of the 6 healthy samples were found to have an elevated level of H3.1 -nucleosomes above 31.7 ng / ml. EDTA blood samples were processed by centrifugation within 4 hours of venipuncture. Streck Cell- Free DNA BCTs were processed as per the manufacturer’s instructions.

[0529] CfDNA was extracted from 1 mL of whole plasma samples using a Qiagen extraction kit. Cf- nucleosomes were precipitated from (another) 1mL of the same plasma samples by ChIP using magnetic beads coated with an anti-H3.1 -nucleosome antibody. The beads were washed and nucleosome-bound cf-DNA was extracted using a Qiagen extraction kit. The extracted cfDNA was measured by Qubit. The amount of cfDNA present in the ChIP isolated cf-nucleosomes, as a proportion of that present in the respective whole plasma was estimated as the recovery of cfDNA after ChIP

[0530] The results are shown in Figure 20. In healthy subjects (Figure 20(a)), cf-DNA recovery was inversely related to the concentration of cf-nucleosomes present in the sample. The recovery of native cf-nucleosomes (in EDTA plasma) and crosslinked cf-nucleosomes (in Streck plasma) was similar. This indicates that the stability of cf-nucleosomes in healthy samples was not increased by crosslinking.

[0531] In cancer patients (Figure 20(b)), cfDNA recovery was similar to that observed for healthy subjects for samples containing low (normal) H3.1 -nucleosome levels. For cancer samples with higher cf-nucleosome concentrations, cfDNA recovery of cross-linked, but not native, cf- nucleosomes was positively related to cf-nucleosome concentration. The recovery of cfDNA by cf-nucleosome ChIP from samples containing elevated cf-nucleosome concentrations was increased by crosslinking of cf-nucleosomes. This demonstrates that the stability of cf- nucleosomes in cancer samples is increased by intra-nucleosomal crosslinking.

[0532] In patients with an inflammatory condition (Figure 20(c)), cfDNA recovery from native cf- nucleosomes (in EDTA plasma) was greater than that from crosslinked cf-nucleosomes (in Streck plasma) at all cf-nucleosome levels tested. Thus, recovery of cf-DNA from isolated cf- nucleosomes in inflammatory patient samples was reduced by crosslinking of cf-nucleosomes.

[0533] An elevated cf-nucleosome level may reflect cf-nucleosome release from inflammatory cells (e.g. by NETosis) or from tumour cells. This lack of disease specificity complicates the interpretation of elevated cf-nucleosome results and has been a longstanding difficulty in the clinical use of cf-nucleosome measurements. Thus, there is a need for a simple method to identify the underlying cause of an elevated cf-nucleosome level as inflammatory or cancerous in nature.

[0534] Figure 21 shows the recovery of native and crosslinked cfDNA from isolated cf-nucleosomes in samples with an elevated cf-nucleosome concentration. The results for healthy, cancer and inflammatory patient groups are shown in ascending order of cf-nucleosome concentration. In addition, the numerical concentrations of each sample are displayed on the figure. In summary Figure 21 shows that recovery of cf-DNA from isolated cf-nucleosomes in samples from healthy subjects is largely unaffected by crosslinking of cf-nucleosomes. Recovery of cf-DNA from isolated cf-nucleosomes in cancer patient samples is increased by crosslinking of cf- nucleosomes. Recovery of cf-DNA from isolated cf-nucleosomes in inflammatory patient samples is reduced by crosslinking of cf-nucleosomes.

[0535] We further checked the data by correlating H3.1 -nucleosome levels measured by automated immunoassay in Streck plasma samples with cfDNA levels measured by Qubit for EDTA and Streck plasma samples as well as for the cfDNA extracted from cf-nucleosomes isolated from both plasma types by ChlP. The results showed that cfDNA levels measured in samples from healthy subjects and patients with cancer correlate well with H3.1 -nucleosome measurements for the same samples (Figure 22(a) and 22(b)), but showed poor correlation for samples obtained from patients with an inflammatory condition (Figure 23(a) and 23(b)). This further illustrates the difference in nucleosome structures in these sample types.

[0536] The cfDNA levels measured by Qubit on cfDNA extracts from H3.1 -nucleosomes isolated from samples by ChlP was also correlated with the H3.1 -nucleosome levels measured by automated immunoassay in Streck plasma samples. The results again showed that cfDNA levels isolated by ChlP correlated well with plasma cf-nucleosome levels for cancer patients and most healthy subjects, but not for healthy subjects for which a false positive result was obtained by immunoassay of crosslinked cf-nucleosomes (Figure 22(c) and 22(d)). Again, poor correlation was observed for samples obtained from patients with an inflammatory condition (Figure 23(c) and 23(d)).

