WO2017027379A1 - Method for diagnosisng cancer or cancer-associated thrombosis by measuring levels of h3cit in plasma - Google Patents

Method for diagnosisng cancer or cancer-associated thrombosis by measuring levels of h3cit in plasma Download PDF

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WO2017027379A1
WO2017027379A1 PCT/US2016/045781 US2016045781W WO2017027379A1 WO 2017027379 A1 WO2017027379 A1 WO 2017027379A1 US 2016045781 W US2016045781 W US 2016045781W WO 2017027379 A1 WO2017027379 A1 WO 2017027379A1
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cancer
h3cit
plasma
biomarker
individual
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PCT/US2016/045781
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French (fr)
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Charlotte THÅLIN
Mélanie DEMERS
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Thomas Helledays Stiftelse För Medicinsk Forskning
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Priority to EP16751130.2A priority Critical patent/EP3332257A1/en
Priority to CA2993624A priority patent/CA2993624A1/en
Priority to AU2016307448A priority patent/AU2016307448A1/en
Publication of WO2017027379A1 publication Critical patent/WO2017027379A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse

Definitions

  • the present invention relates to a method for screening for the presence of cancer and/or cancer-associated thrombosis in an individual by measuring the presence and/or quantity in a plasma sample of said individual a biomarker chosen from citrullinated histone H3 (H3Cit).
  • the presence and/or quantity in the plasma sample of said citrullinated histone H3 (H3Cit) is indicating cancer and/or cancer-associated thrombosis in said individual.
  • the invention also relates to a kit for use in said method.
  • biomarkers used for a variety of tumors, such as prostate-specific antigen (PSA) for prostate cancer, carcinoembryonic antigen (CEA) for colorectal cancer and cancer antigen 125 (CA-125) for ovarian cancer.
  • PSA prostate- specific antigen
  • CEA carcinoembryonic antigen
  • CA-125 cancer antigen 125
  • the estimated sensitivity from pooled analyses is 21 % for the detection of any prostate cancer, and 51 % for the detection of high-grade cancer.
  • the specificity is 91 % (Wolf AM, et al.
  • the early stage sensitivity of CA-125 is approximated to 45% (Skates SJ et al., Preoperative sensitivity and specificity for early-stage ovarian cancer when combining cancer antigen CA- 12511, CA 15-3, CA 72-4, and macrophage colony-stimulating factor using mixtures of multivariate normal distributions, J Clin Oncol 2004).
  • NETs Neutrophil extracellular traps
  • neutrophils Upon activation, neutrophils can release chromatin (DNA and histones) coated with antimicrobial granular proteins.
  • chromatin DNA and histones coated with antimicrobial granular proteins.
  • PAD4 peptidylarginine deiminase 4
  • an enzyme that is primarily expressed in neutrophils translocate to the nucleus and citrullinates the core histones H3, H2A and H2B initiating chromatin decondensation by preventing the binding of heterochromatin binding protein 1 (HP1 ).
  • Citrullinated histone H3 (H3Cit) is thereby considered a NET specific biomarker.
  • NETs have since then been implicated in several non-infectious diseases such as autoimmune diseases, thrombosis and cancer.
  • NETs/PAD4 have been shown to be crucial for deep vein thrombosis in mice and studies have shown markers of NETs in both arterial and venous thrombosis, as well as in surrounding infarcted tissues.
  • NETs/PAD4 Although the role of NETs/PAD4 is becoming clearer in thrombosis an additional role in cancer is emerging, as some cancers express PAD4.
  • the first implication of NETs in cancer- associated thrombosis was reported by Demers et al in 2012 (Demers M, et al. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci USA
  • G-CSF granulocyte-colony stimulating factor
  • PAD4 is highly expressed in neutrophils but is also upregulated in many cancers where it negatively regulates tumor suppressor genes of the p53 pathway and thus promote tumor growth. In vitro, its overexpression in cancer cells results in histone citrullination and chromatin decondensation. The release of NET-like structures by the cancer cells may thus also be a potential source of the NET burden in cancer.
  • NETs such as the enzymes coating the chromatin in NETs; neutrophil elastase (NE), myeloperoxidase (MPO) and cell free DNA.
  • NE neutrophil elastase
  • MPO myeloperoxidase
  • cell free DNA may be released from necrotic tissues following tissue injury and vessel wall damage.
  • MPO-DNA complexes Some studies also used MPO-DNA complexes but MPO is also highly expressed in monocyte/macrophages and since MPO is a secreted protein and it is highly positively charged, it is possible that the electrical attraction cause MPO to bind to any negatively charged DNA that could be released in the plasma, thus questioning its specificity as a NET marker. Clear clinical need of a better method for analyzing and quantifying a NET specific biomarker
  • H3Cit Detection and quantification of the NET specific biomarker H3Cit would be of better diagnostic value than the surrogate biomarkers of NETs. H3Cit in plasma has so far only been detected qualitatively using Western Blot analysis. No method has thus far been able to quantify H3Cit in plasma. Such a method would add a clear clinical value since the quantity of plasma H3Cit is a biomarker for the quantity of plasma NETs. The quantity/level of NETs in plasma can be a useful tool in the diagnosis of a variety of diseases.
  • cancer In spite of many advances in treatments of cancer, cancer remains a leading cause of death. The disease is often diagnosed at a too late stage to be able to use treatments with curative intent and there is a large unmet clinical need to detect cancers earlier, to be able to increase cure rates. A new method for screening for cancer disease in general is needed.
  • the present invention is related to a method for screening for the presence of cancer in an individual comprising the steps:
  • screening for the presence of cancer we include testing to identify whether the individual has cancer. Therefore, by “indicating cancer” we include that a result obtained from the method is used to identify the presence of cancer in the individual.
  • plasma sample we include a blood plasma sample. It will be appreciated that plasma is a general term for the liquid component of blood. Those skilled in the art of medicine, in particular a general practitioner, oncologist or nurse, will be familiar with what is required to provide a plasma sample, such as obtaining a blood sample from a patient and then isolating a plasma sample from the cellular component of the blood, for example by centrifugation.
  • H3Cit histone 3
  • PADI peptidylarginine deiminase
  • PAD1 PAD2
  • PAD3 PAD3
  • PAD4 PAD4
  • citrullination is a general term for the conversion of the amino acid arginine to the amino acid citrulline.
  • the N-terminus of H3 is citrullinated at arginine residues at amino acid positions 2, 8 and 17.
  • H3 has the amino acid sequence A RT KQTA R KSTG G KA P R KQ L AT KA A R KS (SEQ ID NO: 1 ) (Wang Y, et al. Human PAD4 Regulates Histone Arginine Methylation Levels via Demethylimination. Science 2004;306:279-283).
  • H3Cit is a biomarker that can be used to detect cancer, cancer associated thrombosis, and thrombosis associated with cancer.
  • the association between those conditions and H3Cit is particularly surprising as H3Cit had previously not been associated with cancer, cancer associated thrombosis, or thrombosis associated with cancer.
  • the methods described herein are based upon those new findings.
  • the present invention is related to a method for screening for cancer-associated thrombosis or an elevated risk for cancer-associated thrombosis in cancer individuals or for screening for cancer in individuals with idiopathic thrombosis, comprising the steps:
  • Screening for cancer-associated thrombosis we include testing to identify whether the individual has thrombosis associated, in full or in part, by cancer in the individual. Therefore, by "indicating cancer-associated thrombosis” we include that a result obtained from the method is used to identify the presence of cancer-associated thrombosis in the individual.
  • screening for elevated risk for cancer-associated thrombosis in cancer individuals we include testing to identify whether the individual with cancer has an increased risk of developing thrombosis. Therefore, by "indicating an elevated risk for cancer-associated thrombosis” we include that a result obtained from the method is used to establish that the individual has an increased risk of cancer-associated thrombosis.
  • screening for cancer in individuals with idiopathic thrombosis we include testing to identify whether an individual already diagnosed with an idiopathic thrombosis (such as a de novo thrombosis and/or a thrombosis that occurred spontaneously and/or a thrombosis that has an unknown cause) also has cancer. Therefore, by "indicating cancer in said individuals with idiopathic thrombosis” we include that a result obtained from the method is used to identify the presence of cancer in the individual with idiopathic thrombosis.
  • thrombosis thrombosis
  • cancer-associated thrombosis thrombosis
  • idiopathic thrombosis a method for screening for thrombosis or an elevated risk for thrombosis in cancer individuals following chemotherapy, comprising the steps:
  • thrombosis we include that a result obtained from the method is used to identify the presence of thrombosis in the individual with cancer who has received chemotherapy.
  • screening for elevated risk for thrombosis in cancer individuals following chemotherapy we include testing to identify whether the individual with cancer that has received chemotherapy is at risk of developing thrombosis. Therefore, by "elevated risk for thrombosis in said cancer individuals” we include that a result obtained from the method is used to establish that the cancer individual has an increased risk of thrombosis.
  • a method for screening for tumour progression in individuals or screening for recurrence of cancer in individuals with previously diagnosed cancer comprising the steps:
  • said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating tumour progression in said individuals or cancer in said individuals with previously diagnosed cancer.
  • tumour prognosis are general terms that can relate to ascertaining an improved tumour prognosis (such as a reduction in the number of tumour symptoms and/or a reduction in the severity of tumour symptoms and/or a reduction in tumour metastasis and/or a reduced risk of the tumour being fatal) or ascertaining a worsened tumour prognosis (such as an increase in the number of tumour symptoms and/or an increase in the severity of tumour symptoms and/or an increase in tumour metastasis and/or an increased risk of the tumour being fatal). Therefore, by "indicating tumour progression” we include that a result obtained from the method is used to provide a tumour prognosis, such as an improved tumour prognosis or a worsened tumour prognosis.
  • screening for recurrence of cancer in individuals with previously diagnosed cancer we include testing to identify a relapsed cancer, such as identifying whether an individual that has previously recovered from cancer again has cancer. Accordingly, by “recovered from said cancer” we include that the individual previously fully recovered from said cancer (for example, the individual no longer exhibited cancer symptoms) or that the individual partially previously recovered from said cancer (for example, the individual exhibited a reduced number of symptoms and/or exhibited a reduced severity of symptoms). If the individual partially previously recovered from said cancer by "has cancer again” we include that the individual has an increased number of symptoms and/or an increased severity in symptoms. Therefore, by "indicating cancer in said individuals with previously diagnosed cancer” we include that a result obtained from the method is used to identify the presence of relapsed cancer in the individual.
  • a method for screening for cancer progression in individuals comprising the steps: a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or the quantity in the plasma sample of a biomarker chosen from citrullinated histone H3 (H3Cit),
  • screening for cancer progression we include testing to ascertain a cancer prognosis.
  • screening for cancer progression and “cancer prognosis” are general terms that can relate to ascertaining an improved cancer prognosis (such as a reduction in the number of cancer symptoms and/or a reduction in the severity of cancer symptoms and/or a reduction in cancer metastasis and/or a reduced risk of the cancer being fatal) or ascertaining a worsened cancer prognosis (such as an increase in the number of cancer symptoms and/or an increase in the severity of cancer symptoms and/or an increase in cancer metastasis and/or an increased risk of the cancer being fatal). Therefore, by "indicating cancer progression” we include that a result obtained from the method is used to provide a cancer prognosis, such as an improved cancer prognosis or a worsened cancer prognosis.
  • a method for screening for cancer, an elevated risk for cancer-associated thrombosis and/or cancer-associated thrombosis in individuals comprising the steps:
  • biomarkers chosen from citrullinated histone H3 (H3Cit) and granulocyte colony stimulating factor (G-CSF),
  • said presence and/or quantity in the test sample of said biomarkers chosen from citrullinated histone H3 (H3Cit) and granulocyte colony stimulating factor (G-CSF) is indicating cancer, an elevated risk for cancer-associated thrombosis and/or cancer- associated thrombosis in said individuals.
  • G-CSF Granulocyte colony stimulating factor
  • GCSF colony-stimulating factor 3
  • Human G-CSF is a 19.6 kDa protein with 174 amino acid residues:
  • the DNA sequence of G-CSF is: ctgccgcttc caggcgtcta tcagcggctc agcctttgtt cagctgttct gttcaaacac tctggggcca ttcaggcctg ggtggggcag cgggaggaag ggagtttgag gggggcaagg cgacgtcaaa ggaggatcag agattccaca atttcacaaa actttcgcaa acagcttttt gtccaaccc cctgcattg tcttggacac caaatttgca taaatcctgg gaagttatta ctaagcctta gtggccc caggtaattttcccaggc ctccatgggggg tgg tgg t
  • G-CSF granulocyte colony stimulating factor
  • said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating adverse effects in individuals receiving granulocyte colony stimulating factor (G-CSF) following chemotherapy.
  • G-CSF is administered after chemotherapy in order to reduce chemotherapy associated neutropenia (ie a low concentration of neutrophils) and the incidence of febrile neutropenia (ie a fever associated with a low concentration of neutrophils).
  • G-CSF granulocyte colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • Fig 1 Depicts plasma H3Cit measured with our tailor-made ELISA, demonstrating plasma H3Cit in patients with ischemic stroke with and without an underlying active cancer.
  • Fig 2. depicts Western Blot analysis with qualitative detection of H3Cit in plasma. Ischemic stroke patients with cancer had detectable H3Cit in plasma, whereas ischemic stroke patients without cancer had no detectable H3Cit in plasma. This clearly demonstrates that our tailor made ELISA was successful as it was done on the same samples. The added value of the tailor made ELISA compared to the Western Blot analysis is the
  • the tailored made method not only detects H3Ct in plasma (as do Western Blot Analysis) but also quantifies the level of H3Cit in plasma, which we believe is key in achieving substantial clinical value with our method.
  • Fig 3. Depicts plasma G-CSF in patients with and without cancer.
  • Ischemic stroke patients with cancer had a seven fold increase in plasma G- CSF compared to ischemic stroke patients without cancer.
  • the boxplot depicts a min-max value with median.
  • Fig 4. depicts correlation between plasma H3Cit and plasma G-CSF.
  • the positive correlation between elevations of plasma G-CSF and plasma H3Cit in patients with cancer supports a cancer-released G-CSF-induced NET burden. Significance of correlation was analyzed with Spearman's rank correlation using ST ATA 12.1 software (STATA, Texas, USA).
  • Fig 5. depicts the stability of the standard (STD) in our tailor-made H3Cit ELISA-based assay.
  • STD standard
  • Fig 6. depicts the stability of the standard (STD) in our tailor-made H3Cit ELISA-based assay.
  • Fig 7. depicts standard (STD) curves generated from in vitro PAD4- citrullinated histone H3 and from histone H3 incubated in the same
  • Fig 8. depicts concentrations of H3Cit, measured with tailor-made H3Cit ELISA-based assay, in plasma samples taken from healthy individuals before and after LPS injection. The quantification of H3Cit in plasma of healthy volunteers before LPS injections were under the detection limit of approximately 5 ng/mL.
  • H3Cit was measured with our tailor-made H3Cit ELISA-based assay.
  • Fig 9. depicts the age and sex distribution of the study population in Example 3.
  • the boxplot depicts a min-max value with median. There was no significant differences in age or sex distribution between the patients with active cancer (median age 70.5, 41 .7% men), hospitalized patients with other diseases but no known cancer (median age 77, 40% men), and the healthy individuals (median age 70.4, 42% men).
  • Fig 10. depicts plasma levels of H3Cit in Example 3.
  • Cancer patients had a 5-fold increase in plasma H3Cit levels compared to healthy individuals (mean 36.1 ng/mL vs. 6.6 ng/mL, *** p ⁇ 0.001 ) and a 3-fold increase compared to hospitalized patients with other diseases but no known cancer (mean 36.1 ng/mL vs. 1 1 .5 ng/mL, *** p ⁇ 0.001 ).
  • “Other” comprises gingival, liposarcoma, sarcoma, acute myeloid leukemia, lymphoma, neuroendocrine and unknown origin, see also Table 2 under Example 3).
  • Plasma H3Cit was measured with an ELISA of the invention, demonstrating a clear elevation in several malignant tumor types (both epithelial and other solid malignant tumors as well as hematological cancers), suggesting the potential of H3Cit as a biomarker in screening for several cancer types (ie cancer in general).
  • Fig 12. depicts the Charlson comorbidity index scores (Charlson ME, et al. A new method of classifying prognostic comorbidity in longitudinal studies: Development and validation. 1987 Journal of Chronic Diseases. 1987;40:373-83) for cancer patients and hospitalized patients with other diseases but no known cancer (referred to as multisick in the figure).
  • the boxplot depicts a min-max value with median.
  • the cancer patients had a significantly lower comorbidity index scores compared to hospitalized patients with other diseases but no known cancer (median 3 vs. 6, p ⁇ 0.001 ), suggesting that plasma H3Cit is not a biomarker for disease burden in general.
  • Detailed description of the invention depicts the Charlson comorbidity index scores (Charlson ME, et al. A new method of classifying prognostic comorbidity in longitudinal studies: Development and validation. 1987 Journal of Chronic Diseases. 1987;40:373-83) for cancer patients and hospitalized patients with other diseases but
  • the present invention is related to a method for diagnosing several different conditions as described above by measuring the presence and/or quantity of H3Cit in a plasma sample.
  • H3Cit such as in urine, cerebrospinal fluid, sputum and nasopharynx, wherein said presence and/or quantity in the sample tested indicates that the individual has the condition as tested for.
  • the plasma sample is provided from venous blood.
  • Venous blood is a term that will be well understood by the person skilled in medicine, in particular an oncologist.
  • the H3Cit is a histone 3 that is citrullinated at one or more arginine residues at amino acid positions 2, 8 or 17.
  • the H3Cit is a histone 3 that is citrullinated at arginine residues at amino acid positions 2, 8 and 17.
  • step b) comprises measuring the optical density of the plasma sample, which is further described in the experimental part.
  • the optical density represents the presence and/or quantity of the biomarker (such as H3Cit) - for example, the optical density is proportional to the presence and/or the quantity of the biomarker. Put in another way, if the optical density is high the presence and/or the quantity of the biomarker is also high.
  • Methods of measuring optical density will be well known to those skilled in the art of biology and chemistry, in particular a molecular biologist or biochemist, such as measuring optical density using spectrometry.
  • a high optical density of the plasma sample is indicating cancer ⁇ i.e. is indicative of cancer) in said individual.
  • a high optical density of the plasma sample is indicating cancer-associated thrombosis ⁇ i.e. is indicative of cancer-associated thrombosis) or an elevated risk for cancer- associated thrombosis in said cancer individuals or cancer in said individuals with idiopathic thrombosis.
  • a high optical density of the plasma sample is indicating thrombosis ⁇ i.e. is indicative of thrombosis) or an elevated risk for thrombosis in said cancer individuals following chemotherapy.
  • a high optical density of the plasma sample is indicating tumour progression ⁇ i.e. is indicative of tumour progression) in said individuals or cancer in said individuals with previously diagnosed cancer.
  • a high optical density of the plasma sample is indicating cancer progression ⁇ i.e. is indicative of cancer progression).
  • a high optical density of the plasma sample is indicative of cancer, an elevated risk for cancer-associated thrombosis and/or cancer-associated thrombosis in said individuals.
  • a high optical density of the plasma sample is indicating adverse effects ⁇ i.e. is indicative of adverse effects) in individuals receiving granulocyte colony stimulating factor (G-CSF) following chemotherapy.