[0537] The results clearly show that this cf-DNA recovery can be used to determine the cause of an elevated cf-nucleosome level as tumour or inflammatory in nature. In a subject with cancer the cfDNA recovery will be higher from crosslinked than from native cf-nucleosomes. In a subject with an inflammatory condition the recovery of cfDNA will be higher from native than from crosslinked cf-nucleosomes. This measurement of DNA recovery of ChIP isolated from native and crosslinked nucleosomes can be used to identify the cause of an elevated cf- nucleosome level or an elevated cfDNA level.

[0538] Without being bound by theory, the results are consistent with an interpretation that native cancer derived cf-nucleosomes, are relatively unstable and are stabilised by intra- nucleosomal crosslinking. Native inflammatory cf-nucleosomes, are relatively stable and are destabilised by crosslinking. This may be due to inter-nucleosome crosslinking leading to clumping of polynucleosomes strings.

[0539] EXAMPLE 18

[0540] The methods described herein are not related to a change in assay chemistry but to a change in sample collection and hence also to a change in preanalytics. The preanalytical effects of cfDNA measurements in plasma samples collected in EDTA or cfDNA BCTs are well known in the art. In brief, whole blood samples collected for cfDNA analysis in EDTA BCTs must be processed promptly (for example, within 2-4 hours) after venipuncture. Failure to process promptly leads to artifactually elevated cfDNA results due to contamination of plasma with white cell chromatin and DNA. cfDNA BCTs are designed to stabilise cfDNA levels in whole blood by prevention of contamination. Multiple authors have shown that a variety of cfDNA stabilising tubes, including Streck and Sarstedt cfDNA BCTs, are effective for the stabilisation of measured cfDNA levels with little or no increase in measured cfDNA after several days of storage of whole blood prior to processing.

[0541] We investigated the stability of cross-linked cf-nucleosome levels in 4 whole blood samples collected from each of 10 healthy volunteers in Streck BCTs. The 4 tubes of blood collected were stored for 4, 6, 24 or 72 hours at room temperature before processing by centrifugation. Plasma was separated from the samples and the H3.1 -nucleosome level of each plasma sample was measured. The results (Figure 24), surprisingly, show that the cf-nucleosome level measured decreased over 72 hours for all the samples. The consistently low cf-nucleosome levels we observed in healthy control subjects may be related to this effect.

[0542] We repeated the preanalytical experiment several times with plasma samples from different volunteers (usually 10 volunteers) and obtained reproducible results. As prevention of plasma contamination by cellular chromatin may prevent an increase in cf-nucleosome levels but cannot cause a decrease, the cause of this effect must lie elsewhere. Whilst the cause is not clear, and without being bound by any theory, it may be related to nucleosome crosslinking. One possibility is a slow removal of cf-nucleosomes from plasma by sequestration of cf- nucleosomes onto cell surfaces. In EDTA plasma, nucleosome binding to cell surfaces may be a reversible or transient, equilibrium process. In a whole blood sample containing a crosslinking agent, or crosslinking releasing agent, any nucleosome that binds to a cell surface may be permanently fixed to the cell by crosslinking, thus effectively removing it from solution.

[0543] In conclusion, there may be multiple preanalytical effects of cf-nucleosome crosslinking in whole blood. Some preanalytical effects may lead to an apparent increase in measured cf- nucleosome levels and others may lead to an apparent decrease. The culmination of these effects when used for sample collection leads to a surprisingly accurate and specific method that is able to identify persons with an early stage cancer by means of a low cost, rapid blood test.

[0544] REFERENCES

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[0556] Yang and Hayes (2011) Biochemistry 50(46): 9973-81. doi: 10.1021 / bi201377x.

[0557] EMBODIMENTS

[0558] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paras).

[0559] 1 . A method of the identification of a characteristic of cancer, comprising the steps of:

[0560] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent; and (ii) analysing the cell free nucleosomes to identify a cancer characteristic. A method for stabilising, storing and analysing cell free nucleosomes for a characteristic of cancer, comprising the steps of:

[0561] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;

[0562] (ii) separating plasma from the blood sample; and

[0563] (iii) analysing the plasma for cell free nucleosomes having a characteristic of cancer. A method for diagnosing or detecting a cancer, comprising the steps of:

[0564] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;

[0565] (ii) separating plasma from the blood sample;

[0566] (iii) contacting the plasma sample with a binding agent to detect or measure cell free nucleosomes; and

[0567] (iv) using the cell free nucleosomes detected to diagnose the subject with cancer. A method for determining the prognosis of a subject with a cancer, comprising the steps of:

[0568] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;

[0569] (ii) separating plasma from the blood sample;

[0570] (iii) contacting the plasma sample with a binding agent to detect or measure cell free nucleosomes; and

[0571] (iv) using the cell free nucleosomes detected as indicative of the prognosis of said cancer. A method for monitoring the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer, comprising the steps of:

[0572] (i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;

[0573] (ii) separating plasma from the blood sample;

[0574] (iii) contacting the plasma sample with a binding agent to detect or measure cell free nucleosomes; and (iv) comparing the cell free nucleosomes detected with an earlier plasma sample taken from said subject to determine the efficacy of said therapy. The method of any preceding para, wherein the blood sample is contacted with the stabilising agent in a blood collection tube. The method of any preceding para, wherein the stabilising agent is a cross-linking agent. The method of para 7, wherein the cross-linking agent is selected from formaldehyde, paraformaldehyde, formalin, glutaraldehyde, Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE). The method of para 7 or 8, wherein in the cross-linking agent is a cross-linking agent releasing agent such as one selected from the group consisting of: diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5dimethylhydantoin, dimethylol urea, 2-bromo-2.- nitropropane-1 ,3-diol, oxazolidines, sodium hydroxymethyl glycinate, 5- hydroxymethoxymethyl-1-1aza-3,7-dioxabicyclo[3.3.0]octane, 5-hydroxymethyl-1- 1aza-3,7dioxabicyclo[3.3.0]octane, 5-hydroxypoly[methyleneoxy]methyl-1-1aza-3, 7dioxabicyclo[3.3.0]octane, quaternary adamantine and any combination thereof. The method of any preceding para, wherein in step (i) the blood sample is also contacted with an anticoagulant, such as heparin, ethylenediamine tetraacetic acid (EDTA), citrate or oxalate. The method of any preceding para, further including the step of isolating the cell free nucleosomes from the blood sample before analysis. The method of any preceding para, wherein (a) either or both of the isolating or analysing steps occurs up to 7 days after the blood sample is drawn from a subject, (b) either or both of the isolating or analysing steps occurs without freezing the blood sample; or both (a) and (b). The method of any preceding para, wherein the method of analysing, detecting or measuring cell free nucleosomes comprises an immunoassay, immunochemical, mass spectroscopy, chromatographic, chromatin immunoprecipitation or biosensor method. 14. The method of any preceding para, wherein the method of analysing, detecting or measuring cell free nucleosomes comprises contacting the sample with a solid phase comprising a binding agent that detects cell free nucleosomes or a component thereof, and detecting binding to said binding agent.

[0575] 15. The method of any preceding para, wherein the blood sample is obtained from a human or an animal subject.

[0576] 16. The method of para 15, wherein the subject is suspected of relapse to a cancer.

[0577] 17. The method of any one of paras 2 to 16, comprising comparing a level of said cell free nucleosomes in said plasma sample with one or more controls.

[0578] 18. The method of para 17, wherein the control is a healthy subject or a subject with a noncancer disease.

[0579] 19. The method of para 17 or para 18, wherein the level of cell free nucleosomes is elevated compared to the control.

[0580] 20. The method of any preceding para, wherein the cancer is a cancer of the bladder, breast, colon, cervix, oesophagus, kidney, large intestine, liver, lung, oral cavity, ovary, pancreas, prostate, rectum, skin or stomach or a vascular or haematological cancer.

[0581] 21. The method of any preceding para, wherein the cell free nucleosome is analysed by detecting or measuring a component of the nucleosome, such as a histone variant or histone isoform.

[0582] 22. The method of para 21 , wherein the cell free nucleosome is analysed by detecting or measuring an epigenetic feature of the cell free nucleosome.

[0583] 23. The method of para 22, wherein the epigenetic feature of the cell free nucleosome is a histone isoform, such as a histone isoform of a core nucleosome, in particular a histone H3 isoform, such as H3.1. 24. The method of para 22, wherein the epigenetic feature of the cell free nucleosome is a histone post translational modification (PTM), such as a histone PTM of a core nucleosome, in particular a histone H3 PTM.

[0584] 25. Use of a stabilised nucleosome in a body fluid sample as a biomarker for cancer.

[0585] 26. The use of para 25, wherein the stabilised nucleosome is analysed, detected or measured by an immunoassay, immunochemical, mass spectroscopy, chromatographic, chromatin immunoprecipitation or biosensor method.

[0586] 27. The use of para 25 or para 26, wherein the stabilised nucleosome is analysed, detected or measured by contacting the body fluid sample with a solid phase comprising a binding agent that detects cell free nucleosomes or a component thereof, and detecting binding to said binding agent.

[0587] 28. The use of any one of paras 25 to 27, wherein the body fluid sample is obtained from a human or an animal subject.

[0588] 29. The use of any one of paras 25 to 28, wherein the body fluid sample is obtained from a subject who is suspected of relapse to a cancer.

[0589] 30. The use of any one of paras 25 to 28, comprising comparing a level of said stabilised nucleosomes with one or more controls.

[0590] 31. The use of para 30, wherein the control is a healthy subject or a subject with a noncancer disease.

[0591] 32. The use of para 30 or para 31 , wherein the level of stabilised nucleosomes is elevated compared to the control.

[0592] 33. The use of any one of paras 25 to 32, wherein the cancer is a cancer of the bladder, breast, colon, cervix, oesophagus, kidney, large intestine, liver, lung, oral cavity, ovary, pancreas, prostate, rectum, skin or stomach or a vascular or haematological cancer. 34. The use of any one of paras 25 to 33, wherein the stabilised nucleosome is analysed as a biomarker for cancer by detecting or measuring a component of the nucleosome, such as a histone variant or histone isoform.

[0593] 35. The use of para 34, wherein the stabilised nucleosome is analysed as a biomarker for cancer by detecting or measuring an epigenetic feature of the cell free nucleosome.

[0594] 36. The use of para 35, wherein the epigenetic feature of the stabilised nucleosome is a histone isoform, such as a histone isoform of a core nucleosome, in particular a histone H3 isoform, such as H3.1.

[0595] 37. The use of para 35, wherein the epigenetic feature of the stabilised nucleosome is a histone PTM, such as a histone PTM of a core nucleosome, in particular a histone H3 PTM.

[0596] 38. A kit comprising one or more reagents for carrying out the method as defined according to any one of paras 1 to 24.

[0597] 39. Use of a kit comprising: (i) one or more reagents to detect or measure the level of cell free nucleosomes or a component thereof, and (ii) a blood collection tube comprising a stabilising agent, to detect, monitor or diagnose cancer.

[0598] 40. A kit to detect, monitor or diagnose cancer in a subject, wherein said kit comprises (i) a first binding agent which specifically binds to an epigenetic feature of a cell free nucleosome (e.g. H3.1) and (ii) a second binding agent which specifically binds to cell free nucleosomes, for use with : (iii) a blood collection tube comprising a stabilising agent.

[0599] 41. A blood collection tube comprising a stabilising agent for a cell free nucleosome for use in the method of any one of paras 1 to 24.

[0600] 42. Use of a blood collection tube comprising a stabilising agent for collecting a cell free nucleosome in a body fluid sample as a biomarker for cancer.

Claims

CLAIMS1. A method of the identification of a characteristic of cancer, comprising the steps of:(i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent; and(ii) analysing the cell free nucleosomes to identify a cancer characteristic.

2. A method for stabilising, storing and analysing cell free nucleosomes for a characteristic of cancer, comprising the steps of:(i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;(ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains stabilised cell free nucleosomes; and(iii) analysing the plasma sample or serum sample for cell free nucleosomes having a characteristic of cancer.

3. A method for diagnosing or detecting a cancer, comprising the steps of:(i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;(ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains stabilised cell free nucleosomes;(iii) contacting the plasma sample or serum sample with a nucleosome binding agent to detect or measure cell free nucleosomes; and(iv) using the cell free nucleosomes detected or measured to diagnose the subject with cancer.

4. A method for determining the prognosis of a subject with a cancer, comprising the steps of:(i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;(ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains stabilised cell free nucleosomes;(iii) contacting the plasma sample or serum sample with a nucleosome binding agent to detect or measure cell free nucleosomes; and(iv) using the cell free nucleosomes detected or measured as indicative of the prognosis of said cancer.

5. A method for monitoring the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer, comprising the steps of:(i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;(ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains stabilised cell free nucleosomes;(iii) contacting the plasma sample or serum sample with a nucleosome binding agent to detect or measure cell free nucleosomes; and(iv) comparing the cell free nucleosomes detected or measured with an earlier plasma sample or serum sample taken from said subject to determine the efficacy of said therapy.

6. A method for quantifying cell free nucleosomes in a blood sample, comprising the steps of:(i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;(ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains stabilised cell free nucleosomes;(iii) contacting the plasma sample or serum sample with a nucleosome binding agent; and(iv) quantifying the concentration of cell free nucleosomes in the plasma sample or serum sample.

7. The method of claim 6, wherein the concentration of cell free nucleosomes in the plasma sample or serum sample is used to diagnose cancer in a subject, determine the prognosis of a subject with a cancer, or monitor the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer.