  • G-CSF granulocyte colony stimulating factor
  • the high optical density is an optical density in the range of 0.1 to 4 - for example, an optical density of 0.15 to 4, an optical density of 0.2 to 4, an optical density of 0.22 to 4, an optical density of 0.25 to 4, an optical density of 0.3 to 4, an optical density of 0.35 to 4, an optical density of 0.4 to 4, an optical density of 0.45 to 4, an optical density of 0.5 to 4, an optical density of 0.6 to 4, an optical density of 0.7 to 4, an optical density of 0.8 to 4, an optical density of 0.9 to 4, an optical density of 1 .0 to 4, an optical density of 1 .1 to 4, an optical density of 1 .2 to 4, an optical density of 1 .3 to 4, an optical density of 1 .4 to 4, an optical density of 1 .5 to 4, an optical density of 1 .6 to 4, an optical density of 1 .7 to 4, an optical density of 1 .8 to 4, an optical density of 1 .9 to 4, an optical density of 2 to 4, an optical density of 2.1 to 4, an optical density of 2.2 to 4, an optical density of 0.15
  • the high optical density is an optical density of 0.1 or more - for example, an optical density of 0.15 or more, an optical density of 0.2 or more, an optical density of 0.25 or more, an optical density of 0.3 or more, an optical density of 0.35 or more, an optical density of 0.4 or more, an optical density of 0.45 or more, an optical density of 0.5 or more, an optical density of 0.6 or more, an optical density of 0.7 or more, an optical density of 0.8 or more, an optical density of 0.9 or more, an optical density of 1 .0 or more, an optical density of 1 .1 or more, an optical density of 1 .2 or more, an optical density of 1 .3 or more, an optical density of 1 .4 or more, an optical density of 1 .5 or more, an optical density of 1 .6 or more, an optical density of 1 .7 or more, an optical density of 1 .8 or more, an optical density of 1 .9 or more, an optical density of 2 or more, an optical density of 2.1 or
  • the high optical density is an optical density of 0.22 or more.
  • step b) comprises comparing the presence and/or the quantity of the H3Cit biomarker in the plasma sample with the presence and/or the quantity of the H3Cit biomarker in one or more plasma samples from healthy individual(s) (for example, a healthy individual) having known concentrations of citrullinated histone H3 (H3Cit) in said plasma samples.
  • healthy individual we include an individual that does not have cancer and/or an individual that does not have thrombosis and/or an individual that does not have cancer-associated thrombosis and/or an individual with no known cancer - for example, the individual does not have cancer or thrombosis.
  • the healthy individual is one or more healthy individual(s) - for example a population of a number of healthy individuals, such as 10 or more healthy individuals, 20 or more healthy individuals, 30 or more healthy individuals, 40 or more healthy individuals, 50 or more healthy individuals, 60 or more healthy individuals, 70 or more healthy individuals, 80 or more healthy individuals, 90 or more healthy individuals, 100 or more healthy individuals, 200 or more healthy individuals, 300 or more healthy individuals, 400 or more healthy individuals, 500 or more healthy individuals, 1000 or more, 2000 or more healthy individuals, or 3000 or more healthy individuals.
  • healthy individuals such as 10 or more healthy individuals, 20 or more healthy individuals, 30 or more healthy individuals, 40 or more healthy individuals, 50 or more healthy individuals, 60 or more healthy individuals, 70 or more healthy individuals, 80 or more healthy individuals, 90 or more healthy individuals, 100 or more healthy individuals, 200 or more healthy individuals, 300 or more healthy individuals, 400 or more healthy individuals, 500 or more healthy individuals, 1000 or more, 2000 or more healthy individuals, or 3000 or more healthy individuals.
  • the healthy individual is a population of 1000 or more healthy individuals.
  • step b) comprises measuring the optical density of the plasma sample and comparing to the optical density of one or more plasma samples from healthy individual(s) having known concentrations of citrullinated histone H3 (H3Cit) in said plasma samples.
  • a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of cancer in said individual.
  • a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of cancer-associated thrombosis or an elevated risk for cancer-associated thrombosis in said cancer individuals or cancer in said individuals with idiopathic thrombosis.
  • a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of thrombosis or an elevated risk for thrombosis in said cancer individuals following
  • a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of tumour progression in said individuals or cancer in said individuals with previously diagnosed cancer.
  • a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of cancer progression in said individuals.
  • a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of cancer, an elevated risk for cancer-associated thrombosis and/or cancer-associated thrombosis in said individuals.
  • a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of adverse effects in individuals receiving granulocyte colony stimulating factor (G-CSF) following chemotherapy.
  • G-CSF granulocyte colony stimulating factor
  • a manner in which to define whether the optical density in the test sample is higher than the optical density plasma samples from healthy individual(s) is by calculating a fold change. How to calculate a fold change for this purpose will be well known to those skilled in biology, in particular a molecular biologist or a computational biologist. Generally, when a fold change is calculated it is based upon a comparison between a test sample and the mean or median of the plasma samples from a healthy individual(s).
  • the optical density of the plasma sample is two-fold higher or more than the mean or median optical density of one or more plasma samples from healthy individual(s) - for example, three-fold higher or more, four-fold higher or more, five-fold higher or more, six-fold higher or more, seven-fold higher or more, eight-fold higher or more, nine-fold higher or more, or ten-fold higher or more than the optical density of one or more plasma samples from healthy individual(s).
  • the optical density of the plasma sample is three-fold higher or more than the mean or median optical density of one or more plasma samples from healthy individual(s).
  • the optical density of the plasma sample is two- fold higher or more than the mean or median optical density of one or more plasma samples from healthy individual(s).
  • the optical density of the plasma sample is fivefold higher or more than the mean or median optical density of one or more plasma samples from healthy individual(s).
  • step b) further comprises comparing the presence and/or the quantity of the H3Cit biomarker in the plasma sample with one or more standard sample(s) having known concentrations of citrullinated histone H3 (H3Cit), to identify the concentration of the H3Cit biomarker in the plasma sample.
  • H3Cit citrullinated histone H3
  • step b) further comprises comparing the presence and/or the quantity of the H3Cit biomarker in the plasma samples from healthy individual(s) with one or more standard sample(s) having known
  • H3Cit citrullinated histone H3
  • the standard sample is histone H3 citrullinated in vitro by a PAD enzyme, such as PAD4, to form H3Cit.
  • a PAD enzyme such as PAD4
  • the histone H3 is human recombinant histone H3.
  • the PAD4 is human recombinant PAD4.
  • An exemplary method is to incubate human recombinant histone H3 and human recombinant PAD4 at 37 °C for 1 hour in a reaction buffer (50 imM Trizma base with 4 mM CaCI2, pH 7.6, 4 mM DTT and 1 mM PMSF), and make a final concentration of 10,000 ng/mL H3Cit by adding PBS-1 % BSA.
  • a reaction buffer 50 imM Trizma base with 4 mM CaCI2, pH 7.6, 4 mM DTT and 1 mM PMSF
  • the one or more standard samples are one or more standard samples having different known concentrations of H3Cit.
  • the standard samples having different known concentrations of H3Cit are used to calculate a standard curve.
  • concentrations of H3Cit to calculate a standard curve will be known to those skilled in molecular biology.
  • An exemplary method could be to make an original standard sample with a known concentration of H3Cit, and then to serially dilute that original standard sample to produce different standard samples with known concentrations of H3Cit.
  • the experimental read-out (for example, an optical density) of those different standard samples with known concentrations of H3Cit can be measured, and plotted onto a graph to calculate a standard curve.
  • the experimental read-out of the H3Cit in the plasma sample can then be measured and compared to the standard curve, to provide a measurement of the quantity of the H3Cit in that plasma sample.
  • H3Cit concentrations of H3Cit are used to calculate a standard curve, with an original standard sample (for example, with a H3Cit concentration of 10,000 ng/ml) being serially diluted each time (for example, at a ratio of 1 :2).
  • step b) further comprises comparing the presence and/or the quantity of the H3Cit biomarker in the plasma sample, and/or the presence and/or the quantity the plasma samples from healthy individual(s), with a standard curve of known concentrations of citrullinated histone H3 (H3Cit), to identify the concentration of the H3Cit biomarker.
  • said presence and/or quantity of the H3Cit biomarker in the test sample is a concentration in the range of 5 ng/ml to 350 ng/ml - for example, 10 ng/ml to 350 ng/ml, 15 ng/ml to 350 ng/ml, 20 ng/ml to 350 ng/ml, 25 ng/ml to 350 ng/ml, 30 ng/ml to 350 ng/ml, 35 ng/ml to 350 ng/ml, 40 ng/ml to 350 ng/ml, 45 ng/ml to 350 ng/ml, 50 ng/ml to 350 ng/ml, 60 ng/ml to 350 ng/ml, 70 ng/ml to 350 ng/ml, 80 ng/ml to 350 ng/ml, 90 ng/ml to 350 ng/ml, 100 ng/ml to 350 ng/ml, 1 10 ng/ml to 350 -ml
  • said presence and/or quantity of the H3Cit biomarker in the test sample is a concentration of 5 ng/ml or more - for example, 10 ng/ml or more, 15 ng/ml or more, 20 ng/ml or more, 25 ng/ml or more, 30 ng/ml or more, 35 ng/ml or more, 40 ng/ml or more, 45 ng/ml or more, 50 ng/ml or more, 60 ng/ml or more, 70 ng/ml or more, 80 ng/ml or more, 90 ng/ml or more, 100 ng/ml or more, 1 10 ng/ml or more, 120 ng/ml or more, 130 ng/ml or more, 140 ng/ml or more, 150 ng/ml or more, 200 ng/ml or more, 250 ng/ml or more, 300 ng/ml or more, or 350 ng/ml or more.
  • said presence and/or quantity of the H3Cit biomarker in the test sample is a concentration of 20 ng/ml or more.
  • the presence and/or the quantity of the H3Cit biomarker in the plasma sample is higher than the presence and/or the quantity of the H3Cit biomarker in one or more plasma samples from a healthy individual(s).
  • the presence and/or the quantity of the H3Cit biomarker in the one or more plasma samples from healthy individual(s) is a concentration in the range of 0 ng/ml to 50 ng/ml - for example, 0 ng/ml to 1 ng/ml, 0 ng/ml to 2 ng/ml, 0 ng/ml to 3 ng/ml, 0 ng/ml to 4 ng/ml, 0 ng/ml to 5 ng/ml, 0 ng/ml to 6 ng/ml, 0 ng/ml to 7 ng/ml, 0 ng/ml to 8 ng/ml, 0 ng/ml to 9 ng/ml, 0 ng/ml to 10 ng/ml, 0 ng/ml to 12 ng/ml, 0 ng/ml to 14 ng/ml, 0 ng/ml to 16 ng/ml,
  • the presence and/or the quantity of the H3Cit biomarker in the one or more plasma samples from healthy individual(s) is a concentration of 50 ng/ml or less - for example, 1 ng/ml or less, 2 ng/ml or less, 3 ng/ml or less, 4 ng/ml or less, 5 ng/ml or less, 6 ng/ml or less, 7 ng/ml or less, 8 ng/ml or less, 9 ng/ml or less, 10 ng/ml or less, 12 ng/ml or less, 14 ng/ml or less, 16 ng/ml or less, 18 ng/ml or less, 20 ng/ml or less, 25 ng/ml or less, 30 ng/ml or less, 35 ng/ml or less, 40 ng/ml or less, or 45 ng/ml or less. Even more preferably, the presence and/or the quantity of the H3Cit biomarker in the
  • the presence and/or the quantity of the H3Cit biomarker in the plasma sample is two-fold higher or more than the presence and/or the mean or median quantity of the H3Cit biomarker in one or more plasma samples from healthy individual(s) - for example, three-fold higher or more, four-fold higher or more, five-fold higher or more, six-fold higher or more, seven-fold higher or more, eight-fold higher or more, nine-fold higher or more, or ten-fold higher or more than the presence and/or the quantity of the H3Cit biomarker in one or more plasma samples from a healthy individual(s).
  • the presence and/or the quantity of the H3Cit biomarker in the plasma sample is three-fold higher or more than the presence and/or the mean or median quantity of the H3Cit biomarker in one or more plasma samples from a healthy individual(s).
  • the presence and/or the quantity of the H3Cit biomarker in the plasma sample is two-fold higher or more than the presence and/or the mean or median quantity of the H3Cit biomarker in one or more plasma samples from a healthy individual(s).
  • the presence and/or the quantity of the H3Cit biomarker in the plasma sample is five-fold higher or more than the presence and/or the mean or median quantity of the H3Cit biomarker in one or more plasma samples from a healthy individual(s).
  • step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time;
  • step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a
  • step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time; and step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of cancer-associated thrombosis or an elevated risk for cancer-associated thrombosis in said cancer individuals or cancer in said individuals with idiopathic thrombosis.
  • H3Cit citrullinated histone H3
  • step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time;
  • step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of thrombosis or an elevated risk for thrombosis in said cancer individuals following chemotherapy.
  • H3Cit citrullinated histone H3
  • step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time;
  • step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of tumour progression in said individuals or cancer in said individuals with previously diagnosed cancer.
  • step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time;
  • step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of cancer progression in said individuals.
  • step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time;
  • step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of cancer, an elevated risk for cancer-associated thrombosis and/or cancer-associated thrombosis in said individuals.
  • H3Cit citrullinated histone H3
  • step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time;
  • step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of adverse effects in individuals receiving granulocyte colony stimulating factor (G-CSF) following chemotherapy.
  • G-CSF granulocyte colony stimulating factor
  • the number of plasma samples is two or more - for example, three or more, four or more, five or move, six or more, seven or more, eight or more, nine or more, ten or more, 15 or more, 20 or more, 30 or more 40 or more, or 50 or more.
  • the period of time is one week or more - for example, two weeks or more, three weeks or more, four weeks or more, one month or more, two months or more, three months of more, four months or more, five months or more, six months or move, seven months or more, eight months or more, nine months or more, or one year or more.
  • the period of time is one month.
  • the number of plasma samples are provided at equal time intervals during the period of time - for example, when two plasma samples are provided in a one week period of time, one plasma sample is provided at the first day of the week and a second plasma sample is provided at the seventh day of the week.
  • a consistent presence and/or quantity of citrullinated histone H3 is a presence and/or quantity of citrullinated histone H3 (H3Cit) not changing by 1 % or more when compared to the presence and/or quantity of citrullinated histone H3 (H3Cit) in the preceding plasma sample - for example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.
  • a consistent presence and/or quantity of citrullinated histone H3 is a presence and/or quantity of citrullinated histone H3 (H3Cit) not changing by 20% or more when compared to the presence and/or quantity of citrullinated histone H3 (H3Cit) in the preceding plasma sample.
  • step b) further comprises measuring the presence and/or the quantity in the plasma sample of G-CSF,
  • step b) further comprises measuring the presence and/or the quantity in the plasma sample of G-CSF,
  • said presence and/or quantity in the test sample of G-CSF is indicative of cancer-associated thrombosis or an elevated risk for cancer- associated thrombosis in said cancer individuals or cancer in said individuals with idiopathic thrombosis.
  • step b) further comprises measuring the presence and/or the quantity in the plasma sample of G-CSF,
  • step b) further comprises measuring the presence and/or the quantity in the plasma sample of G-CSF, wherein said presence and/or quantity in the test sample of G-CSF is indicative of tumour progression in said individuals or cancer in said
  • step b) further comprises measuring the presence and/or the quantity in the plasma sample of G-CSF,
  • step b) further comprises measuring the presence and/or the quantity in the plasma sample of G-CSF,
  • the concentration of G-CSF in the plasma sample can be calculated using methods similar to those outlined above for H3Cit, as will be known to those skilled in molecular biology.
  • said presence and/or quantity of the G-CSF biomarker in the test sample is a concentration in the range of 5 pg/ml to 100 pg/ml - for example, 10 pg/ml to 100 pg/ml, 15 pg/ml to 100 pg/ml, 20 pg/ml to 100 pg/ml, 21 pg/ml to 100 pg/ml, 25 pg/ml to 100 pg/ml, 30 pg/ml to 100 pg/ml, 35 pg/ml to 100 pg/ml, 40 pg/ml to 100 pg/ml, 45 pg/ml to 100 pg/ml, 50 pg/ml to 100 pg/ml, 60 pg/ml to 100 pg/ml, 70 pg/ml to 100 pg/ml, 80 pg/ml to 100 pg
  • said presence and/or quantity of the G-CSF biomarker in the test sample is a concentration of 5 pg/ml or more - for example, 10 pg/ml or more, 15 pg/ml or more, 20 pg/ml or more, 25 pg/ml or more, 30 pg/ml or more, 35 pg/ml or more, 40 pg/ml or more, 45 pg/ml or more, 50 pg/ml or more, 60 pg/ml or more, 70 pg/ml or more, 80 pg/ml or more, 90 pg/ml or more, or 100 pg/ml or more. Even more preferably, said presence and/or quantity of the G-CSF biomarker in the test sample is a concentration of 21 pg/ml or more.
  • step b) comprises comparing the presence and/or the quantity of the G-CSF biomarker in the plasma sample with the presence and/or the quantity of the biomarker in one or more plasma samples from healthy individual(s) having known concentrations of G-CSF in said plasma samples.
  • the presence and/or the quantity of the G-CSF biomarker in the plasma sample is higher than the presence and/or the quantity of the G- CSF biomarker in one or more plasma samples from a healthy individual.
  • the presence and/or the quantity of the G-CSF biomarker in the one or more plasma samples from healthy individual(s) is a concentration of 5 pg/ml or less - for example, 1 pg/ml or less, 2 pg/ml or less, 3 pg/ml or less, or 4 pg/ml or less.
  • the presence and/or the quantity of the G-CSF biomarker in the one or more plasma samples from healthy individual(s) is a concentration in the range of 0 pg/ml to 5 pg/ml - for example, 0 pg/ml to 1 pg/ml, 0 pg/ml to 2 pg/ml, 0 pg/ml to 3 pg/ml to 0 pg/ml, or 0 pg/ml to 4 pg/ml.
  • the presence and/or the quantity of the G-CSF biomarker in the one or more plasma samples from healthy individual(s) is a concentration of 2 ng/ml or less.
  • the presence and/or the quantity of the G-CSF biomarker in the one or more plasma samples from healthy individual(s) is a concentration of 3 pg/ml or less.
  • the presence and/or the quantity of the G-CSF biomarker in the plasma sample is two-fold higher or more than the presence and/or the median or mean quantity of the G-CSF biomarker in one or more plasma samples from healthy individual(s) - for example, three-fold higher or more, four-fold higher or more, five-fold higher or more, six-fold higher or more, seven-fold higher or more, eight-fold higher or more, nine-fold higher or more, or ten-fold higher or more than the presence and/or the quantity of the G-CSF biomarker in one or more plasma samples from a healthy individual.
  • the presence and/or the median or mean quantity of the G-CSF biomarker in the plasma sample is seven-fold higher or more than the presence and/or the quantity of the G-CSF biomarker in one or more plasma samples from a healthy individual.
  • cancer screening methods for example screening for prostate cancer using prostate specific antigen (PSA).
  • PSA prostate specific antigen
  • the method further comprises step a') conducting a further cancer screening method
  • step b) further comprises that the result of the cancer screening method is also indicative of cancer in said individual.
  • the method further comprises step a') conducting a further cancer screening method
  • step b) further comprises that the result of the cancer screening method is also indicative of cancer-associated thrombosis or an elevated risk for cancer-associated thrombosis in said cancer individuals or cancer in said individuals with idiopathic thrombosis.