8. The method of any preceding claim, wherein the blood sample is contacted with the stabilising agent in a blood collection tube.

9. The method of any preceding claim, wherein the stabilising agent is a cross-linking agent.

10. The method of claim 9, wherein the cross-linking agent is selected from formaldehyde, paraformaldehyde, formalin, glutaraldehyde, Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE).

11. The method of claim 9 or 10, wherein in the cross-linking agent is a cross-linking agent releasing agent such as one selected from the group consisting of: diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5dimethylhydantoin, dimethylol urea, 2-bromo-2.- nitropropane-1,3-diol, oxazolidines, sodium hydroxymethyl glycinate, 5- hydroxymethoxymethyl-1-1aza-3,7-dioxabicyclo[3.3.0]octane, 5-hydroxymethyl-1- 1aza-3,7dioxabicyclo[3.3.0]octane, 5-hydroxypoly[methyleneoxy]methyl-1-1aza-3, 7dioxabicyclo[3.3.0]octane, quaternary adamantine and any combination thereof.

12. The method of any preceding claim, wherein in step (i) the blood sample is also contacted with an anticoagulant, such as heparin, ethylenediamine tetraacetic acid (EDTA), citrate or oxalate.

13. The method of any preceding claim, further including the step of isolating the cell free nucleosomes from the blood sample before analysis.

14. The method of any preceding claim, wherein (a) either or both of the isolating or analysing steps occurs up to 7 days after the blood sample is drawn from a subject, (b) either or both of the isolating or analysing steps occurs without freezing the blood sample; or both (a) and (b).

15. The method of any preceding claim, wherein the method comprises storing the blood sample, plasma sample or serum sample at room temperature for up to 7 days.

16. The method of any preceding claim, wherein the method comprises storing the blood sample, plasma or serum sample at about -80°C for up to two years.

17. The method of any preceding claim, wherein the method of analysing, detecting, measuring or quantifying cell free nucleosomes comprises an immunoassay, immunochemical, mass spectroscopy, chromatographic, chromatin immunoprecipitation or biosensor method.

18. The method of any preceding claim, wherein the method of analysing, detecting, measuring or quantifying cell free nucleosomes comprises contacting the sample with a solid phase comprising a nucleosome binding agent that detects cell free nucleosomes or a component thereof, and detecting binding to said binding agent.

19. The method of any preceding claim, wherein the blood sample is obtained from a human or an animal subject.

20. The method of claim 19, wherein the subject is suspected of relapse to a cancer.21 . The method of any one of claims 2 to 20, comprising comparing a level of said cell free nucleosomes in said plasma sample or serum sample with one or more controls.

22. The method of claim 21 , wherein the control is a healthy subject or a subject with a non-cancer disease.

23. The method of claim 21 or claim 22, wherein the level of cell free nucleosomes is elevated compared to the control.

24. The method of any preceding claim, wherein the cancer is a cancer of the bladder, brain, breast, colon, cervix, endometrium, oesophagus, kidney, large intestine, liver (such as bile duct), lung, oral cavity, ovary, pancreas, pharynx, prostate, rectum, skin (such as melanoma), stomach or thyroid, or a vascular or haematological cancer (such as AML, NHL or myeloma).

25. The method of any preceding claim, wherein the cell free nucleosome is analysed by detecting or measuring a component of the nucleosome, such as a histone variant or histone isoform.

26. The method of claim 25, wherein the cell free nucleosome is analysed by detecting or measuring an epigenetic feature of the cell free nucleosome.

27. The method of claim 26, wherein the epigenetic feature of the cell free nucleosome is a histone isoform, such as a histone isoform of a core nucleosome, in particular a histone H3 isoform, such as H3.1.

28. The method of claim 26, wherein the epigenetic feature of the cell free nucleosome is a histone post translational modification (PTM), such as a histone PTM of a core nucleosome, in particular a histone H3 PTM.

29. The method of any of claims 3-28, wherein the nucleosome binding agent comprises a detectable label.

30. The method of claim 29, wherein the nucleosome binding agent is an anti-nucleosome antibody, an anti-DNA antibody, or an anti-histone antibody linked to a detectable label.

31. The method of claim 29 or 30, wherein the detectable label is an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP); a luminescent label, such as an acridinium ester derivative; a fluorescent label; or a radioactive label.

32. The method of any of claims 39 to 31 , wherein cell free nucleosomes are analysed, detected, measured or quantified using the detectable label.

33. The method of any of any of claims 2-32, further including the step of isolating the stabilised cell free nucleosomes from the plasma sample or serum sample before analysing, detecting, or measuring or quantifying cell free nucleosomes.