  • the method further comprises step a') conducting a further cancer screening method
  • step b) further comprises that the result of the cancer screening method is also indicative of tumour progression in said individuals or cancer in said individuals with previously diagnosed cancer.
  • the method further comprises step a') conducting a further cancer screening method
  • step b) further comprises that the result of the cancer screening method is also indicative of cancer progression in said individuals.
  • the method further comprises step a') conducting a further cancer screening method;
  • step b) further comprises that the result of the cancer screening method is also indicative of cancer, an elevated risk for cancer- associated thrombosis and/or cancer-associated thrombosis in said
  • the cancer screening method is for detecting prostate cancer, for example the cancer screening method for detecting prostate specific antigen (PSA).
  • PSA prostate specific antigen
  • the cancer screening method is for detecting colorectal cancer, for example the cancer screening method for detecting CEA or the cancer screening method is a colonoscopy.
  • the cancer screening method for detecting ovarian cancer for example the cancer screening method for detecting CA-125.
  • the cancer screening method is for detecting breast cancer, for example the cancer screening method is a mammography.
  • step b) comprises using a binding agent capable of binding to the biomarker citrullinated histone H3 (H3Cit) in the plasma of the individual, such as an anti-H3Cit-antibody or a fragment thereof, i.e. a natural or synthetic binding agent capable of binding to the H3Cit as present in the plasma or other body fluid of the individual.
  • a binding agent capable of binding to the biomarker citrullinated histone H3 (H3Cit) in the plasma of the individual such as an anti-H3Cit-antibody or a fragment thereof, i.e. a natural or synthetic binding agent capable of binding to the H3Cit as present in the plasma or other body fluid of the individual.
  • the binding agent (such as an antibody) has binding specificity for a H3Cit that is citrullinated at one or more arginine residues at amino acid positions 2, 8 or 17.
  • the binding agent (such as an antibody) has binding specificity for a H3Cit that is citrullinated at arginine residues at amino acid positions 2, 8 and 17.
  • step b) comprises measuring the presence and/or the quantity of the biomarker G-CSF
  • step b) further comprises using a binding agent capable of binding to the biomarker G-CSF in the plasma of the individual, such as an anti-G-CSF-antibody or a fragment thereof, i.e. a natural or synthetic binding agent capable of binding to the G-CSF as present in the plasma or other body fluid of the individual.
  • a binding agent capable of binding to the biomarker G-CSF in the plasma of the individual such as an anti-G-CSF-antibody or a fragment thereof, i.e. a natural or synthetic binding agent capable of binding to the G-CSF as present in the plasma or other body fluid of the individual.
  • the presence and/or quantity of the biomarker is detected and/or measured using a method selected from the group consisting of: an enzyme-linked immunosorbent assay
  • the presence and/or quantity of the biomarker is even more preferably, the presence and/or quantity of the biomarker
  • ELISA-based assay for example, H3Cit and/or G-CSF
  • H3Cit and/or G-CSF is detected and/or measured using an ELISA-based assay.
  • ELISA-based assay for example, H3Cit and/or G-CSF
  • How to undertake an ELISA, a Western blot or immunohistochemistry will be well known to those skilled in the art of molecular biology.
  • the presence and/or quantity of the H3Cit biomarker is detected and/or measured using an ELISA-based assay.
  • An ELISA method for detecting H3Cit may use microplate modules and the coating anti-histone and incubation buffer of cell death detection ELISA kit (for example, Roche, cat nr 1 1774425001 ).
  • an ELISA for detecting and/or measuring H3Cit comprises one or more of the following steps:
  • coating solution anti-histone
  • incubating such as adding a 100 ul of coating solution (anti-histone 1 :10) to the pre-coated wells and incubating overnight at 4 degrees; and/or
  • adding incubation buffer such as by adding 200 ul incubation buffer and incubating 1 h at room temperature;
  • incubation buffer such as from Cell Death Detection ELISA PLUS kit, Roche, Cat. No. 1 1 774 425 001
  • incubating such as adding 40 ul incubation buffer and 10 ul plasma
  • reading optical density such as reading the optical density at 450 nm with a reference correction wavelength at 570 nm.
  • an ELISA for detecting and/or measuring H3Cit comprises making a H3Cit standard sample, which comprises one or more of the following steps: ⁇ incubating histone H3 and PAD4, such as incubating human recombinant histone H3 and human recombinant PAD4 at 37 °C for 1 hour in buffer (such as a reaction buffer comprising 50 mM Trizma base with 4 mM CaCI 2 , pH 7.6, 4 mM DTT and 1 mM PMSF); and/or
  • H3Cit • obtaining a final concentration of H3Cit, such as by adding PBS-1 % BSA (such as obtaining a concentration of 10 000 ng/mL).
  • an ELISA for detecting and/or measuring H3Cit comprises one or more of the following steps:
  • adding the anti-histone H3Cit antibody such as adding 100 ⁇ _ of anti- histone H3 (citrulline R2 + R8 + R17; Anti-H3Cit) antibody (1 :2000 in 1 % BSA in PBS) to each well for a 1 hour incubation at room temperature; and/or
  • HRP horseradish peroxidase
  • ⁇ detecting the bound anti-rabbit HRP conjugate antibody such as by adding 100 ⁇ _ TMB to each well and incubating for 20 minutes in the dark at room temperature;
  • an ELISA method for detecting and/or measuring H3Cit comprises one or more of the following equipment: microplates with 96 streptavidin pre-coated wells (such as from Cell Death Detection ELISA PLUS kit, Roche, Cat. No. 1 1 774 425 001 ), and an ELISA reader (such as from Tecan Sunrise).
  • an ELISA method for detecting and/or measuring H3Cit comprises one or more of the following antibodies: monoclonal anti-Histone- Biotin (such as from Cell Death Detection ELISA PLUS kit, Roche, Cat. No. 1 1 774 425 001 ); anti-Histone H3Cit antibody (such as a rabbit polyclonal anti-histone H3 (citrulline R2 + R8 + R17) antibody from Abeam, Cat. No. AB5103); and anti-lgG horseradish-peroxidase conjugate (such as goat anti- rabbit IgG horseradish-peroxidase conjugate from BioRad, Cat. No. 170- 6515).
  • monoclonal anti-Histone- Biotin such as from Cell Death Detection ELISA PLUS kit, Roche, Cat. No. 1 1 774 425 001
  • anti-Histone H3Cit antibody such as a rabbit polyclonal anti-histone H3 (citrulline R2 + R
  • an ELISA method for detecting and/or measuring H3Cit comprises one or more of the following buffers and/or solutions: incubation buffer (such as from Cell Death Detection ELISA PLUS kit, Roche, Cat. No. 1 1 774 425 001 ); phosphate buffered saline (PBS; such as from Life Technologies, Cat. No. 14190-250); tween 20 (such as from Sigma-Aldrich, Cat. No. A9418); bovine serum albumin (BSA, such as from Sigma-Aldrich, Cat. No. A9418); 3,3', 5,5'-tetramethylbenzidine (TMB) liquid substrate (such as from Sigma-Aldrich, Cat. No. T0440); and a stop solution (such as from Thermo Scientific, Cat. No. N600).
  • incubation buffer such as from Cell Death Detection ELISA PLUS kit, Roche, Cat. No. 1 1 774 425 001
  • PBS phosphate buffered s
  • an ELISA method for detecting and/or measuring H3Cit comprises one or more of the following components: trizma base (such as from Sigma-Aldrich, Cat. No. T1503); CaC (such as from Sigma-Aldrich C1016); phenylmethylsulfonyl fluoride (PMSF) protease inhibitor (such as from Life Technologies, Cat. No. 36978); and dithiothreitol (DTT, such as from Invitrogen, Cat. No. P2325).
  • trizma base such as from Sigma-Aldrich, Cat. No. T1503
  • CaC such as from Sigma-Aldrich C1016
  • PMSF phenylmethylsulfonyl fluoride
  • DTT dithiothreitol
  • an ELISA method for detecting H3Cit can comprise one or more of the following proteins: PAD4, such as human recombinant PAD4 (such as from Cayman Chemical, Cat. No. 10500); and Histone H3, such as human recombinant Histone H3 (Cayman Chemical, Cat. No. 10263).
  • PAD4 such as human recombinant PAD4 (such as from Cayman Chemical, Cat. No. 10500)
  • Histone H3 such as human recombinant Histone H3 (Cayman Chemical, Cat. No. 10263).
  • room temperature will vary, and those skilled in biology and chemistry, in particular molecular biology and biochemistry, will understand the temperature ranges in which the aforementioned method steps can be undertaken.
  • the cancer is selected from a group consisting of: leukemia; carcinoma; AIDS-related cancers; lymphoma; anal cancer; appendix cancer; astrocytoma; bile duct cancer, extrahepatic cancer; bladder cancer; bone tumor, osteosarcoma fibrous histiocytoma; malignant fibrous histiocytoma; glioma; brain cancer; brain tumor; breast cancer; bronchial adenomas;
  • myeloproliferative disorders colon cancer; desmoplastic small round cell tumor; endometrial cancer; ependymoma; epitheliod hemangioendothelioma (EHE); esophageal cancer; extrahepatic bile duct cancer; eye cancer;
  • gallbladder cancer gastric (i.e. stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; head and neck cancer; heart cancer; hepatocellular (i.e. liver) cancer; primary hepatocellular (i.e. liver) cancer; hypopharyngeal cancer; kidney cancer; renal cell cancer; laryngeal cancer; lip and oral cavity cancer; Waldenstrom macroglobulinemia; male breast cancer; childhood medulloblastoma;
  • melanoma melanoma
  • merkel cell cancer metastatic squamous neck cancer with occult primary
  • mouth cancer childhood multiple endocrine neoplasia syndrome
  • multiple myeloma plasma cell neoplasm
  • mycosis fungoides myelodysplastic syndromes
  • myelodysplastic diseases myeloproliferative diseases
  • chronic myeloproliferative disorders myxoma; nasal cavity and paranasal sinus cancer
  • neuroblastoma oligodendroglioma
  • oral cancer oropharyngeal cancer
  • ovarian cancer pancreatic cancer
  • pancreatic cancer islet cell pancreatic cancer
  • islet cell pancreatic cancer islet cell pancreatic cancer
  • paranasal sinus and nasal cavity cancer parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal germinoma; pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; pituitary adenoma; pleuropulmonary blastoma; prostate cancer; rectal cancer; renal pelvis and ureter cancer; salivary gland cancer; Sezary syndrome; skin cancer; small intestine cancer; metastatic squamous neck cancer with occult primary; stomach cancer; childhood supratentorial primitive neuroectodermal tumor; fungoides and Sezary syndrome; testicular cancer; throat cancer;
  • childhood thymoma thyroid cancer; childhood thyroid cancer; transitional cell cancer of the renal pelvis and ureter; gestational trophoblastic tumor; urethral cancer; endometrial uterine cancer; vaginal cancer; vulvar cancer; and childhood Wilms tumor.
  • the leukemia is selected from a group consisting of: acute lymphoblastic leukemia (ALL); acute myeloid leukemia; chronic lymphocytic leukemia; chronic myelogenous leukemia; hairy cell leukemia; acute lymphoblastic leukemia (also called acute lymphocytic leukaemia); acute myeloid leukemia (also called acute myelogenous leukemia); chronic lymphocytic leukemia (also called chronic lymphocytic leukemia); chronic myelogenous leukemia (also called chronic myeloid leukemia); hairy cell leukemia; adult acute myeloid leukemia; and childhood acute myeloid leukemia.
  • ALL acute lymphoblastic leukemia
  • acute myeloid leukemia also called acute lymphocytic leukaemia
  • acute myeloid leukemia also called acute myelogenous leukemia
  • chronic lymphocytic leukemia also called chronic lymphocytic leukemia
  • the carcinoma is selected from a group consisting of:
  • adrenocortical carcinoma basal-cell carcinoma
  • carcinoma squamous cell carcinoma
  • thymoma and thymic carcinoma squamous cell carcinoma
  • the astrocytoma is selected from a group consisting of: cerebellar astrocytoma; cerebral astrocytoma; childhood cerebellar
  • astrocytoma childhood cerebral astrocytoma; and pineal astrocytoma.
  • the lymphoma is selected from a group consisting of: AIDS- related lymphoma; Burkitt's lymphoma; primary central nervous system lymphoma; central nervous system lymphoma; cutaneous T-cell lymphoma; Hodgkin lymphoma; and non-Hodgkin lymphoma.
  • the glioma is selected from a group consisting of:
  • brainstem glioma cerebral astrocytoma glioma; malignant glioma; visual pathway glioma; hypothalamic glioma; and visual pathway and hypothalamic glioma.
  • the brain tumor is selected from a group consisting of: cerebellar astrocytoma; ependymoma; cerebral astrocytoma glioma;
  • malignant glioma childhood malignant glioma; medulloblastoma;
  • the carcinoid tumor is selected from a group consisting of: childhood carcinoid tumor; gastrointestinal carcinoid tumor; and gastric carcinoid.
  • the sarcoma is selected from a group consisting of:
  • chondrosarcoma Ewing's sarcoma in the Ewing family of tumors; kaposi sarcoma; liposarcoma; malignant fibrous histiocytoma of osteosarcoma; malignant fibrous histiocytoma of bonesarcoma; rhabdomyosarcoma;
  • the eye cancer is selected from a group consisting of:
  • intraocular melanoma intraocular melanoma
  • retinoblastoma intraocular melanoma
  • the germ cell tumor is selected from a group consisting of: extracranial germ cell tumor; extragonadal germ cell tumor; ovarian germ cell tumor; extragonadal germ cell tumor; and childhood extracranial germ cell tumor.
  • the lung cancer is selected from a group consisting of: non- small cell lung cancer; and small cell lung cancer.
  • the mesothelioma is selected from a group consisting of: adult malignant mesothelioma; and childhood mesothelioma.
  • the ovarian cancer is selected from a group consisting of: ovarian epithelial cancer; surface epithelial-stromal tumor; ovarian germ cell tumor; and ovarian low malignant potential tumor.
  • the adenocarcinoma is selected from the group consisting of: prostate adenocarcinoma; lung adenocarcinoma; hepatocellular adenocarcinoma; pancreatic adenocarcinoma; prostate adenocarcinoma; breast adenocarcinoma; and urothelial adenocarcinoma.
  • the cancer is selected from the group consisting of: lung cancer; pancreatic cancer; breast cancer; prostate cancer; urothelial cancer; liver cancer; colon cancer; colorectal cancer; gastrointestinal cancer; gynecological cancer; glioblastoma; lymphoma; acute myeloid leukemia; gingival cancer; liposarcoma; sarcoma; neuroendocrine cancer and melanoma (such as malignant melanoma).
  • the cancer is selected from a group consisting of: lung cancer; pancreatic cancer; breast cancer; prostate cancer; urotheal cancer; and liver cancer.
  • the cancer is selected from the group consisting of: colon cancer; breast cancer; gastrointestinal cancer; lung cancer; prostate cancer; and gynecological cancer.
  • the thrombosis is an arterial thrombotic disorder or a venous thrombotic disorder.
  • the thrombosis is an arterial thrombotic disorder.
  • the thrombosis is a venous thrombotic disorder.
  • the arterial thrombotic disorder is a stroke or a myocardial infarction.
  • the methods for screening are in vitro methods for screening.
  • the chemotherapy is a chemotherapy drug selected from a group consisting of: 13-cis-Retinoic Acid, 2-CdA, 2-Chlorodeoxyadenosine, 5- Azacitidine, 5-Fluorouracil 5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6- Thioguanine, Abiraterone acetate, Abraxane, Accutane, Actinomycin-D, Adcetris, Ado-Trastuzumab,, Emtansine, Adriamycin, Adrucil, Afatinib, Afinitor, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alimta, Alitretinoin, Alkaban-AQ, Alkeran, All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron,
  • a chemotherapy drug selected from a group consisting
  • Bevacizumab Bexarotene, Bexxar, Bicalutamide, Bicnu, Blenoxane,
  • Daunorubicin Daunorubicin-hydrochloride, Daunorubicin, Liposomal,
  • Lenalidomide Lenvatinib, Lenvima
  • Letrozole Leucovorin, Leukeran
  • Pertuzumab Phenylalanine Mustard, Platinol, Platinol-AQ, Pomalidomide, Pomalyst, Ponatinib, Portrazza, Pralatrexate, Prelone, Procarbazine, Procrit, Proleukin, Prolia, Prolifeprospan 20 with Carmustine Implant, Provenge, Purinethol, Radium 223 Dichloride, Raloxifene, Ramucirumab, Rasuvo, Regorafenib, Revlimid, Rheumatrex, Rituxan, Rituximab, Roferon-A
  • the chemotherapy drugs can be administered at the same time or at different times.
  • the administration of chemotherapy and chemotherapy regimens will be well known to those skilled in medicine, in particular an oncologist.
  • the individual has further been diagnosed as having had a stroke (such as an ischemic stroke) or suspected as having had a stroke (such as an ischemic stroke).
  • a stroke such as an ischemic stroke
  • a stroke such as an ischemic stroke
  • the method for screening is undertaken one or more day following chemotherapy - for example, two or more days following chemotherapy; three or more days following chemotherapy; four or more days following chemotherapy; five or more days following chemotherapy; six or more days following
  • chemotherapy one or more week following chemotherapy; two or more weeks following chemotherapy; three or more weeks following chemotherapy; one or more month following chemotherapy; two or more months following chemotherapy; three or more months following chemotherapy; four or more months following chemotherapy; five or more months following
  • the method for screening is undertaken one day following chemotherapy.
  • the individual is a human individual.
  • the individual is a non-human individual, such as a rodent
  • a mouse, rat or hamster for example a mouse, rat or hamster
  • a rabbit for example a cat, a dog, a sheep, a pig, a goat, a horse, or cattle.
  • the individual is an elderly individual, such as an elderly human individual.
  • an elderly human individual we include an individual that is 50 or more years old - for example, 55 or more years old, 60 or more years old, 65 or more years old, 70 or more years old, 75 or more years old, 80 or more years old, 85 or more years old, or 90 or more years old.
  • the method further comprises step:
  • selecting the treatment in step c) is based on presence and/or quantity in the test sample of said biomarkers.
  • the method further comprises step:
  • the treatment is selected from the group consisting of: cancer treatment; thrombosis treatment; tumour treatment; and treatment for the adverse effects G-CSF.
  • the treatment is cancer treatment.
  • the treatment is thrombosis treatment.
  • the treatment is tumour treatment.
  • the treatment is for the adverse effects G-CSF. It will be appreciated that cancer treatment is often the same as tumour treatment.
  • the cancer treatment or tumour treatment is one or more selected from the group consisting of: surgery; chemotherapy; and
  • the treatment for thrombosis is selected from a list consisting of: heparin (such as unfractionated heparin); low molecular weight heparins (such as enoxaparin, dalteparin, tinzaparin, nadroparin, reviparin, ardeparin, and certoparin); pentasaccharide (such as fondaparinux, which is a synthetic pentasaccharide); antivitamin K agents (such as warfarin, dicoumarol, phenprocoumon, and acenocoumarol); direct acting oral coagulants (such as new oral anticoagulants (NOACs), dabigatran, rivaroxaban, apixaban, betrixaban, and edoxaban); antiplatelet agents (such as acetylsalicylic acid (aspirin)); clopidogrel; prasugrel; ticagrelor; and dipyrida
  • the treatment for a venous thrombotic disorder is selected from a list consisting of: heparin (such as unfractionated heparin); low molecular weight heparins (such as enoxaparin, dalteparin, tinzaparin, nadroparin, reviparin, ardeparin, and certoparin); pentasaccharide (such as fondaparinux, which is a synthetic pentasaccharide); antivitamin K agents (such as warfarin, dicoumarol, phenprocoumon, and acenocoumarol); and direct acting oral coagulants (such as new oral anticoagulants (NOACs), dabigatran, rivaroxaban, apixaban, betrixaban, and edoxaban).
  • NOACs new oral anticoagulants
  • the treatment for an arterial thrombotic disorder is selected from a list consisting of: heparin (such as unfractionated heparin); low molecular weight heparins (such as, enoxaparin, dalteparin, tinzaparin, nadroparin, reviparin, ardeparin, and certoparin); pentasaccharide (such as fondaparinux, which is a synthetic pentasaccharide); antivitamin K agents (such as, warfarin, dicoumarol, phenprocoumon, and acenocoumarol); direct acting oral coagulants (such as new oral anticoagulants (NOACs),
  • NOACs new oral anticoagulants
  • antiplatelet agents such as, acetylsalicylic acid (aspirin)); clopidogrel; prasugrel;
  • the treatment for the adverse effects G-CSF is to stop G- CSF treatment or to decrease G-CSF treatment.
  • the present invention is also related to a diagnostic kit for use in any of the above described methods for diagnosing one or more of the described condition(s).
  • said diagnostic kit comprises at least one binding agent which could be chosen from a binding agent such as an anti-H3Cit-antibody or a fragment thereof capable of binding to the H3Cit.
  • the diagnosis of the one or more conditions is performed in a tailor made ELISA model, as described below, to measure the presence and quantity of said H3Cit.
  • the kit further comprises an additional binding agent, such as an anti-G-CSF-antibody or a fragment thereof capable of binding to the G- CSF.
  • the kit further comprises an additional binding agent capable of binding to the anti-H3Cit-antibody or a fragment thereof and/or an anti-G-CSF-antibody or a fragment thereof (for example, an anti-lgG antibody).
  • the additional binding agent comprises a detectable moiety, such as a fluorescent label, a fluorescein-type label, a rhodamine-type label, VIVOTAG 680 XL FLUOROCHROMETM (Perkin Elmer), phycoerythrin;
  • a detectable moiety such as a fluorescent label, a fluorescein-type label, a rhodamine-type label, VIVOTAG 680 XL FLUOROCHROMETM (Perkin Elmer), phycoerythrin;
  • umbelliferone Lissamine; a cyanine; a phycoerythrin, Texas Red, BODIPY FL-SE® (Invitrogen), a chemoluminescent label, an enzyme, a prosthetic group complex, a paramagnetic label, or a radioactive label.
  • the fluorescent label is a rare earth chelate, such as europium chelate.
  • the fluorescein-type label is selected from the group consisting of: fluorescein; fluorescein isothiocyanate; 5-carboxyfluorescein; 6- carboxy fluorescein; and dichlorotriazinylamine fluorescein.
  • the rhodamine-type label is selected from the group consisting of: ALEXA FLUOR® 568 (Invitrogen); TAMRA®; or dansyl chloride.
  • the chemoluminescent label is selected from the group consisting of: luminol; luciferase; luciferin; and aequorin.
  • the enzyme is selected from the group consisting of:
  • horseradish peroxidase alkaline phosphatase; beta-galactosidase; and or acetylcholinesterase.
  • the prosthetic group complex is selected from the group consisting of: streptavidin/biotin and avidin/biotin.
  • the paramagnetic labels is selected from the list consisting of: paramagnetic ions of Aluminum (Al), Barium (Ba), Calcium (Ca), Cerium (Ce), Dysprosium (Dy), Erbium (Er), Europium (Eu), Gandolinium (Gd), Holmium (Ho), Iridium (Ir), Lithium (Li), Magnesi-um (Mg), Manganese (Mn), Molybdenum (M), Neodymium (Nd), Osmium (Os), Oxygen (O), Palladium (Pd), Platinum (Pt), Rhodium (Rh), Ruthenium (Ru), Samarium (Sm), Sodium (Na), Strontium (Sr), Terbium (Tb), Thulium (Tm), Tin (Sn), Titanium (Ti), Tungsten (W), or Zirconium (Zi), and particularly, Co+2, CR+2, Cr+3, Cu+2, Fe+2, Fe+3, Ga+3,
  • the radioactive label is selected from the list consisting of: bismuth (213Bi); carbon (1 1 C, 13C, 14C); chromium (51 Cr); cobalt (57Co, 60Co); copper (64Cu); dysprosium (165Dy); erbium (169Er); fluorine (18F); gadolinium (153Gd, 159Gd); galli-um (68Ga, 67Ga); germanium (68Ge); gold (198Au); holmium (166Ho); hydrogen (3H); indium (1 1 1 In, 1 12ln, 1 13ln, 1 15ln); iodine (121 1, 123I, 125I, 131 1); iridium (1 92lr); iron (59Fe); krypton (81 mKr); lanthanium (140La); lutelium (177Lu); manganese (54Mn);
  • molybdenum 99Mo
  • nitrogen 13N, 15N
  • oxygen 150
  • palladium 103Pd
  • phosphorus 32P
  • potassium 42K
  • praseodymium 142Pr
  • promethium 149Pm
  • rhenium 186Re, 188Re
  • rhodium 105Rh
  • rubidium 81 Rb, 82Rb
  • ruthenium 82Ru, 97Ru
  • samarium 15Sm
  • scandium 47Sc
  • H3Cit has previously not been quantified in plasma and there is no known or commercial method for doing so. It was therefore developed a tailor made method for the quantification of H3Cit in plasma using the following protocol:
  • This H3Cit ELISA allows the specific detection of histone H3 citrullinated at arginines position 2, 8 and 17, which has been shown to be necessary for decondensation of the chromatin.
  • the method uses the microplate modules and the coating anti-histone and incubation buffer of Cell death detection ELISA kit, Roche, cat nr 1 1774425001 . ⁇ Add 100 ul of coating solution (anti-histone 1 :10) to the pre-coated wells and incubate overnight at 4 degrees
  • results are expressed as the read optical density and are compared to the absorbance of plasma samples from healthy donors.
  • a standard curve using various concentration of in vitro citrullinated histone H3 diluted in pool plasma from healthy donors, is being developed to quantify the exact level of H3Cit in plasma samples.
  • H3Cit in the patients without cancer were diminishingly small and it is believed that these patients had no H3Cit in plasma (ie background signal) as their levels were similar to 10 healthy controls (aged 62-93), and all of these had diminishingly low optical density suggesting no detectable H3Cit in plasma.
  • Western Blot detection of H3Cit in the same patients revealed similar results, with positive signal of plasma H3Cit in the patients with underlying cancer (ie presence of H3Cit), and no signal (ie absence of H3Cit) in the patients without underlying cancer (Fig 2).
  • H3Cit ie NETs
  • NETosis resulting in circulating H3Cit in plasma, as shown in the patients.
  • the circulating NETs may subsequently contribute to the prothrombotic state in cancer patients.
  • NETs in thrombus formation and infarction in patients without cancer may be regarded as locally induced NET burden, whereas NETs in cancer patients may be a systemically induced NET burden.
  • H3Cit may therefore be detectable in plasma of cancer patients only. As the levels of NETs in plasma of cancer patients increase (it is
  • NETs may contribute to cancer-associated thrombosis.
  • the primary tumor sites in the patient group with ischemic stroke and cancer covered lung, pancreas, breast, prostate, urotheal and liver tumors.
  • Comorbidity, age and infarct volume did not differ between the group with ischemic stroke without cancer and the group with ischemic stroke and cancer.
  • all patients were old and had a high burden of comorbidity such as diabetes mellitus, renal insufficiency, hypertension, hearth failure and acute coronary artery disease.
  • H3Cit in plasma could be used as a biomarker for cancer.
  • H3Cit in plasma has not been suggested.
  • biomarkers used for a variety of tumors, such as PSA (prostate cancer), CEA (colorectal cancer) and CA-125 (ovarian cancer). These biomarkers are hampered by low specificity and low sensitivity. On the contrary, H3Cit may have the potential to contribute in screening for cancer in general.
  • H3Cit may also allow for a higher specificity and sensitivity than existing biomarkers. Cancer is one of the leading causes of death in the world. The poor prognosis is often due to late diagnosis. A biomarker, which could aid in diagnosing a wide variety of malignancies would allow for an earlier detection and thus a better prognosis for cancer patients in general. H3Cit in plasma may also be useful in the clinical follow up of tumor progression and screening for recurrence in patients with previously diagnosed cancer.
  • the present tailor made H3Cit ELISA is the first method described able to detect and quantify H3Cit in plasma Quantifying the levels of H3Cit in plasma could furthermore predict the susceptibility to cancer-associated thrombosis in cancer patients as well as cancer in patients with idiopathic thrombosis.
  • a systemically induced NETosis may, however, have to reach a certain level to contribute to the prothrombotic state in cancer.
  • a method to quantify H3Cit in plasma may therefore be crucial in the screening for cancer patients at highest risk for thrombosis.
  • Low-molecular-weight heparin is currently the preferred treatment and prophylaxis of cancer-associated thrombosis.
  • Anticoagulant therapy is, however, associated with a substantial risk of bleeding, especially in patients with active cancer, and the benefit-risk ratio is therefore too unfavourable to consider routine thrombo-prophylaxis. Quantification of H3Cit in plasma could reveal patients with the highest risk of thrombosis and allow for individualized thrombo-prophylaxis strategies.
  • Chemotherapy is known to increase the risk for thrombosis.
  • Oxidative stress such as in chemotherapy, induces NETosis.
  • NETs are highly procoagulant, it is conceivable to believe that quantifying plasma H3Cit before and after chemotherapy could aid in screening for patients at highest risk for chemotherapy-associated thrombosis.
  • recombinant DNA technology is often administered after chemotherapy in order to reduce chemotherapy associated neutropenia and the incidence of febrile neutropenia.
  • chemotherapy associated neutropenia As tumor derived G-CSF is known to induce NETs, Filgrastim could hypothetically also induce NETs, which could be a
  • H3Cit may therefore also be useful in screening patients receiving Filgrastim.
  • Example 2 describes a further characterisation of the assay discussed in Example 1 .
  • Serial experiments to obtain the optimal concentrations of the standard curve, incubation times and dilutions of samples were performed, after which a methodological validation of the assay was performed.
  • Microplates with 96 streptavidin pre-coated wells, Monoclonal Anti-Histone-Biotin antibodies, and incubation buffer (all from Cell Death Detection ELISA PLUS kit, Roche, Cat. No. 1 1 774 425 001 ).
  • H3Cit A working stock solution of H3Cit was made as described previously (Lee P, et al. Mol Cell Biol 2008;28:4745-58). Briefly, human recombinant histone H3 and human recombinant PAD4 were incubated at 37 °C for 1 h in reaction buffer (50 imM Trizma base with 4 imM CaCI 2 , pH 7.6, 4 imM DTT and 1 imM PMSF). A final concentration of 10 000 ng/mL H3Cit was obtained by adding PBS-1 % BSA. The stock solution was aliquoted, frozen on dry ice, and stored at -80 °C until later use.
  • microplate and diluents were kept at room temperature 30 min prior to starting the assay.
  • Stock solution, antibodies, and samples were thawed on ice and kept on ice until loading of microplate. All incubations were at room temperature and washes were repeated 4 times with PBS-Tween (0.05%) with 20 sec soaking for each wash.
  • concentrations of the standard curve, incubation times and dilutions of samples were optimized in preliminary experiments.
  • the assay was performed as follows: 100 ⁇ _ of anti-histone Biotin (1 :10 in incubation buffer) was added to Streptavidin pre-coated wells and incubated for 2 h. After washing, 50 ⁇ _ of standard solutions or samples were added to each well and incubated for 1 .5 h, then washed again. 100 ⁇ _ of anti-histone H3 (citrulline R2 + R8 + R17; Anti-H3Cit) antibody (1 :2000 in 1 % BSA in PBS) was applied to each well for 1 h incubation. After washing, the wells were incubated for another hour with 100 ⁇ _ anti-rabbit HRP conjugate antibody (1 :5000 in 1 % BSA in PBS), followed by washing.
  • the linear interval was defined as the linear section of the best- fit standard curve.
  • Each standard curve was fitted using a four-parameter logistic (4PL) regression, and the 95% confidence interval (95 % CI) was considered.
  • the limit of detection was approximated from the intersection of the lower asymptote of the upper 95% CI with the 4PL fit of the standards data. Specificity was assessed by the ability to detect citrullinated histone H3 but not non-citrullinated histone H3 in similar conditions by preparing a standard without PAD4, thus preventing the citrullination of histone H3.
  • Precision was expressed by the intra- and inter-assay coefficient of variation (%CV, defined as the ratio between standard deviation and mean value).
  • %CV intra- and inter-assay coefficient of variation
  • the maximum accepted %CV for intra- and inter-assay variability were set to 15%.
  • Stability was assessed by comparing the detector response obtained from freshly prepared and frozen aliquots of H3Cit standard, and comparing standard curves from frozen aliquots from three different batches of H3Cit that had been citrullinated on three different days. One vs. two freeze-thaw cycles of plasma samples were also compared.
  • Standard preparation and linearity As no international standard preparation is available for H3Cit, we generated a standard curve using in vitro PAD4- citrullinated H3Cit, as described above.
  • the stock was serially diluted 1 :2 in PBS-1 % BSA to obtain a standard curve and applied to a streptavidin-coated plate using an anti-histone biotin antibody as capture and an anti-H3Cit antibody for detection.
  • To determine the suitable linear interval we interpolated the detected O.D. from the serial dilutions of H3Cit to different regressions.
  • the best-fit curve was a sigmoidal 4PL curve rendering a linear interval of the curve between ⁇ 0.5 and 3.5 O.D., corresponding to concentrations between ⁇ 5 and ⁇ 300 ng/mL (Fig. 5).
  • H3Cit Concentrations of H3Cit in plasma in a human model of LPS-induced inflammation.
  • Surrogate markers of NETs cfDNA, nucleosomes and MPO- DNA complexes
  • LPS lipopolysaccharide
  • H3Cit was detected by western blot in plasma of mice shortly after LPS injection (Li Y et al. Surgery 201 1 ;150:442-51 ).
  • This example establishes an assay allowing for the fast and reliable quantification of the NET specific biomarker H3Cit in human plasma.
  • the validation of the assay revealed a high specificity for H3Cit as well as a high stability of the custom-made standard, rendering a good precision.
  • Example provides a proof-of-principle for the H3Cit assay. From this data, we consider that several measurements for plasma H3Cit over time, for example two measurements with one or a few weeks intervals, may identify those with stable elevations (ie induced by an active cancer) in contrast to those with temporary elevations (ie induced by a severe inflammatory reaction due to for example sepsis). Patients with sepsis will also present with symptoms of sepsis, alerting the clinician of a possible source of H3Cit other than cancer. All samples taken at baseline in the LPS-induced model of inflammation were below the limit of detection, suggesting that healthy individuals do not have a baseline systemic NET burden.
  • H3Cit ELISA-based assay presents a robust, precise and specific assay for the quantification of plasma H3Cit, which may be implicated, alone or in combination with existing screening protocols, in identifying cancer and cancer-associated conditions such as thrombosis.
  • H3Cit As a biomarker in screening for cancer, a study was conducted with a larger cohort of cancer patients with the intention to cover a normal biological variation as well as a broader variation in primary malignant tumor types. In a prospective case-control design, patients with active cancer admitted to Swisss Sjukhem were recruited as case patients. To explore the specificity of H3Cit in cancer, 51 age- and gender matched hospitalized patients with other diseases but without known cancer, admitted to
  • liposarcoma sarcoma
  • neuroendocrine neuroendocrine
  • Patient sample SSH41 is omitted from the above table, as the patient was inadvertently included in the study twice.
  • Cancer patients had a significant 5-fold increase in plasma H3Cit levels compared to healthy individuals (mean 36.1 ng/mL vs. 6.6 ng/mL, p ⁇ 0.001 ) and a significant 3-fold increase compared to hospitalized patients with other diseases but no known cancer (mean 36.1 ng/mL vs. 1 1 .5 ng/mL, p ⁇ 0.001 ).
  • the mean levels of plasma H3Cit in patients with the different malignant tumor types are depicted in Figure 1 1 and in Table 3. Table 3.
  • H3Cit is elevated in cancer patients, and that this elevation can be seen in many different cancer types.
  • H3Cit gives an approximate 75% sensitivity, as depicted in Figure 10 and 1 1 , and therefore it exceeds the sensitivity of the to-date clinically available screening biomarkers, ranging from 20-80% in various studies.
  • H3Cit seems to be elevated in a large variety of cancers, suggesting that it may be applicable as a screening biomarker for cancer in general.
  • biomarkers for screening for cancer in general There are as of today no clinically available biomarkers for screening for cancer in general.
  • H3Cit may also, due to its potential as a biomarker in screening for several malignant tumor types, be implemented in combination with existing biomarkers, such as PSA, CA-125 and CEA, allowing for an increased sensitivity and specificity.
  • Plasma H3Cit was detectable in some hospitalized patients with other diseases but no known cancer (33%) and in some healthy individuals (18%). Although these study participants had no known cancer diagnosis at the time of blood sampling, they were of old age (median age 70.4 years), and there may be a possibility that some of these individuals have an unknown cancer. Furthermore, the hospitalized patients with other diseases but no known cancer had a large amount of comorbidities, with a median comorbidity index score of 6 using the Charlson Comorbidity Index Scoring System (Charlson ME, et al. A new method of classifying prognostic comorbidity in longitudinal studies: Development and validation. 1987 Journal of Chronic Diseases. 1987;40:373-83).

Abstract

The present invention is related to a method for screening for the presence of cancer and several other indications in an individual comprising the steps of providing a plasma sample to be tested from the individual; and measuring the presence and/or quantity in the plasma sample of a biomarker chosen from citrullinated histone H3 (H3Cit), wherein said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating cancer in said individual.

Description

METHOD FOR DIAGNOSISNG CANCER OR CANCER-ASSOCIATED
THROMBOSIS BY MEASURING LEVELS OF H3CIT IN PLASMA
Related Applications
This application claims the benefit of priority to Swedish Patent
Application No. 1500330-4, filed on 07 August 2015 and Swedish Patent Application No. 1551074-6, filed on 14 August 2015. The entire contents of each of the foregoing applications are incorporated herein by reference.
Field of the invention
The present invention relates to a method for screening for the presence of cancer and/or cancer-associated thrombosis in an individual by measuring the presence and/or quantity in a plasma sample of said individual a biomarker chosen from citrullinated histone H3 (H3Cit). The presence and/or quantity in the plasma sample of said citrullinated histone H3 (H3Cit) is indicating cancer and/or cancer-associated thrombosis in said individual. The invention also relates to a kit for use in said method.
Background of the invention
Detecting and diagnosing cancer early is essential when it comes to treatment outcome and survival. There are a number of clinically
implemented biomarkers used for a variety of tumors, such as prostate- specific antigen (PSA) for prostate cancer, carcinoembryonic antigen (CEA) for colorectal cancer and cancer antigen 125 (CA-125) for ovarian cancer. These biomarkers are hampered by low specificity and low sensitivity.