34. The method of claim 33, wherein the stabilised cell free nucleosomes are isolated by a nucleosome binding agent which binds to or is bound to a solid support such as a magnetic particle, a microplate, or a plate.

35. The method of any of claims 3-34, wherein the method comprises contacting the plasma sample or serum sample with a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support, wherein the stabilised cell free nucleosomes are isolated from the plasma sample or serum sample using the second nucleosome binding agent and analysed, detected, measured or quantified using the detectable label.

36. The method of any preceding claim, wherein the method does not comprise a step of contacting the blood sample, plasma sample or serum sample with a binding agent which binds to a transcription factor.

37. Use of a stabilised nucleosome in a body fluid sample as a biomarker for cancer.

38. The use of claim 37, wherein the stabilised nucleosome is analysed, detected, measured or quantified by an immunoassay, immunochemical, mass spectroscopy, chromatographic, chromatin immunoprecipitation or biosensor method.

39. The use of claim 37 or claim 38, wherein the stabilised nucleosome is analysed, detected, measured or quantified by contacting the body fluid sample with a solid phase comprising a nucleosome binding agent that detects cell free nucleosomes or a component thereof, and detecting binding to said binding agent.

40. The use of any one of claims 37 to 39, wherein the body fluid sample is obtained from a human or an animal subject.41 . The use of any one of claims 37 to 40, wherein the body fluid sample is obtained from a subject who is suspected of relapse to a cancer.

42. The use of any one of claims 37 to 41 , comprising comparing a level of said stabilised nucleosomes with one or more controls.

43. The use of claim 42, wherein the control is a healthy subject or a subject with a noncancer disease.

44. The use of claim 42 or claim 43, wherein the level of stabilised nucleosomes is elevated compared to the control.

45. The use of any one of claims 27 to 44, wherein the cancer is a cancer of the bladder, breast, colon, cervix, oesophagus, kidney, large intestine, liver, lung, oral cavity, ovary, pancreas, prostate, rectum, skin or stomach or a vascular or haematological cancer.

46. The use of any one of claims 27 to 45, wherein the stabilised nucleosome is analysed as a biomarker for cancer by detecting or measuring a component of the nucleosome, such as a histone variant or histone isoform.

47. The use of claim 46, wherein the stabilised nucleosome is analysed as a biomarker for cancer by detecting or measuring an epigenetic feature of the cell free nucleosome.

48. The use of claim 47, wherein the epigenetic feature of the stabilised nucleosome is a histone isoform, such as a histone isoform of a core nucleosome, in particular a histone H3 isoform, such as H3.1.

49. The use of claim 47, wherein the epigenetic feature of the stabilised nucleosome is a histone PTM, such as a histone PTM of a core nucleosome, in particular a histone H3 PTM.

50. A kit comprising one or more reagents for carrying out the method as defined according to any one of claims 1 to 36.51 . Use of a kit comprising: (i) one or more reagents to detect or measure the level of cell free nucleosomes or a component thereof, and (ii) a blood collection tube comprising a stabilising agent, to detect, monitor or diagnose cancer.

52. A kit to detect, monitor or diagnose cancer in a subject, wherein said kit comprises (i) a first binding agent which specifically binds to an epigenetic feature of a cell free nucleosome (e.g. H3.1) and (ii) a second binding agent which specifically binds to cell free nucleosomes, for use with (iii) a blood collection tube comprising a stabilising agent.

53. A blood collection tube comprising a stabilising agent for a cell free nucleosome for use in the method of any one of claims 1 to 36.

54. Use of a blood collection tube comprising a stabilising agent for collecting a cell free nucleosome in a body fluid sample as a biomarker for cancer.

55. A nucleosome binding agent for use in a diagnostic method, wherein the nucleosome binding agent is used in combination with a blood collection tube comprising a crosslinking agent.

56. A nucleosome binding agent for use in a diagnostic method, wherein the method comprises obtaining a blood sample that contains cell free nucleosomes from a subject and: (i) contacting the blood sample with a cross-linking agent; (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample thatcontains cross-linked cell free nucleosomes; and (iii) contacting the plasma sample or serum sample with the nucleosome binding agent to analyse, detect, measure or quantify the cross-linked cell free nucleosomes.

57. A nucleosome binding agent for use in a method as defined in any of claims 1 to 36.

58. Use of a nucleosome binding agent to analyse, detect, measure or quantify crosslinked cell free nucleosomes.

59. A kit for analysing, detecting, measuring or quantifying cell free nucleosomes in a blood sample, wherein the kit comprises a nucleosome binding agent and instructions for carrying out the method as defined according to any one of claims 1 to 36.

60. The nucleosome binding agent for use according to any of claims 55-57, the use according to claim 58, or the kit according to claim 59, wherein the nucleosome binding agent comprises a detectable label, optionally wherein the nucleosome binding agent is an anti-nucleosome antibody, an anti-DNA antibody, or an anti-histone antibody linked to a detectable label.

61. The nucleosome binding agent for use according to claim 60, the use according to claim 60, or the kit according to claim 60, wherein the detectable label is an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP); a luminescent label, such as an acridinium ester derivative; a fluorescent label; or a radioactive label.

62. The nucleosome binding agent for use according to any of claims 55-57, the use according to claim 58, or the kit according to claim 59, wherein the nucleosome binding agent binds to or is bound to a solid support such as a magnetic particle, a microplate, or a plate.

63. The nucleosome binding agent for use according to claim 62, the use according to claim 62, or the kit according to claim 62, wherein the nucleosome binding agent is an anti-nucleosome antibody, an anti-DNA antibody, or an anti-histone antibody that binds to or is bound to a solid support, optionally wherein the nucleosome binding agent is a magnetic particle coated with an anti-nucleosome antibody, an anti-DNA antibody or an anti-histone antibody, ora biotinylated anti-nucleosome antibody, anti-DNA antibody or anti-histone antibody.

64. A combination of a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support, for use in a diagnostic method, wherein the first and second nucleosome binding agents are used in combination with a blood collection tube comprising a cross-linking agent.

65. A combination of a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support, for use in a diagnostic method, wherein the method comprises obtaining a blood sample that contains cell free nucleosomes from a subject and: (i) contacting the blood sample with a cross-linking agent; (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains cross-linked cell free nucleosomes; and (iii) contacting the plasma sample or serum sample with the first and second nucleosome binding agents to isolate the cross-linked cell free nucleosomes from the plasma sample or serum sample and analyse, detect, measure or quantify the cross-linked cell free nucleosomes.

66. A combination of a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support for use in a method as defined in any of claims 1 to 36.

67. Use of a combination of a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support to isolate and analyse, detect, measure or quantify cross-linked cell free nucleosomes.

68. A kit for analysing, detecting, measuring or quantifying cell free nucleosomes in a blood sample, wherein the kit comprises a first nucleosome binding agent comprising a detectable label, a second nucleosome binding agent which binds to or is bound to a solid support, and instructions for carrying out the method as defined according to any one of claims 1 to 36.

69. The combination for use according to any of claims 64 to 66, the use according to claim 67, or the kit according to claim 68, wherein the first nucleosome binding agent is an anti-nucleosome antibody, an anti-DNA antibody, or an anti-histone antibody linked to a detectable label.

70. The combination for use according to claim 69, the use according to claim 69, or the kit according to claim 69, wherein the detectable label is an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP); a luminescent label, such as an acridinium ester derivative; a fluorescent label; or a radioactive label.71 . The combination for use according to any of claims 64-66 or 69-70, the use according to any of claims 67 or 69-70, or the kit according to any of claims 68 or 69-70, wherein the second nucleosome binding agent is an anti-nucleosome antibody, an anti-DNA antibody, or an anti-histone antibody that binds to or is bound to a solid support.

72. The combination for use according to claim 71 , the use according to claim 71 , or the kit according to claim 71 , wherein the second nucleosome binding agent is a magnetic particle coated with an anti-nucleosome antibody, an anti-DNA antibody or an anti- histone antibody, or a biotinylated anti-nucleosome antibody, anti-DNA antibody or anti- histone antibody.

73. A cross-linking agent for use in a diagnostic method, wherein the cross-linking agent is used in combination with a nucleosome binding agent.

74. A cross-linking agent for use in a diagnostic method, wherein the method comprises obtaining a blood sample that contains cell free nucleosomes from a subject and: (i) contacting the blood sample with the cross-linking agent; (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains cross-linked cell free nucleosomes; and (iii) contacting the plasma sample or serum sample with a nucleosome binding agent to analyse, detect, measure or quantify the cross-linked cell free nucleosomes.

75. A cross-linking agent for use in a diagnostic method, wherein the cross-linking agent is used in combination with a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support.

76. A cross-linking agent for use in a diagnostic method, wherein the method comprises obtaining a blood sample that contains cell free nucleosomes from a subject and: (i) contacting the blood sample with the cross-linking agent; (ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample thatcontains cross-linked cell free nucleosomes; and (iii) contacting the plasma sample or serum sample with a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support, to isolate the cross-linked cell free nucleosomes from the plasma sample or serum sample and analyse, detect, measure or quantify the cross-linked cell free nucleosomes.