Several studies have attempted to assess the sensitivity and specificity of these biomarkers, with varying results.
With a cut-off of 4 ng/mL for a normal PSA-level (the traditional cut-off in the majority of screening studies), the estimated sensitivity from pooled analyses is 21 % for the detection of any prostate cancer, and 51 % for the detection of high-grade cancer. The specificity is 91 % (Wolf AM, et al.
American Cancer Society guideline for the early detection of prostate cancer; update 2010. CA Cancer J Clin;2010:60-70). However, the specificity is poorer in men with symptomatic benign prostate hyperplasia, which is very common in older men. The sensitivity of CEA for early colorectal cancer is only 40% (Duffy MJ et al. Clin. Chem. 2001 ;47:624-630), and CEA can be elevated in the absence of malignancy. The early stage sensitivity of CA-125 is approximated to 45% (Skates SJ et al., Preoperative sensitivity and specificity for early-stage ovarian cancer when combining cancer antigen CA- 12511, CA 15-3, CA 72-4, and macrophage colony-stimulating factor using mixtures of multivariate normal distributions, J Clin Oncol 2004).
Neutrophil extracellular traps (NETs)
NETs were discovered by Brinkman et al in 2004 (Brinkmann V, et al.
Neutrophil extracellular traps kill bacteria. Science 2004;303:1532-5) as a mechanism for trapping and killing bacteria by the innate immune system. Upon activation, neutrophils can release chromatin (DNA and histones) coated with antimicrobial granular proteins. Prior to releasing NETs, peptidylarginine deiminase 4 (PAD4), an enzyme that is primarily expressed in neutrophils, translocate to the nucleus and citrullinates the core histones H3, H2A and H2B initiating chromatin decondensation by preventing the binding of heterochromatin binding protein 1 (HP1 ). Citrullinated histone H3 (H3Cit) is thereby considered a NET specific biomarker. NETs have since then been implicated in several non-infectious diseases such as autoimmune diseases, thrombosis and cancer.
NETs/PAD4 in thrombosis
The link between NETs and thrombosis was established in 2010 by Fuchs et al (Fuchs TA, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci USA 2010;107:15880-5). By activating and binding platelets, exposing tissue factor, inhibiting tissue factor pathway inhibitor, activating coagulation factors (factor XII), and by promoting thrombin generation, NETs are believed to be highly procoagulant. Moreover, NETs/PAD4 have been shown to be crucial for deep vein thrombosis in mice and studies have shown markers of NETs in both arterial and venous thrombosis, as well as in surrounding infarcted tissues.
NETs/PAD4 in cancer and cancer-associated thrombosis
Although the role of NETs/PAD4 is becoming clearer in thrombosis an additional role in cancer is emerging, as some cancers express PAD4. The presence of NETs in diverse types of murine and human tumors has been observed. NETs have also been associated with cancer progression and metastasis where they are hypothezised to protect and anchor cancer cells as well as promote tumor growth. The first implication of NETs in cancer- associated thrombosis was reported by Demers et al in 2012 (Demers M, et al. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci USA
2012;109:13076-81 ). The study showed that different types of murine cancer systemically released granulocyte-colony stimulating factor (G-CSF), which primes neutrophils toward NETosis. This priming resulted in NET formation that was associated with spontaneous lung thrombosis, suggesting a link between NETs and cancer-associated thrombosis.
PAD4 is highly expressed in neutrophils but is also upregulated in many cancers where it negatively regulates tumor suppressor genes of the p53 pathway and thus promote tumor growth. In vitro, its overexpression in cancer cells results in histone citrullination and chromatin decondensation. The release of NET-like structures by the cancer cells may thus also be a potential source of the NET burden in cancer.
Detection and quantification of PAD4/NETs
Previous studies have provided data on the detection and quantification of surrogate biomarkers of NETs, such as the enzymes coating the chromatin in NETs; neutrophil elastase (NE), myeloperoxidase (MPO) and cell free DNA. These biomarkers are, however, not specific for NETs. NE and MPO merely suggest a neutrophil activation, and cell free DNA may be released from necrotic tissues following tissue injury and vessel wall damage. Some studies also used MPO-DNA complexes but MPO is also highly expressed in monocyte/macrophages and since MPO is a secreted protein and it is highly positively charged, it is possible that the electrical attraction cause MPO to bind to any negatively charged DNA that could be released in the plasma, thus questioning its specificity as a NET marker. Clear clinical need of a better method for analyzing and quantifying a NET specific biomarker
Detection and quantification of the NET specific biomarker H3Cit would be of better diagnostic value than the surrogate biomarkers of NETs. H3Cit in plasma has so far only been detected qualitatively using Western Blot analysis. No method has thus far been able to quantify H3Cit in plasma. Such a method would add a clear clinical value since the quantity of plasma H3Cit is a biomarker for the quantity of plasma NETs. The quantity/level of NETs in plasma can be a useful tool in the diagnosis of a variety of diseases.
In spite of many advances in treatments of cancer, cancer remains a leading cause of death. The disease is often diagnosed at a too late stage to be able to use treatments with curative intent and there is a large unmet clinical need to detect cancers earlier, to be able to increase cure rates. A new method for screening for cancer disease in general is needed.
The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
Summary of the invention
In a first aspect, the present invention is related to a method for screening for the presence of cancer in an individual comprising the steps:
a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or quantity in the plasma sample of a biomarker chosen from citrullinated histone H3 (H3Cit), wherein said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating cancer in said individual.
By "screening for the presence of cancer" we include testing to identify whether the individual has cancer. Therefore, by "indicating cancer" we include that a result obtained from the method is used to identify the presence of cancer in the individual.
By "plasma sample" we include a blood plasma sample. It will be appreciated that plasma is a general term for the liquid component of blood. Those skilled in the art of medicine, in particular a general practitioner, oncologist or nurse, will be familiar with what is required to provide a plasma sample, such as obtaining a blood sample from a patient and then isolating a plasma sample from the cellular component of the blood, for example by centrifugation.
By "citrullinated histone H3 (H3Cit)" we include histone 3 (H3) that has been citrullinated by a peptidylarginine deiminase (PAD or PADI), such as PAD1 , PAD2, PAD3 and/or PAD4 enzymes. It will be appreciated that citrullination (which is the process that leads to a histone being citrullinated) is a general term for the conversion of the amino acid arginine to the amino acid citrulline. In some circumstances, the N-terminus of H3 is citrullinated at arginine residues at amino acid positions 2, 8 and 17. The N-terminus of H3 has the amino acid sequence A RT KQTA R KSTG G KA P R KQ L AT KA A R KS (SEQ ID NO: 1 ) (Wang Y, et al. Human PAD4 Regulates Histone Arginine Methylation Levels via Demethylimination. Science 2004;306:279-283).
As discussed here, the present inventors have surprisingly identified that H3Cit is a biomarker that can be used to detect cancer, cancer associated thrombosis, and thrombosis associated with cancer. The association between those conditions and H3Cit is particularly surprising as H3Cit had previously not been associated with cancer, cancer associated thrombosis, or thrombosis associated with cancer. The methods described herein are based upon those new findings.
In a second aspect, the present invention is related to a method for screening for cancer-associated thrombosis or an elevated risk for cancer-associated thrombosis in cancer individuals or for screening for cancer in individuals with idiopathic thrombosis, comprising the steps:
a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or the quantity in the plasma sample of a biomarker chosen from citrullinated histone H3 (H3Cit), wherein said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating cancer-associated thrombosis or an elevated risk for cancer- associated thrombosis in said cancer individuals or cancer in said individuals with idiopathic thrombosis. By "screening for cancer-associated thrombosis" we include testing to identify whether the individual has thrombosis associated, in full or in part, by cancer in the individual. Therefore, by "indicating cancer-associated thrombosis" we include that a result obtained from the method is used to identify the presence of cancer-associated thrombosis in the individual.
By "screening for elevated risk for cancer-associated thrombosis in cancer individuals" we include testing to identify whether the individual with cancer has an increased risk of developing thrombosis. Therefore, by "indicating an elevated risk for cancer-associated thrombosis" we include that a result obtained from the method is used to establish that the individual has an increased risk of cancer-associated thrombosis.
By "screening for cancer in individuals with idiopathic thrombosis" we include testing to identify whether an individual already diagnosed with an idiopathic thrombosis (such as a de novo thrombosis and/or a thrombosis that occurred spontaneously and/or a thrombosis that has an unknown cause) also has cancer. Therefore, by "indicating cancer in said individuals with idiopathic thrombosis" we include that a result obtained from the method is used to identify the presence of cancer in the individual with idiopathic thrombosis. Those skilled in the art of medicine, in particular a general practitioner or oncologist, will be familiar with the symptoms and pathology of the conditions "thrombosis", "cancer-associated thrombosis" and "idiopathic thrombosis". In a third aspect, there is provided a method for screening for thrombosis or an elevated risk for thrombosis in cancer individuals following chemotherapy, comprising the steps:
a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or the quantity in the plasma sample of a biomarker chosen from citrullinated histone H3 (H3Cit), wherein said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating thrombosis or an elevated risk for thrombosis in said cancer individuals
(such as when following chemotherapy).
By "screening for thrombosis in cancer individuals following chemotherapy" we include testing to identify whether an individual with cancer that has received chemotherapy has thrombosis. Therefore, by "indicating
thrombosis" we include that a result obtained from the method is used to identify the presence of thrombosis in the individual with cancer who has received chemotherapy. By "screening for elevated risk for thrombosis in cancer individuals following chemotherapy" we include testing to identify whether the individual with cancer that has received chemotherapy is at risk of developing thrombosis. Therefore, by "elevated risk for thrombosis in said cancer individuals" we include that a result obtained from the method is used to establish that the cancer individual has an increased risk of thrombosis. In a fourth aspect, there is provided a method for screening for tumour progression in individuals or screening for recurrence of cancer in individuals with previously diagnosed cancer, comprising the steps:
a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or the quantity in the plasma sample of a biomarker chosen from citrullinated histone H3 (H3Cit),
wherein said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating tumour progression in said individuals or cancer in said individuals with previously diagnosed cancer.
By "screening for tumour progression" we include testing to ascertain a tumour prognosis. It will be appreciated that "screening for tumour
progression" and "tumour prognosis" are general terms that can relate to ascertaining an improved tumour prognosis (such as a reduction in the number of tumour symptoms and/or a reduction in the severity of tumour symptoms and/or a reduction in tumour metastasis and/or a reduced risk of the tumour being fatal) or ascertaining a worsened tumour prognosis (such as an increase in the number of tumour symptoms and/or an increase in the severity of tumour symptoms and/or an increase in tumour metastasis and/or an increased risk of the tumour being fatal). Therefore, by "indicating tumour progression" we include that a result obtained from the method is used to provide a tumour prognosis, such as an improved tumour prognosis or a worsened tumour prognosis. By "screening for recurrence of cancer in individuals with previously diagnosed cancer" we include testing to identify a relapsed cancer, such as identifying whether an individual that has previously recovered from cancer again has cancer. Accordingly, by "recovered from said cancer" we include that the individual previously fully recovered from said cancer (for example, the individual no longer exhibited cancer symptoms) or that the individual partially previously recovered from said cancer (for example, the individual exhibited a reduced number of symptoms and/or exhibited a reduced severity of symptoms). If the individual partially previously recovered from said cancer by "has cancer again" we include that the individual has an increased number of symptoms and/or an increased severity in symptoms. Therefore, by "indicating cancer in said individuals with previously diagnosed cancer" we include that a result obtained from the method is used to identify the presence of relapsed cancer in the individual.
In an alternative embodiment of the fourth aspect, there is provided a method for screening for cancer progression in individuals, comprising the steps: a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or the quantity in the plasma sample of a biomarker chosen from citrullinated histone H3 (H3Cit),
wherein said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating cancer progression in said individuals.
By "screening for cancer progression" we include testing to ascertain a cancer prognosis. It will be appreciated that "screening for cancer progression" and "cancer prognosis" are general terms that can relate to ascertaining an improved cancer prognosis (such as a reduction in the number of cancer symptoms and/or a reduction in the severity of cancer symptoms and/or a reduction in cancer metastasis and/or a reduced risk of the cancer being fatal) or ascertaining a worsened cancer prognosis (such as an increase in the number of cancer symptoms and/or an increase in the severity of cancer symptoms and/or an increase in cancer metastasis and/or an increased risk of the cancer being fatal). Therefore, by "indicating cancer progression" we include that a result obtained from the method is used to provide a cancer prognosis, such as an improved cancer prognosis or a worsened cancer prognosis.
In a fifth aspect, there is provided a method for screening for cancer, an elevated risk for cancer-associated thrombosis and/or cancer-associated thrombosis in individuals, comprising the steps:
a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or the quantity in the plasma sample of biomarkers chosen from citrullinated histone H3 (H3Cit) and granulocyte colony stimulating factor (G-CSF),
wherein said presence and/or quantity in the test sample of said biomarkers chosen from citrullinated histone H3 (H3Cit) and granulocyte colony stimulating factor (G-CSF) is indicating cancer, an elevated risk for cancer-associated thrombosis and/or cancer- associated thrombosis in said individuals.
Granulocyte colony stimulating factor (G-CSF) is a cytokine glycoprotein that is produced by a number of cells of the immune system, and which acts in hematopoiesis by controlling the production, differentiation, and function of granulocytes. G-CSF is normally synthesized by leukocytes and endothelial cells, but may also be overexpressed in cancer cells. G-CSF is also known by the abbreviation GCSF and as colony-stimulating factor 3 (CSF 3).
Human G-CSF is a 19.6 kDa protein with 174 amino acid residues:
TPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGH SLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQ ALEGISPELGP TLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGV LVASHLQSFLEVSYRVLRHLAQP (SEQ ID NO: 2)
(Hill CP., et al. 1993 PNAS 1 ;90(1 1 ):5167-71 ).
The DNA sequence of G-CSF is: ctgccgcttc caggcgtcta tcagcggctc agcctttgtt cagctgttct gttcaaacac tctggggcca ttcaggcctg ggtggggcag cgggaggaag ggagtttgag gggggcaagg cgacgtcaaa ggaggatcag agattccaca atttcacaaa actttcgcaa acagcttttt gttccaaccc ccctgcattg tcttggacac caaatttgca taaatcctgg gaagttatta ctaagcctta gtcgtggccc caggtaattt cctcccaggc ctccatgggg ttatgtataa agggccccct agagctgggc cccaaaacag cccggagcct gcagcccagc cccacccaga cccatggctg gacctgccac ccagagcccc atgaagctga tgggtgagtg tcttggccca ggatgggaga gccgcctgcc ctggcatggg agggaggctg gtgtgacaga ggggctgggg atccccgttc tgggaatggg gattaaaggc acccagtgtc cccgagaggg cctcaggtgg tagggaacag catgtctcct gagcccgctc tgtccccagc cctgcagctg ctgctgtggc acagtgcact ctggacagtg caggaagcca cccccctggg ccctgccagc tccctgcccc agagcttcct gctcaagtgc ttagagcaag tgaggaagat ccagggcgat ggcgcagcgc tccaggagaa gctggtgagt gaggtgggtg agagggctgt ggagggaagc ccggtgggga gagctaaggg ggatggaact gcagggccaa catcctctgg aagggacatg ggagaatatt aggagcagtg gagctgggga aggctgggaa gggacttggg gaggaggacc ttggtgggga cagtgctcgg gagggctggc tgggatggga gtggaggcat cacattcagg agaaagggca agggcccctg tgagatcaga gagtgggggt gcagggcaga gaggaactga acagcctggc aggacatgga gggaggggaa agaccagaga gtcggggagg acccgggaag gagcggcgac ccggccacgg cgagtctcac tcagcatcct tccatcccca gtgtgccacc tacaagctgt gccaccccga ggagctggtg ctgctcggac actctctggg catcccctgg gctcccctga gcagctgccc cagccaggcc ctgcagctgg tgagtgtcag gaaaggataa ggctaatgag gagggggaag gagaggagga acacccatgg gctcccccat gtctccaggt tccaagctgg gggcctgacg tatctcaggc agcaccccct aactcttccg ctctgtctca caggcaggct gcttgagcca actccatagc ggccttttcc tctaccaggg gctcctgcag gccctggaag ggatctcccc cgagttgggt cccaccttgg acacactgca gctggacgtc gccgactttg ccaccaccat ctggcagcag gtgagccttg ttgggcaggg tggccaaggt cgtgctggca ttctgggcac cacagccggg cctgtgtatg ggccctgtcc atgctgtcag cccccagcat ttcctcattt gtaataacgc ccactcagaa gggcccaacc actgatcaca gctttccccc acagatggaa gaactgggaa tggcccctgc cctgcagccc acccagggtg ccatgccggc cttcgcctct gctttccagc gccgggcagg aggggtcctg gttgcctccc atctgcagag cttcctggag gtgtcgtacc gcgttctacg ccaccttgcc cagccctgag ccaagccctc cccatcccat gtatttatct ctatttaata tttatgtcta tttaagcctc atatttaaag acagggaaga gcagaacgga gccccaggcc tctgtgtcct tccctgcatt tctgagtttc attctcctgc ctgtagcagt gagaaaaagc tcctgtcctc ccatcccctg gactgggagg tagataggta aataccaagt atttattact atgactgctc cccagccctg gctctgcaat gggcactggg atgagccgct gtgagcccct ggtcctgagg gtccccacct gggacccttg agagtatcag gtctcccacg tgggagacaa gaaatccctg tttaatattt aaacagcagt gttccccatc tgggtccttg cacccctcac tctggcctca gccgactgca cagcggcccc tgcatcccct tggctgtgag gcccctggac aagcagaggt ggccagagct gggaggcatg gccctggggt cccacgaatt tgctggggaa tctcgttttt cttcttaaga cttttgggac atggtttgac tcccgaacat caccgacgtg tctcctgttt ttctgggtgg cctcgggaca cctgccctgc ccccacgagg gtcaggactg tgactctttt tagggccagg caggtgcctg gacatttgcc ttgctggatg gggactgggg atgtgggagg gagcagacag gaggaatcat gtcaggcctg tgtgtgaaag gaagctccac tgtcaccctc cacctcttca ccccccactc accagtgtcc cctccactgt cacattgtaa ctgaacttca ggataataaa gtgtttgcct ccagtcacgt ccttcctcct tcttgagtcc agctggtgcc tggccagggg ctggggaggt ggctgaaggg tgggagaggc cagagggagg tcggggagga ggtctgggga ggaggtccag ggaggaggag gaaagttctc aagttcgtct gacattcatt ccgttagcac atatttatct gagcacctac tctgtgcaga cgctgggcta agtgctgggg acacagcagg gaacaaggca gacatggaat ctgcactcga (SEQ ID NO: 3)
In a sixth aspect, there is provided a method for screening for adverse effects in individuals receiving granulocyte colony stimulating factor (G-CSF) following chemotherapy, comprising the steps:
a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or the quantity in the plasma sample of a biomarker chosen from citrullinated histone H3 (H3Cit),
wherein said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating adverse effects in individuals receiving granulocyte colony stimulating factor (G-CSF) following chemotherapy.
In some circumstances, G-CSF is administered after chemotherapy in order to reduce chemotherapy associated neutropenia (ie a low concentration of neutrophils) and the incidence of febrile neutropenia (ie a fever associated with a low concentration of neutrophils).