77. A cross-linking agent for use in a method as defined in any one of claims 1 to 36.

78. Use of a cross-linking agent to cross-link cell free nucleosomes prior to analysing, detecting, measuring or quantifying the cross-linked cell free nucleosomes with a nucleosome binding agent.

79. Use of a cross-linking agent to cross-link cell free nucleosomes prior to isolating and analysing, detecting, measuring or quantifying the cross-linked cell free nucleosomes with a first nucleosome binding agent comprising a detectable label and a second nucleosome binding agent which binds to or is bound to a solid support.

80. A kit for quantifying cell free nucleosomes in a blood sample, wherein the kit comprises a cross-linking agent and instructions for carrying out the method as defined according to any one of claims 1 to 36.81 . A kit for quantifying cell free nucleosomes in a blood sample, wherein the kit comprises a nucleosome binding agent and a cross-linking agent, and optionally instructions for carrying out the method as defined according to any one of claims 1 to 36.

82. A kit for quantifying cell free nucleosomes in a blood sample, wherein the kit comprises a first nucleosome binding agent comprising a detectable label, a second nucleosome binding agent which binds to or is bound to a solid support, and a cross-linking agent, and optionally instructions for carrying out the method as defined according to any one of claims 1 to 36.

83. The cross-linking agent for use according to any of claims 73-77, the use according to claim 78 or 79, or the kit according to any of claims 80-82, wherein the cross-linking agent is comprised in a blood collection tube.

84. A plasma sample or serum sample comprising cross-linked cell free nucleosomes and a nucleosome binding agent comprising a detectable label.

85. The plasma sample or serum sample according to claim 84, wherein the plasma sample or serum sample further comprises a nucleosome binding agent which binds to or is bound to a solid support.

86. A method for analysing DNA fragments which are bound to or associated with the cell free nucleosomes in a blood sample, comprising the steps of:(i) contacting a blood sample that contains cell free nucleosomes with a stabilising agent;(ii) separating plasma or serum from the blood sample, to provide a plasma sample or serum sample that contains stabilised cell free nucleosomes;(iii) analysing, detecting, measuring or quantifying the DNA fragments which are bound to or associated with the stabilised cell free nucleosomes.

87. The method of claim 86, further including the step of isolating the stabilised cell free nucleosomes from the plasma sample or serum sample before analysing the DNA fragments.

88. The method of claim 87, wherein the stabilised cell free nucleosomes are isolated by a nucleosome binding agent which binds to or is bound to a solid support such as a magnetic particle, a microplate, or a plate.

89. The method of any of claims 86 to 88, further including the step of extracting the DNA fragments which are bound to or associated with the stabilised cell free nucleosomes.

90. The method of any of claims 86 to 89, wherein the method comprises sequencing the DNA fragments.

91. The method of any of claims 86 to 90, wherein the method comprises amplifying the DNA fragments.

92. The method of any of claims 86 to 91 , wherein analysing, detecting, measuring or quantifying the DNA fragments is used to diagnose cancer in a subject, determine theprognosis of a subject with a cancer, or monitor the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a cancer.

93. A method for determining cell free nucleosome stability in a blood sample, comprising the steps of:(i) providing a first blood sample from a subject containing stabilised cell free nucleosomes and a second blood sample from the subject containing native cell free nucleosomes;(ii) separating plasma or serum from the blood, to provide a first plasma or serum sample containing stabilised cell free nucleosomes and a second plasma or serum sample containing native cell free nucleosomes; and(iii) analysing the DNA fragments which are bound to or associated with the stabilised cell free nucleosomes in the first plasma or serum sample and the DNA fragments which are bound to or associated with the native cell free nucleosomes in the second plasma or serum sample.

94. The method of claim 93, further including the step of isolating the cell free nucleosomes from the plasma or serum samples before analysing the DNA fragments.

95. The method of claim 94, wherein the cell free nucleosomes are isolated by a nucleosome binding agent which binds to or is bound to a solid support such as a magnetic particle, a microplate, or a plate.

96. The method of any of claims 93 to 95, further including the step of extracting the DNA fragments which are bound to or associated with the cell free nucleosomes.

97. The method of any of claims 93 to 96, wherein the method comprises quantifying the DNA fragments which are bound to or associated with the cell free nucleosomes, optionally by a fluorometer using a fluorescent DNA intercalating dye.

98. The method of claim 97, wherein the difference in DNA fragments quantified from the first sample and the second sample is used as a biomarker for health or disease.

99. Use of a blood collection tube comprising a stabilising agent for collecting a body fluid sample and analysing, detecting, measuring or quantifying stabilised cell free nucleoproteins.

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