By "screening for adverse effects in individuals receiving granulocyte colony stimulating factor (G-CSF)" we include testing to identify whether the individual is exhibiting an adverse reaction to the G-CSF and/or the G-CSF is having an undesired effect in the individual and/or the G-CSF is not reducing neutropenia in the individual and/or the G-CSF is not reducing febrile neutropenia in the individual. G-CSF may induce NETs from neutrophils, which might lead to cancer progression, cancer-associated thrombosis or thrombosis. It will generally be appreciated that the adverse effect would negatively affect the health of the individual. Therefore, by "indicating adverse effects in individuals receiving granulocyte colony stimulating factor (G-CSF) following chemotherapy" we include that a result obtained from the method is used to identify that the individual is exhibiting an adverse reaction to the G-CSF and/or the G-CSF is having an undesired effect in the individual and/or the G-CSF is not reducing neutropenia in the individual and/or the G- CSF is not reducing febrile neutropenia in the individual and/or the individual having an elevated risk of having an adverse reaction to G-CSF and/or cancer progression and/or the individual has cancer-associated thrombosis and/or the individual has thrombosis.
In a seventh aspect, there is provided a diagnostic kit for use in a method according to any one of the above mentioned methods for comprising;
a) a binding agent capable of binding to the biomarker citrullinated histone H3 (H3Cit); and
b) instructions for performing any of the above described methods. Brief description of drawings
Fig 1 . Depicts plasma H3Cit measured with our tailor-made ELISA, demonstrating plasma H3Cit in patients with ischemic stroke with and without an underlying active cancer. These data clearly show that patients with ischemic stroke only (without underlying cancer) have diminishing or no levels of circulating H3Cit, whereas patients with ischemic stroke and an underlying active cancer have detectable and quantifiable levels of circulating H3Cit. Notably, the outlier among the patients in the group without cancer (with H3Cit 0.55 O.D.) died shortly after the stroke onset. There was no autopsy, and, although the patient had not been diagnosed with cancer, there were strong clinical indications that this patient actually had an underlying active cancer. The boxplot depicts a min-max value with median.
Fig 2. depicts Western Blot analysis with qualitative detection of H3Cit in plasma. Ischemic stroke patients with cancer had detectable H3Cit in plasma, whereas ischemic stroke patients without cancer had no detectable H3Cit in plasma. This clearly demonstrates that our tailor made ELISA was successful as it was done on the same samples. The added value of the tailor made ELISA compared to the Western Blot analysis is the
important fact that the tailored made method not only detects H3Ct in plasma (as do Western Blot Analysis) but also quantifies the level of H3Cit in plasma, which we believe is key in achieving substantial clinical value with our method.
Fig 3. Depicts plasma G-CSF in patients with and without cancer.
Ischemic stroke patients with cancer had a seven fold increase in plasma G- CSF compared to ischemic stroke patients without cancer. The boxplot depicts a min-max value with median.
Fig 4. depicts correlation between plasma H3Cit and plasma G-CSF. The positive correlation between elevations of plasma G-CSF and plasma H3Cit in patients with cancer supports a cancer-released G-CSF-induced NET burden. Significance of correlation was analyzed with Spearman's rank correlation using ST ATA 12.1 software (STATA, Texas, USA).
Fig 5. depicts the stability of the standard (STD) in our tailor-made H3Cit ELISA-based assay. The detector response when preparing STD curves from frozen aliquots from three different batches of in vitro PAD4- citrullinated histone H3 on three different days (STD 1 -3) were not
significantly different (F (DFn, DFd)= 2.6 (8,9); p= 0.088).
Fig 6. depicts the stability of the standard (STD) in our tailor-made H3Cit ELISA-based assay. STD curves prepared from freshly made in vitro PAD4-citrullinated histone H3 and frozen aliquots of in vitro PAD4- citrullinated histone H3. No significant difference was observed when comparing the detector response of the freshly made versus frozen standards (F (DFn, DFd)= 0.2 (4, 52); p= 0.916.
Fig 7. depicts standard (STD) curves generated from in vitro PAD4- citrullinated histone H3 and from histone H3 incubated in the same
conditions, but without PAD4, rendering non-citrullinated histones. The STD curves are representative of three different experiments. There was a low amount of antibody antigen detection when large amounts of non-citrullinated histone H3 were present, but the antibody antigen detection was specific for H3Cit in the linear interval of the assay. Fig 8. depicts concentrations of H3Cit, measured with tailor-made H3Cit ELISA-based assay, in plasma samples taken from healthy individuals before and after LPS injection. The quantification of H3Cit in plasma of healthy volunteers before LPS injections were under the detection limit of approximately 5 ng/mL. An increase in the levels of H3Cit in all plasma samples taken from the same individuals 3-4 h after LPS was observed, ranging from 28.7 ng/mL to 93.2 ng/mL. Plasma H3Cit was measured with our tailor-made H3Cit ELISA-based assay.
Fig 9. depicts the age and sex distribution of the study population in Example 3. The boxplot depicts a min-max value with median. There was no significant differences in age or sex distribution between the patients with active cancer (median age 70.5, 41 .7% men), hospitalized patients with other diseases but no known cancer (median age 77, 40% men), and the healthy individuals (median age 70.4, 42% men).
Fig 10. depicts plasma levels of H3Cit in Example 3. Plasma H3Cit was measured with our tailor-made ELISA, demonstrating plasma H3Cit levels in patients with active cancer (n=60), hospitalized patients with other diseases but no known cancer (n=51 ) and age and gender matched healthy individuals (n=50). Cancer patients had a 5-fold increase in plasma H3Cit levels compared to healthy individuals (mean 36.1 ng/mL vs. 6.6 ng/mL, *** p<0.001 ) and a 3-fold increase compared to hospitalized patients with other diseases but no known cancer (mean 36.1 ng/mL vs. 1 1 .5 ng/mL, *** p<0.001 ). There was no significant difference in plasma H3Cit levels between healthy individuals and hospitalized patients with other diseases but no known cancer (NS p=0.510), suggesting that plasma H3Cit is not a marker for disease burden. Comparison and p-values were obtained with the two- sample Kolmogorov-Smirnov test using ST ATA 12.1 software (STATA, Texas, USA). The scatter-plot depicts all concentrations and mean value (box). The boxplot depicts a min-max value with median (line). The table shows the percentage of positive (ie detectable), the median and mean plasma H3Cit levels in the three groups. Fig 1 1 . depicts plasma H3Cit levels in patients with various primary malignant tumors. "Other" comprises gingival, liposarcoma, sarcoma, acute myeloid leukemia, lymphoma, neuroendocrine and unknown origin, see also Table 2 under Example 3). Plasma H3Cit was measured with an ELISA of the invention, demonstrating a clear elevation in several malignant tumor types (both epithelial and other solid malignant tumors as well as hematological cancers), suggesting the potential of H3Cit as a biomarker in screening for several cancer types (ie cancer in general).
Fig 12. depicts the Charlson comorbidity index scores (Charlson ME, et al. A new method of classifying prognostic comorbidity in longitudinal studies: Development and validation. 1987 Journal of Chronic Diseases. 1987;40:373-83) for cancer patients and hospitalized patients with other diseases but no known cancer (referred to as multisick in the figure). The boxplot depicts a min-max value with median. The cancer patients had a significantly lower comorbidity index scores compared to hospitalized patients with other diseases but no known cancer (median 3 vs. 6, p<0.001 ), suggesting that plasma H3Cit is not a biomarker for disease burden in general. Detailed description of the invention
Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For example, any method disclosed for detecting a specific biomarker can be applied to each of the biomarkers disclosed herein.
As described above, the present invention is related to a method for diagnosing several different conditions as described above by measuring the presence and/or quantity of H3Cit in a plasma sample.
In the present inventive method other body fluid samples of an individual may also be tested for the presence and/or quantity of H3Cit, such as in urine, cerebrospinal fluid, sputum and nasopharynx, wherein said presence and/or quantity in the sample tested indicates that the individual has the condition as tested for.
Preferably, the plasma sample is provided from venous blood. Venous blood is a term that will be well understood by the person skilled in medicine, in particular an oncologist.
Preferably, the H3Cit is a histone 3 that is citrullinated at one or more arginine residues at amino acid positions 2, 8 or 17.
Even more preferably, the H3Cit is a histone 3 that is citrullinated at arginine residues at amino acid positions 2, 8 and 17.
In an embodiment of the described inventive method, step b) comprises measuring the optical density of the plasma sample, which is further described in the experimental part.
It will be appreciated that the optical density represents the presence and/or quantity of the biomarker (such as H3Cit) - for example, the optical density is proportional to the presence and/or the quantity of the biomarker. Put in another way, if the optical density is high the presence and/or the quantity of the biomarker is also high. Methods of measuring optical density will be well known to those skilled in the art of biology and chemistry, in particular a molecular biologist or biochemist, such as measuring optical density using spectrometry.
In an embodiment of the first aspect of the invention, a high optical density of the plasma sample is indicating cancer {i.e. is indicative of cancer) in said individual.
In an embodiment of the second aspect of the invention, a high optical density of the plasma sample is indicating cancer-associated thrombosis {i.e. is indicative of cancer-associated thrombosis) or an elevated risk for cancer- associated thrombosis in said cancer individuals or cancer in said individuals with idiopathic thrombosis.
In an embodiment of the third aspect of the invention, a high optical density of the plasma sample is indicating thrombosis {i.e. is indicative of thrombosis) or an elevated risk for thrombosis in said cancer individuals following chemotherapy. In an embodiment of the fourth aspect of the invention, a high optical density of the plasma sample is indicating tumour progression {i.e. is indicative of tumour progression) in said individuals or cancer in said individuals with previously diagnosed cancer.
In an embodiment of the alternative fourth aspect of the invention, a high optical density of the plasma sample is indicating cancer progression {i.e. is indicative of cancer progression).
In an embodiment of the fifth aspect of the invention, a high optical density of the plasma sample is indicative of cancer, an elevated risk for cancer-associated thrombosis and/or cancer-associated thrombosis in said individuals.
In an embodiment of the sixth aspect of the invention, a high optical density of the plasma sample is indicating adverse effects {i.e. is indicative of adverse effects) in individuals receiving granulocyte colony stimulating factor (G-CSF) following chemotherapy.
Preferably, the high optical density is an optical density in the range of 0.1 to 4 - for example, an optical density of 0.15 to 4, an optical density of 0.2 to 4, an optical density of 0.22 to 4, an optical density of 0.25 to 4, an optical density of 0.3 to 4, an optical density of 0.35 to 4, an optical density of 0.4 to 4, an optical density of 0.45 to 4, an optical density of 0.5 to 4, an optical density of 0.6 to 4, an optical density of 0.7 to 4, an optical density of 0.8 to 4, an optical density of 0.9 to 4, an optical density of 1 .0 to 4, an optical density of 1 .1 to 4, an optical density of 1 .2 to 4, an optical density of 1 .3 to 4, an optical density of 1 .4 to 4, an optical density of 1 .5 to 4, an optical density of 1 .6 to 4, an optical density of 1 .7 to 4, an optical density of 1 .8 to 4, an optical density of 1 .9 to 4, an optical density of 2 to 4, an optical density of 2.1 to 4, an optical density of 2.2 to 4, an optical density of 2.3 to 4, an optical density of 2.4 to 4, an optical density of 2.5 to 4, an optical density of 2.6 to 4, an optical density of 2.7 to 4, an optical density of 2.8 to 4, an optical density of 2.9 to 4, an optical density of 3 to 4 an optical density of 3.1 to 4, an optical density of 3.2 to 4, an optical density of 3.3 to 4, an optical density of 3.4 to 4, an optical density of 3.5 to 4, an optical density of 3.6 to 4, an optical density of 3.7 to 4, an optical density of 3.8 to 4, or an optical density of 3.9 to 4. Even more preferably, the high optical density is an optical density in the range of 0.22 to 4.
Preferably, the high optical density is an optical density of 0.1 or more - for example, an optical density of 0.15 or more, an optical density of 0.2 or more, an optical density of 0.25 or more, an optical density of 0.3 or more, an optical density of 0.35 or more, an optical density of 0.4 or more, an optical density of 0.45 or more, an optical density of 0.5 or more, an optical density of 0.6 or more, an optical density of 0.7 or more, an optical density of 0.8 or more, an optical density of 0.9 or more, an optical density of 1 .0 or more, an optical density of 1 .1 or more, an optical density of 1 .2 or more, an optical density of 1 .3 or more, an optical density of 1 .4 or more, an optical density of 1 .5 or more, an optical density of 1 .6 or more, an optical density of 1 .7 or more, an optical density of 1 .8 or more, an optical density of 1 .9 or more, an optical density of 2 or more, an optical density of 2.1 or more, an optical density of 2.2 or more, an optical density of 2.3 or more, an optical density of 2.4 or more, an optical density of 2.5 or more, an optical density of 2.6 or more, an optical density of 2.7 or more, an optical density of 2.8 or more, an optical density of 2.9 or more, an optical density of 3 or more, an optical density of 3.1 or more, an optical density of 3.2 or more, an optical density of 3.3 or more, an optical density of 3.4 or more, an optical density of 3.5 or more, an optical density of 3.6 or more, an optical density of 3.7 or more, an optical density of 3.8 or more, an optical density of 3.9 or more, or an optical density of 4 or more.
Even more preferably, the high optical density is an optical density of 0.22 or more.
Preferably, step b) comprises comparing the presence and/or the quantity of the H3Cit biomarker in the plasma sample with the presence and/or the quantity of the H3Cit biomarker in one or more plasma samples from healthy individual(s) (for example, a healthy individual) having known concentrations of citrullinated histone H3 (H3Cit) in said plasma samples. By "healthy individual", we include an individual that does not have cancer and/or an individual that does not have thrombosis and/or an individual that does not have cancer-associated thrombosis and/or an individual with no known cancer - for example, the individual does not have cancer or thrombosis.
Preferably, the healthy individual is one or more healthy individual(s) - for example a population of a number of healthy individuals, such as 10 or more healthy individuals, 20 or more healthy individuals, 30 or more healthy individuals, 40 or more healthy individuals, 50 or more healthy individuals, 60 or more healthy individuals, 70 or more healthy individuals, 80 or more healthy individuals, 90 or more healthy individuals, 100 or more healthy individuals, 200 or more healthy individuals, 300 or more healthy individuals, 400 or more healthy individuals, 500 or more healthy individuals, 1000 or more, 2000 or more healthy individuals, or 3000 or more healthy individuals.
Even more preferably, the healthy individual is a population of 1000 or more healthy individuals.
In another embodiment of the invention, step b) comprises measuring the optical density of the plasma sample and comparing to the optical density of one or more plasma samples from healthy individual(s) having known concentrations of citrullinated histone H3 (H3Cit) in said plasma samples.
In an embodiment of the first aspect of the invention, a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of cancer in said individual.
In an embodiment of the second aspect of the invention, a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of cancer-associated thrombosis or an elevated risk for cancer-associated thrombosis in said cancer individuals or cancer in said individuals with idiopathic thrombosis.
In an embodiment of the third aspect of the invention, a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of thrombosis or an elevated risk for thrombosis in said cancer individuals following
chemotherapy. In an embodiment of the fourth aspect of the invention, a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of tumour progression in said individuals or cancer in said individuals with previously diagnosed cancer.
In an embodiment of the alternative fourth aspect of the invention, a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of cancer progression in said individuals.
In an embodiment of the fifth aspect of the invention, a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of cancer, an elevated risk for cancer-associated thrombosis and/or cancer-associated thrombosis in said individuals.
In an embodiment of the sixth aspect of the invention, a higher optical density of the plasma sample when compared to the optical density of one or more plasma samples from healthy individual(s) is indicative of adverse effects in individuals receiving granulocyte colony stimulating factor (G-CSF) following chemotherapy.
A manner in which to define whether the optical density in the test sample is higher than the optical density plasma samples from healthy individual(s) is by calculating a fold change. How to calculate a fold change for this purpose will be well known to those skilled in biology, in particular a molecular biologist or a computational biologist. Generally, when a fold change is calculated it is based upon a comparison between a test sample and the mean or median of the plasma samples from a healthy individual(s).
Preferably, the optical density of the plasma sample is two-fold higher or more than the mean or median optical density of one or more plasma samples from healthy individual(s) - for example, three-fold higher or more, four-fold higher or more, five-fold higher or more, six-fold higher or more, seven-fold higher or more, eight-fold higher or more, nine-fold higher or more, or ten-fold higher or more than the optical density of one or more plasma samples from healthy individual(s). Even more preferably, the optical density of the plasma sample is three-fold higher or more than the mean or median optical density of one or more plasma samples from healthy individual(s).
Even more preferably, the optical density of the plasma sample is two- fold higher or more than the mean or median optical density of one or more plasma samples from healthy individual(s).
Even more preferably, the optical density of the plasma sample is fivefold higher or more than the mean or median optical density of one or more plasma samples from healthy individual(s).
It is possible to calculate the concentration of the H3Cit in the test sample by comparing that sample (for example, the optical density of that sample) with a sample having a known concentration of H3Cit. How to calculate such a concentration will be well known to those skilled in biology, in particular a molecular biologist or a computational biologist.
Preferably, step b) further comprises comparing the presence and/or the quantity of the H3Cit biomarker in the plasma sample with one or more standard sample(s) having known concentrations of citrullinated histone H3 (H3Cit), to identify the concentration of the H3Cit biomarker in the plasma sample.
Preferably, step b) further comprises comparing the presence and/or the quantity of the H3Cit biomarker in the plasma samples from healthy individual(s) with one or more standard sample(s) having known
concentrations of citrullinated histone H3 (H3Cit), to identify the concentration of the H3Cit biomarker in the plasma sample samples from the healthy individual(s).
Preferably, the standard sample is histone H3 citrullinated in vitro by a PAD enzyme, such as PAD4, to form H3Cit.
Preferably, the histone H3 is human recombinant histone H3.
Preferably, the PAD4 is human recombinant PAD4.
Methods of making an in vitro standard sample with a known concentration of H3Cit will be known to those skilled in molecular biology (for example, as described in Li P, et al. Regulation of p53 Target Gene
Expression by Peptidylarginine Deiminase 4. Mol Cell Biol 2008;28:4745-58). An exemplary method is to incubate human recombinant histone H3 and human recombinant PAD4 at 37 °C for 1 hour in a reaction buffer (50 imM Trizma base with 4 mM CaCI2, pH 7.6, 4 mM DTT and 1 mM PMSF), and make a final concentration of 10,000 ng/mL H3Cit by adding PBS-1 % BSA.
Preferably, the one or more standard samples are one or more standard samples having different known concentrations of H3Cit.
Even more preferably, the standard samples having different known concentrations of H3Cit are used to calculate a standard curve.
Methods of using standard samples having different known
concentrations of H3Cit to calculate a standard curve will be known to those skilled in molecular biology. An exemplary method could be to make an original standard sample with a known concentration of H3Cit, and then to serially dilute that original standard sample to produce different standard samples with known concentrations of H3Cit. The experimental read-out (for example, an optical density) of those different standard samples with known concentrations of H3Cit can be measured, and plotted onto a graph to calculate a standard curve. The experimental read-out of the H3Cit in the plasma sample can then be measured and compared to the standard curve, to provide a measurement of the quantity of the H3Cit in that plasma sample.
Preferably, eight standard samples having different known
concentrations of H3Cit are used to calculate a standard curve, with an original standard sample (for example, with a H3Cit concentration of 10,000 ng/ml) being serially diluted each time (for example, at a ratio of 1 :2).
Accordingly, most preferably step b) further comprises comparing the presence and/or the quantity of the H3Cit biomarker in the plasma sample, and/or the presence and/or the quantity the plasma samples from healthy individual(s), with a standard curve of known concentrations of citrullinated histone H3 (H3Cit), to identify the concentration of the H3Cit biomarker.
Preferably, said presence and/or quantity of the H3Cit biomarker in the test sample is a concentration in the range of 5 ng/ml to 350 ng/ml - for example, 10 ng/ml to 350 ng/ml, 15 ng/ml to 350 ng/ml, 20 ng/ml to 350 ng/ml, 25 ng/ml to 350 ng/ml, 30 ng/ml to 350 ng/ml, 35 ng/ml to 350 ng/ml, 40 ng/ml to 350 ng/ml, 45 ng/ml to 350 ng/ml, 50 ng/ml to 350 ng/ml, 60 ng/ml to 350 ng/ml, 70 ng/ml to 350 ng/ml, 80 ng/ml to 350 ng/ml, 90 ng/ml to 350 ng/ml, 100 ng/ml to 350 ng/ml, 1 10 ng/ml to 350 ng/ml, 120 ng/ml to 350 ng/ml, 130 ng/ml to 350 ng/ml, 140 ng/ml to 350 ng/ml, 150 ng/ml to 350 ng/ml, 200 ng/ml to 350 ng/ml, 250 ng/ml to 350 ng/ml, or 300 ng/ml to 350 ng/ml.
Preferably, said presence and/or quantity of the H3Cit biomarker in the test sample is a concentration of 5 ng/ml or more - for example, 10 ng/ml or more, 15 ng/ml or more, 20 ng/ml or more, 25 ng/ml or more, 30 ng/ml or more, 35 ng/ml or more, 40 ng/ml or more, 45 ng/ml or more, 50 ng/ml or more, 60 ng/ml or more, 70 ng/ml or more, 80 ng/ml or more, 90 ng/ml or more, 100 ng/ml or more, 1 10 ng/ml or more, 120 ng/ml or more, 130 ng/ml or more, 140 ng/ml or more, 150 ng/ml or more, 200 ng/ml or more, 250 ng/ml or more, 300 ng/ml or more, or 350 ng/ml or more.
Even more preferably, said presence and/or quantity of the H3Cit biomarker in the test sample is a concentration of 20 ng/ml or more.
Preferably, the presence and/or the quantity of the H3Cit biomarker in the plasma sample is higher than the presence and/or the quantity of the H3Cit biomarker in one or more plasma samples from a healthy individual(s).
Preferably, the presence and/or the quantity of the H3Cit biomarker in the one or more plasma samples from healthy individual(s) is a concentration in the range of 0 ng/ml to 50 ng/ml - for example, 0 ng/ml to 1 ng/ml, 0 ng/ml to 2 ng/ml, 0 ng/ml to 3 ng/ml, 0 ng/ml to 4 ng/ml, 0 ng/ml to 5 ng/ml, 0 ng/ml to 6 ng/ml, 0 ng/ml to 7 ng/ml, 0 ng/ml to 8 ng/ml, 0 ng/ml to 9 ng/ml, 0 ng/ml to 10 ng/ml, 0 ng/ml to 12 ng/ml, 0 ng/ml to 14 ng/ml, 0 ng/ml to 16 ng/ml, 0 ng/ml to 18 ng/ml, 0 ng/ml to 20 ng/ml, 0 ng/ml to 25 ng/ml, 0 ng/ml to 30 ng/ml, 0 ng/ml to 35 ng/ml, 0 ng/ml to 40 ng/ml, or 0 ng/ml to 45 ng/ml.
Preferably, the presence and/or the quantity of the H3Cit biomarker in the one or more plasma samples from healthy individual(s) is a concentration of 50 ng/ml or less - for example, 1 ng/ml or less, 2 ng/ml or less, 3 ng/ml or less, 4 ng/ml or less, 5 ng/ml or less, 6 ng/ml or less, 7 ng/ml or less, 8 ng/ml or less, 9 ng/ml or less, 10 ng/ml or less, 12 ng/ml or less, 14 ng/ml or less, 16 ng/ml or less, 18 ng/ml or less, 20 ng/ml or less, 25 ng/ml or less, 30 ng/ml or less, 35 ng/ml or less, 40 ng/ml or less, or 45 ng/ml or less. Even more preferably, the presence and/or the quantity of the H3Cit biomarker in the one or more plasma samples from healthy individual(s) is a concentration of 2 ng/ml or less.
Preferably, the presence and/or the quantity of the H3Cit biomarker in the plasma sample is two-fold higher or more than the presence and/or the mean or median quantity of the H3Cit biomarker in one or more plasma samples from healthy individual(s) - for example, three-fold higher or more, four-fold higher or more, five-fold higher or more, six-fold higher or more, seven-fold higher or more, eight-fold higher or more, nine-fold higher or more, or ten-fold higher or more than the presence and/or the quantity of the H3Cit biomarker in one or more plasma samples from a healthy individual(s).
Even more preferably, the presence and/or the quantity of the H3Cit biomarker in the plasma sample is three-fold higher or more than the presence and/or the mean or median quantity of the H3Cit biomarker in one or more plasma samples from a healthy individual(s).
Even more preferably, the presence and/or the quantity of the H3Cit biomarker in the plasma sample is two-fold higher or more than the presence and/or the mean or median quantity of the H3Cit biomarker in one or more plasma samples from a healthy individual(s).
Even more preferably, the presence and/or the quantity of the H3Cit biomarker in the plasma sample is five-fold higher or more than the presence and/or the mean or median quantity of the H3Cit biomarker in one or more plasma samples from a healthy individual(s).
In an embodiment of the first aspect of the invention, step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time;
and step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a
consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of cancer in said individual.
In an embodiment of the second aspect of the invention, step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time; and step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of cancer-associated thrombosis or an elevated risk for cancer-associated thrombosis in said cancer individuals or cancer in said individuals with idiopathic thrombosis.
In an embodiment of the third aspect of the invention, step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time;
and step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of thrombosis or an elevated risk for thrombosis in said cancer individuals following chemotherapy.
In an embodiment of the fourth aspect of the invention, step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time;
and step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of tumour progression in said individuals or cancer in said individuals with previously diagnosed cancer.
In an embodiment of the alternative fourth aspect of the invention, step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time;
and step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of cancer progression in said individuals. In an embodiment of the fifth aspect of the invention, step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time;
and step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of cancer, an elevated risk for cancer-associated thrombosis and/or cancer-associated thrombosis in said individuals.
In an embodiment of the sixth aspect of the invention, step a) further comprises providing a number of plasma samples to be tested from the individual, over a period of time;
and step b) further comprises measuring the presence and/or quantity of citrullinated histone H3 (H3Cit) in the plasma samples, wherein a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) in the test samples over the period of time is indicative of adverse effects in individuals receiving granulocyte colony stimulating factor (G-CSF) following chemotherapy.
Preferably, the number of plasma samples is two or more - for example, three or more, four or more, five or move, six or more, seven or more, eight or more, nine or more, ten or more, 15 or more, 20 or more, 30 or more 40 or more, or 50 or more.
Preferably, the period of time is one week or more - for example, two weeks or more, three weeks or more, four weeks or more, one month or more, two months or more, three months of more, four months or more, five months or more, six months or move, seven months or more, eight months or more, nine months or more, or one year or more.
Even more preferably, the period of time is one month.
Preferably, the number of plasma samples are provided at equal time intervals during the period of time - for example, when two plasma samples are provided in a one week period of time, one plasma sample is provided at the first day of the week and a second plasma sample is provided at the seventh day of the week. Preferably a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) is a presence and/or quantity of citrullinated histone H3 (H3Cit) not changing by 1 % or more when compared to the presence and/or quantity of citrullinated histone H3 (H3Cit) in the preceding plasma sample - for example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.
Even more preferably, a consistent presence and/or quantity of citrullinated histone H3 (H3Cit) is a presence and/or quantity of citrullinated histone H3 (H3Cit) not changing by 20% or more when compared to the presence and/or quantity of citrullinated histone H3 (H3Cit) in the preceding plasma sample.
In an embodiment of the first aspect of the invention, step b) further comprises measuring the presence and/or the quantity in the plasma sample of G-CSF,
wherein said presence and/or quantity in the test sample of G-CSF is indicative of cancer in said individual.
In an embodiment of the second aspect of the invention, step b) further comprises measuring the presence and/or the quantity in the plasma sample of G-CSF,
wherein said presence and/or quantity in the test sample of G-CSF is indicative of cancer-associated thrombosis or an elevated risk for cancer- associated thrombosis in said cancer individuals or cancer in said individuals with idiopathic thrombosis.
In an embodiment of the third aspect of the invention, step b) further comprises measuring the presence and/or the quantity in the plasma sample of G-CSF,
wherein said presence and/or quantity in the test sample of G-CSF is indicative of thrombosis or an elevated risk for thrombosis in said cancer individuals following chemotherapy.
In an embodiment of the fourth aspect of the invention, step b) further comprises measuring the presence and/or the quantity in the plasma sample of G-CSF, wherein said presence and/or quantity in the test sample of G-CSF is indicative of tumour progression in said individuals or cancer in said
individuals with previously diagnosed cancer.
In an embodiment of the alternative fourth aspect of the invention, step b) further comprises measuring the presence and/or the quantity in the plasma sample of G-CSF,
wherein said presence and/or quantity in the test sample of G-CSF is indicative of cancer progression in said individuals.
In an embodiment of the sixth aspect of the invention, step b) further comprises measuring the presence and/or the quantity in the plasma sample of G-CSF,
wherein said presence and/or quantity in the test sample of G-CSF is indicative of adverse effects in individuals receiving G-CSF following chemotherapy.
The concentration of G-CSF in the plasma sample can be calculated using methods similar to those outlined above for H3Cit, as will be known to those skilled in molecular biology.
Preferably, said presence and/or quantity of the G-CSF biomarker in the test sample is a concentration in the range of 5 pg/ml to 100 pg/ml - for example, 10 pg/ml to 100 pg/ml, 15 pg/ml to 100 pg/ml, 20 pg/ml to 100 pg/ml, 21 pg/ml to 100 pg/ml, 25 pg/ml to 100 pg/ml, 30 pg/ml to 100 pg/ml, 35 pg/ml to 100 pg/ml, 40 pg/ml to 100 pg/ml, 45 pg/ml to 100 pg/ml, 50 pg/ml to 100 pg/ml, 60 pg/ml to 100 pg/ml, 70 pg/ml to 100 pg/ml, 80 pg/ml to 100 pg/ml, or 90 pg/ml to 100 pg/ml.
Preferably, said presence and/or quantity of the G-CSF biomarker in the test sample is a concentration of 5 pg/ml or more - for example, 10 pg/ml or more, 15 pg/ml or more, 20 pg/ml or more, 25 pg/ml or more, 30 pg/ml or more, 35 pg/ml or more, 40 pg/ml or more, 45 pg/ml or more, 50 pg/ml or more, 60 pg/ml or more, 70 pg/ml or more, 80 pg/ml or more, 90 pg/ml or more, or 100 pg/ml or more. Even more preferably, said presence and/or quantity of the G-CSF biomarker in the test sample is a concentration of 21 pg/ml or more.
Preferably, step b) comprises comparing the presence and/or the quantity of the G-CSF biomarker in the plasma sample with the presence and/or the quantity of the biomarker in one or more plasma samples from healthy individual(s) having known concentrations of G-CSF in said plasma samples.
Preferably, the presence and/or the quantity of the G-CSF biomarker in the plasma sample is higher than the presence and/or the quantity of the G- CSF biomarker in one or more plasma samples from a healthy individual.
Preferably, the presence and/or the quantity of the G-CSF biomarker in the one or more plasma samples from healthy individual(s) is a concentration of 5 pg/ml or less - for example, 1 pg/ml or less, 2 pg/ml or less, 3 pg/ml or less, or 4 pg/ml or less.
Preferably, the presence and/or the quantity of the G-CSF biomarker in the one or more plasma samples from healthy individual(s) is a concentration in the range of 0 pg/ml to 5 pg/ml - for example, 0 pg/ml to 1 pg/ml, 0 pg/ml to 2 pg/ml, 0 pg/ml to 3 pg/ml to 0 pg/ml, or 0 pg/ml to 4 pg/ml.
Even more preferably, the presence and/or the quantity of the G-CSF biomarker in the one or more plasma samples from healthy individual(s) is a concentration of 2 ng/ml or less.
Even more preferably, the presence and/or the quantity of the G-CSF biomarker in the one or more plasma samples from healthy individual(s) is a concentration of 3 pg/ml or less.
Preferably, the presence and/or the quantity of the G-CSF biomarker in the plasma sample is two-fold higher or more than the presence and/or the median or mean quantity of the G-CSF biomarker in one or more plasma samples from healthy individual(s) - for example, three-fold higher or more, four-fold higher or more, five-fold higher or more, six-fold higher or more, seven-fold higher or more, eight-fold higher or more, nine-fold higher or more, or ten-fold higher or more than the presence and/or the quantity of the G-CSF biomarker in one or more plasma samples from a healthy individual. Even more preferably, the presence and/or the median or mean quantity of the G-CSF biomarker in the plasma sample is seven-fold higher or more than the presence and/or the quantity of the G-CSF biomarker in one or more plasma samples from a healthy individual.
It will be appreciated that the methods described herein can be used in combination with other cancer screening methods, for example screening for prostate cancer using prostate specific antigen (PSA). The specificity of those cancer screening methods is likely to be improved when used in combination with the methods described herein.
In an embodiment of the first aspect of the invention, the method further comprises step a') conducting a further cancer screening method;
and, wherein step b) further comprises that the result of the cancer screening method is also indicative of cancer in said individual.
In an embodiment of the second aspect of the invention, the method further comprises step a') conducting a further cancer screening method;
and, wherein step b) further comprises that the result of the cancer screening method is also indicative of cancer-associated thrombosis or an elevated risk for cancer-associated thrombosis in said cancer individuals or cancer in said individuals with idiopathic thrombosis.
In an embodiment of the fourth aspect of the invention, the method further comprises step a') conducting a further cancer screening method;
and, wherein step b) further comprises that the result of the cancer screening method is also indicative of tumour progression in said individuals or cancer in said individuals with previously diagnosed cancer.
In an embodiment of the alternative fourth aspect of the invention, the method further comprises step a') conducting a further cancer screening method;
and, wherein step b) further comprises that the result of the cancer screening method is also indicative of cancer progression in said individuals. In an embodiment of the fifth aspect of the invention, the method further comprises step a') conducting a further cancer screening method;
and, wherein step b) further comprises that the result of the cancer screening method is also indicative of cancer, an elevated risk for cancer- associated thrombosis and/or cancer-associated thrombosis in said
individuals.
Preferably, the cancer screening method is for detecting prostate cancer, for example the cancer screening method for detecting prostate specific antigen (PSA).
Preferably, the cancer screening method is for detecting colorectal cancer, for example the cancer screening method for detecting CEA or the cancer screening method is a colonoscopy.
Preferably, the cancer screening method for detecting ovarian cancer, for example the cancer screening method for detecting CA-125.
Preferably, the cancer screening method is for detecting breast cancer, for example the cancer screening method is a mammography.
The person skilled in medicine, in particular an oncologist, will have
knowledge of various cancer screening methods, and the manner in which they are conducted.
In another embodiment of the invention step b) comprises using a binding agent capable of binding to the biomarker citrullinated histone H3 (H3Cit) in the plasma of the individual, such as an anti-H3Cit-antibody or a fragment thereof, i.e. a natural or synthetic binding agent capable of binding to the H3Cit as present in the plasma or other body fluid of the individual.
Preferably, the binding agent (such as an antibody) has binding specificity for a H3Cit that is citrullinated at one or more arginine residues at amino acid positions 2, 8 or 17.
Even more preferably, the binding agent (such as an antibody) has binding specificity for a H3Cit that is citrullinated at arginine residues at amino acid positions 2, 8 and 17.
Preferably, where step b) comprises measuring the presence and/or the quantity of the biomarker G-CSF, step b) further comprises using a binding agent capable of binding to the biomarker G-CSF in the plasma of the individual, such as an anti-G-CSF-antibody or a fragment thereof, i.e. a natural or synthetic binding agent capable of binding to the G-CSF as present in the plasma or other body fluid of the individual. Preferably, the presence and/or quantity of the biomarker (for example, H3Cit and/or G-CSF) is detected and/or measured using a method selected from the group consisting of: an enzyme-linked immunosorbent assay
(ELISA); a Western blot; and, immunohistochemistry.
Even more preferably, the presence and/or quantity of the biomarker
(for example, H3Cit and/or G-CSF) is detected and/or measured using an ELISA-based assay. How to undertake an ELISA, a Western blot or immunohistochemistry will be well known to those skilled in the art of molecular biology.
Most preferably, the presence and/or quantity of the H3Cit biomarker is detected and/or measured using an ELISA-based assay.
An ELISA-based assay for detecting and/or measuring H3Cit is described in Examples 1 , 2 and 3.
An ELISA method for detecting H3Cit may use microplate modules and the coating anti-histone and incubation buffer of cell death detection ELISA kit (for example, Roche, cat nr 1 1774425001 ).
Preferably, an ELISA for detecting and/or measuring H3Cit comprises one or more of the following steps:
• adding coating solution (anti-histone) and incubating, such as adding a 100 ul of coating solution (anti-histone 1 :10) to the pre-coated wells and incubating overnight at 4 degrees; and/or
• removing the coating, such as by tapping dry the wells; and/or
· adding incubation buffer, such as by adding 200 ul incubation buffer and incubating 1 h at room temperature;
• removing solution, such as by washing 3X with PBS-tween 0.05%; and/or
• adding addition incubation buffer (such as from Cell Death Detection ELISA PLUS kit, Roche, Cat. No. 1 1 774 425 001 ) and incubating, such as adding 40 ul incubation buffer and 10 ul plasma and
incubating for 1 hour and 30 minutes at room temperature; and/or
• removing solution, such as washing 3X with PBS-tween 0.05%; and/or • adding anti-H3Cit antibody and incubating, such as adding 100 ul of anti-H3Cit antibody in PBS-BSA 1 % and incubating for 1 hour and 30 minutes at room temperature; and/or
• removing solution, such as washing 3X with PBS-tween 0.05%; and/or · adding anti-rabbit horseradish peroxidase (HRP) and incubating, such as adding 100 ul of anti-rabbit HRP in PBS-BSA 1 % incubating 1 hour room temperature; and/or
• removing solution, such as washing 3X with PBS-tween 0.05%; and/or
• adding 3,3', 5,5'-tetramethylbenzidine (TMB) liquid substrate and
letting the reaction develop, such as adding 100 ul TMB and let develop the reaction; and/or
• stopping the reaction, such as with 50 ul 2% sulfuric acid; and/or
• reading optical density, such as reading the optical density at 450 nm with a reference correction wavelength at 570 nm.
Preferably, an ELISA for detecting and/or measuring H3Cit comprises making a H3Cit standard sample, which comprises one or more of the following steps: · incubating histone H3 and PAD4, such as incubating human recombinant histone H3 and human recombinant PAD4 at 37 °C for 1 hour in buffer (such as a reaction buffer comprising 50 mM Trizma base with 4 mM CaCI2, pH 7.6, 4 mM DTT and 1 mM PMSF); and/or
• obtaining a final concentration of H3Cit, such as by adding PBS-1 % BSA (such as obtaining a concentration of 10 000 ng/mL).
Preferably, an ELISA for detecting and/or measuring H3Cit comprises one or more of the following steps:
• adding anti-histone Biotin and incubating, such as adding 100 μΙ_ of anti-histone biotin (1 :10 in incubation buffer) to streptavidin pre-coated wells and incubating for 2 hour at room temperature; and/or
• washing; and/or • adding test samples or samples from healthily individuals and incubating, such as adding 50 μΙ_ of test samples or samples from healthly individuals to each well and incubating for 1 .5 hour; and/or
• washing; and/or
· adding the anti-histone H3Cit antibody, such as adding 100 μΙ_ of anti- histone H3 (citrulline R2 + R8 + R17; Anti-H3Cit) antibody (1 :2000 in 1 % BSA in PBS) to each well for a 1 hour incubation at room temperature; and/or
• washing; and/or
· incubating the wells with horseradish peroxidase (HRP) conjugate antibody, such as with 100 μΙ_ anti-rabbit HRP conjugate antibody (1 :5000 in 1 % BSA in PBS) for a 1 hour incubation at room temperature; and/or
• washing; and/or
· detecting the bound anti-rabbit HRP conjugate antibody, such as by adding 100 μΙ_ TMB to each well and incubating for 20 minutes in the dark at room temperature; and/or
• stopping the reaction, such as by adding 50 μΙ_ stop solution; and/or
• measuring the optical density, such as by measuring at a wavelength of 450 nm with a reference correction wavelength at 620 nm using an automatic plate reader.
Preferably, an ELISA method for detecting and/or measuring H3Cit comprises one or more of the following equipment: microplates with 96 streptavidin pre-coated wells (such as from Cell Death Detection ELISA PLUS kit, Roche, Cat. No. 1 1 774 425 001 ), and an ELISA reader (such as from Tecan Sunrise).
Preferably, an ELISA method for detecting and/or measuring H3Cit comprises one or more of the following antibodies: monoclonal anti-Histone- Biotin (such as from Cell Death Detection ELISA PLUS kit, Roche, Cat. No. 1 1 774 425 001 ); anti-Histone H3Cit antibody (such as a rabbit polyclonal anti-histone H3 (citrulline R2 + R8 + R17) antibody from Abeam, Cat. No. AB5103); and anti-lgG horseradish-peroxidase conjugate (such as goat anti- rabbit IgG horseradish-peroxidase conjugate from BioRad, Cat. No. 170- 6515).
Preferably, an ELISA method for detecting and/or measuring H3Cit comprises one or more of the following buffers and/or solutions: incubation buffer (such as from Cell Death Detection ELISA PLUS kit, Roche, Cat. No. 1 1 774 425 001 ); phosphate buffered saline (PBS; such as from Life Technologies, Cat. No. 14190-250); tween 20 (such as from Sigma-Aldrich, Cat. No. A9418); bovine serum albumin (BSA, such as from Sigma-Aldrich, Cat. No. A9418); 3,3', 5,5'-tetramethylbenzidine (TMB) liquid substrate (such as from Sigma-Aldrich, Cat. No. T0440); and a stop solution (such as from Thermo Scientific, Cat. No. N600).
Preferably, an ELISA method for detecting and/or measuring H3Cit comprises one or more of the following components: trizma base (such as from Sigma-Aldrich, Cat. No. T1503); CaC (such as from Sigma-Aldrich C1016); phenylmethylsulfonyl fluoride (PMSF) protease inhibitor (such as from Life Technologies, Cat. No. 36978); and dithiothreitol (DTT, such as from Invitrogen, Cat. No. P2325).
Preferably, an ELISA method for detecting H3Cit can comprise one or more of the following proteins: PAD4, such as human recombinant PAD4 (such as from Cayman Chemical, Cat. No. 10500); and Histone H3, such as human recombinant Histone H3 (Cayman Chemical, Cat. No. 10263).
It will be appreciate that room temperature will vary, and those skilled in biology and chemistry, in particular molecular biology and biochemistry, will understand the temperature ranges in which the aforementioned method steps can be undertaken.
An ELISA to detect G-CSF is available from R&D systems (catalogue number DCS50).
Preferably, the cancer is selected from a group consisting of: leukemia; carcinoma; AIDS-related cancers; lymphoma; anal cancer; appendix cancer; astrocytoma; bile duct cancer, extrahepatic cancer; bladder cancer; bone tumor, osteosarcoma fibrous histiocytoma; malignant fibrous histiocytoma; glioma; brain cancer; brain tumor; breast cancer; bronchial adenomas;
bronchial carcinoids; cervical cancer; childhood cancers; chronic
myeloproliferative disorders; colon cancer; desmoplastic small round cell tumor; endometrial cancer; ependymoma; epitheliod hemangioendothelioma (EHE); esophageal cancer; extrahepatic bile duct cancer; eye cancer;
gallbladder cancer; gastric (i.e. stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; head and neck cancer; heart cancer; hepatocellular (i.e. liver) cancer; primary hepatocellular (i.e. liver) cancer; hypopharyngeal cancer; kidney cancer; renal cell cancer; laryngeal cancer; lip and oral cavity cancer; Waldenstrom macroglobulinemia; male breast cancer; childhood medulloblastoma;
melanoma; merkel cell cancer; metastatic squamous neck cancer with occult primary; mouth cancer; childhood multiple endocrine neoplasia syndrome; multiple myeloma; plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myelodysplastic diseases; myeloproliferative diseases; chronic myeloproliferative disorders; myxoma; nasal cavity and paranasal sinus cancer; neuroblastoma; oligodendroglioma; oral cancer; oropharyngeal cancer; ovarian cancer; pancreatic cancer; islet cell pancreatic cancer;
paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal germinoma; pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; pituitary adenoma; pleuropulmonary blastoma; prostate cancer; rectal cancer; renal pelvis and ureter cancer; salivary gland cancer; Sezary syndrome; skin cancer; small intestine cancer; metastatic squamous neck cancer with occult primary; stomach cancer; childhood supratentorial primitive neuroectodermal tumor; fungoides and Sezary syndrome; testicular cancer; throat cancer;
childhood thymoma; thyroid cancer; childhood thyroid cancer; transitional cell cancer of the renal pelvis and ureter; gestational trophoblastic tumor; urethral cancer; endometrial uterine cancer; vaginal cancer; vulvar cancer; and childhood Wilms tumor. Preferably, the leukemia is selected from a group consisting of: acute lymphoblastic leukemia (ALL); acute myeloid leukemia; chronic lymphocytic leukemia; chronic myelogenous leukemia; hairy cell leukemia; acute lymphoblastic leukemia (also called acute lymphocytic leukaemia); acute myeloid leukemia (also called acute myelogenous leukemia); chronic lymphocytic leukemia (also called chronic lymphocytic leukemia); chronic myelogenous leukemia (also called chronic myeloid leukemia); hairy cell leukemia; adult acute myeloid leukemia; and childhood acute myeloid leukemia.
Preferably, the carcinoma is selected from a group consisting of:
adrenocortical carcinoma; basal-cell carcinoma; islet cell carcinoma
(endocrine pancreas); nasopharyngeal carcinoma; merkel cell skin
carcinoma; squamous cell carcinoma; and thymoma and thymic carcinoma.
Preferably, the astrocytoma is selected from a group consisting of: cerebellar astrocytoma; cerebral astrocytoma; childhood cerebellar
astrocytoma; childhood cerebral astrocytoma; and pineal astrocytoma.
Preferably, the lymphoma is selected from a group consisting of: AIDS- related lymphoma; Burkitt's lymphoma; primary central nervous system lymphoma; central nervous system lymphoma; cutaneous T-cell lymphoma; Hodgkin lymphoma; and non-Hodgkin lymphoma.
Preferably, the glioma is selected from a group consisting of:
brainstem glioma; cerebral astrocytoma glioma; malignant glioma; visual pathway glioma; hypothalamic glioma; and visual pathway and hypothalamic glioma.
Preferably, the brain tumor is selected from a group consisting of: cerebellar astrocytoma; ependymoma; cerebral astrocytoma glioma;
malignant glioma; childhood malignant glioma; medulloblastoma;
supratentorial primitive neuroectodermal tumor; visual pathway glioma;
hypothalamic glioma; visual pathway and hypothalamic glioma; childhood visual pathway glioma; childhood hypothalamic glioma; childhood visual pathway and hypothalamic glioma; glioma of the brain stem; and childhood cerebral astrocytoma glioma. Preferably, the carcinoid tumor is selected from a group consisting of: childhood carcinoid tumor; gastrointestinal carcinoid tumor; and gastric carcinoid.
Preferably, the sarcoma is selected from a group consisting of:
chondrosarcoma; Ewing's sarcoma in the Ewing family of tumors; kaposi sarcoma; liposarcoma; malignant fibrous histiocytoma of osteosarcoma; malignant fibrous histiocytoma of bonesarcoma; rhabdomyosarcoma;
childhood rhabdomyosarcoma; soft tissue sarcoma; and uterine sarcoma.
Preferably, the eye cancer is selected from a group consisting of:
intraocular melanoma; and retinoblastoma.
Preferably, the germ cell tumor is selected from a group consisting of: extracranial germ cell tumor; extragonadal germ cell tumor; ovarian germ cell tumor; extragonadal germ cell tumor; and childhood extracranial germ cell tumor.
Preferably, the lung cancer is selected from a group consisting of: non- small cell lung cancer; and small cell lung cancer.
Preferably, the mesothelioma is selected from a group consisting of: adult malignant mesothelioma; and childhood mesothelioma.
Preferably, the ovarian cancer is selected from a group consisting of: ovarian epithelial cancer; surface epithelial-stromal tumor; ovarian germ cell tumor; and ovarian low malignant potential tumor.
Preferably, the adenocarcinoma is selected from the group consisting of: prostate adenocarcinoma; lung adenocarcinoma; hepatocellular adenocarcinoma; pancreatic adenocarcinoma; prostate adenocarcinoma; breast adenocarcinoma; and urothelial adenocarcinoma.
Even more preferably, the cancer is selected from the group consisting of: lung cancer; pancreatic cancer; breast cancer; prostate cancer; urothelial cancer; liver cancer; colon cancer; colorectal cancer; gastrointestinal cancer; gynecological cancer; glioblastoma; lymphoma; acute myeloid leukemia; gingival cancer; liposarcoma; sarcoma; neuroendocrine cancer and melanoma (such as malignant melanoma). Even more preferably, the cancer is selected from a group consisting of: lung cancer; pancreatic cancer; breast cancer; prostate cancer; urotheal cancer; and liver cancer.
Most preferably, the cancer is selected from the group consisting of: colon cancer; breast cancer; gastrointestinal cancer; lung cancer; prostate cancer; and gynecological cancer.
Preferably, the thrombosis is an arterial thrombotic disorder or a venous thrombotic disorder.
Even more preferably, the thrombosis is an arterial thrombotic disorder.
Even more preferably, the thrombosis is a venous thrombotic disorder. Preferably, the arterial thrombotic disorder is a stroke or a myocardial infarction.
Preferably, the methods for screening are in vitro methods for screening.
Preferably, the chemotherapy is a chemotherapy drug selected from a group consisting of: 13-cis-Retinoic Acid, 2-CdA, 2-Chlorodeoxyadenosine, 5- Azacitidine, 5-Fluorouracil 5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6- Thioguanine, Abiraterone acetate, Abraxane, Accutane, Actinomycin-D, Adcetris, Ado-Trastuzumab,, Emtansine, Adriamycin, Adrucil, Afatinib, Afinitor, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alimta, Alitretinoin, Alkaban-AQ, Alkeran, All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron,
Anastrozole, Arabinosylcytosine, Ara-C, Aranesp, Aredia, Arimidex,
Aromasin, Arranon, Arsenic Trioxide, Arzerra, Asparaginase, Atra, Avastin, Axitinib, Azacitidine, Beg, Beleodaq, Belinostat, Bendamustine,
Bevacizumab, Bexarotene, Bexxar, Bicalutamide, Bicnu, Blenoxane,
Bleomycin, Blinatumomab, Blincyto, Bortezomib, Bosulif, Bosutinib,
Brentuximab Vedotin, Busulfan, Busulfex, C225, Cabazitaxel, Cabozantinib, Calcium Leucovorin, Campath, Camptosar, Camptothecin-1 1 , Capecitabine, Caprelsa, Carac, Carboplatin, Carfilzomib, Carmustine, Carmustine Wafer, Casodex, CCI-779, Ccnu, Cddp, Ceenu, Ceritinib, Cerubidine, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Clofarabine, Clolar, Cobimetinib, Cometriq, Cortisone, Cosmegen, Cotellic, Cpt-1 1 , Crizotinib, Cyclophosphamide, Cyramza, Cytadren, Cytarabine, Cytarabine Liposomal, Cytosar-U, Cytoxan, Dabrafenib, Dacarbazine, Dacogen, Dactinomycin, Daratumumab, Darbepoetin Alfa, Darzalex, Dasatinib, Daunomycin,
Daunorubicin, daunorubicin-hydrochloride, Daunorubicin, Liposomal,
DaunoXome, Decadron, Decitabine, Delta-Cortef, Deltasone, Denileukin Diftitox, Denosumab, DepoCyt, Dexamethasone, Dexamethasone Acetate, Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, Dhad, Die, Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin Liposomal, Droxia, DTIC, Dtic-Dome, Duralone, Eculizumab, Efudex, Ellence, Elotuzumab, Eloxatin, Elspar, Emcyt, Empliciti, Enzalutamide, Epirubicin, Epoetin Alfa, Erbitux, Eribulin, Erivedge, Erlotinib, Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos Etoposide, Etoposide Phosphate, Eulexin, Everolimus, Evista, Exemestane, Fareston, Farydak, Faslodex, Femara, Filgrastim, Floxuridine, Fludara, Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream),
Fluoxymesterone, Flutamide, Folinic Acid, Folotyn, Fudr, Fulvestrant, , Gazyva, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gilotrif, Gleevec, Gliadel Wafer, Goserelin, Halaven, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, Ibrance, Ibritumomab, Ibritumomab Tiuxetan, Ibrutinib, Idamycin, Idarubicin, Idelalisib, lclusig, Ifex, IFN-alpha, Ifosfamide, IL-1 1 , IL-2, Imbruvica, Imatinib Mesylate, Imidazole, Carboxamide, Inlyta, Interferon-Alfa, Interferon Alfa-2b (PEG Conjugate), lnterleukin-2, Interleukin- 1 1 , Intron A (interferon alfa-2b), Ipilimumab, Iressa, Irinotecan, Isotretinoin, Istodax, Ixabepilone, Ixazomib, Ixempra, Jakafi, Jevtana, Kadcyla, Keytruda, Kidrolase, Kyprolis, Lanreotide, Lapatinib, L-Asparaginase, Lbrance,
Lenalidomide, Lenvatinib, Lenvima, Letrozole, Leucovorin, Leukeran,
Leukine, Leuprolide, Leurocristine, Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, L-PAM, L-Sarcolysin, Lupron, Lupron Depot, Lynparza, Marqibo, Matulane, Maxidex, Mechlorethamine, Mechlorethamine, Medralone, Medrol, Megace, Megestrol, Megestrol Acetate, Mekinist, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate Sodium, Methylprednisolone, Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX, Mustargen, Mustine, Mutamycin, Myleran, Mylocel, Mylotarg, Navelbine, Necitumumab, Nelarabine, Neosar, Neulasta, Neumega, Neupogen,
Nexavar, Nilandron, Nilotinib, Nilutamide, Ninlaro, Nipent, Nivolumab,
Novladex, Novantrone, Nplate, Obinutuzumab, Octreotide, Octreotide
Acetate, Odomzo, Ofatumumab, Olaparib, Omacetaxine, Oncospar, Oncovin, Ontak, Onxal, Opdivo, Oprelvekin, Orapred, Orasone, Osimertinib, Otrexup, Oxaliplatin, Paclitaxel, Paclitaxel Protein-bound, Palbociclib, Pamidronate, Panitumumab,,, Panobinostat, Panretin, Paraplatin, Pazopanib, Pediapred, Peg Interferon, Pegaspargase, Pegfilgrastim, Peg-lntron, PEG-L- asparaginase, Pembrolizumab, Pemetrexed, Pentostatin, Perjeta,
Pertuzumab, Phenylalanine Mustard, Platinol, Platinol-AQ, Pomalidomide, Pomalyst, Ponatinib, Portrazza, Pralatrexate, Prelone, Procarbazine, Procrit, Proleukin, Prolia, Prolifeprospan 20 with Carmustine Implant, Provenge, Purinethol, Radium 223 Dichloride, Raloxifene, Ramucirumab, Rasuvo, Regorafenib, Revlimid, Rheumatrex, Rituxan, Rituximab, Roferon-A
(Interferon Alfa-2a), Romidepsin, Romiplostim, Rubex, Rubidomycin
Hydrochloride, Ruxolitinib, Sandostatin, Sandostatin, LAR, Sargramostim, Siltuximab, Sipuleucel-T, Soliris, Somatuline, Sonidegib, Sorafenib, Sprycel, Sti-571 , Stivarga, Streptozocin, SU1 1248, Sunitinib, Sutent, Sylvant,
Synribo,, Tafinlar, Tagrisso, Tamoxifen, Tarceva, Targretin, Tasigna, Taxol, Taxotere, Temodar, Temozolomide, Temsirolimus, Teniposide, Tespa, Thalidomide, Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide, Thioplex, Thiotepa, Tice, Toposar, Topotecan,
Toremifene, Torisel, Tositumomab, Trabectedin, Trametinib, Trastuzumab, Treanda, Trelstar, Tretinoin, Trexall, Triptorelin pamoate, Trisenox, Tspa, Tykerb, Valrubicin, Valstar, Vandetanib, VCR, Vectibix, Velban, Velcade, Vemurafenib, Venclexta, Venetoclax, VePesid, Vesanoid, Viadur, Vidaza, Vinblastine, Vinblastine Sulfate, Vincasar Pfs, Vincristine, Vincristine
Liposomal, Vinorelbine, Vinorelbine, Tartrate, Vismodegib, Vlb, VM-26, Vorinostat, Votrient, VP-16, Vumon, Xalkori Capsules, Xeloda, Xgeva, Xofigo, Xtandi, Yervoy, Yondelis, Zaltrap, Zanosar, Zelboraf, Zevalin, Zinecard, Ziv- aflibercept, Zoladex, Zoledronic Acid, Zolinza, Zometa, Zydelig, Zykadia, and Zytiga. It will be appreciated that the aforementioned chemotherapy drugs can be administered individually or in combination. When administered in
combination (a so-called chemotherapy regime), the chemotherapy drugs can be administered at the same time or at different times. The administration of chemotherapy and chemotherapy regimens will be well known to those skilled in medicine, in particular an oncologist.
Preferably, the individual has further been diagnosed as having had a stroke (such as an ischemic stroke) or suspected as having had a stroke (such as an ischemic stroke).
In an embodiment of the third and sixth aspects of the invention, the method for screening is undertaken one or more day following chemotherapy - for example, two or more days following chemotherapy; three or more days following chemotherapy; four or more days following chemotherapy; five or more days following chemotherapy; six or more days following
chemotherapy; one or more week following chemotherapy; two or more weeks following chemotherapy; three or more weeks following chemotherapy; one or more month following chemotherapy; two or more months following chemotherapy; three or more months following chemotherapy; four or more months following chemotherapy; five or more months following
chemotherapy; six or more months following chemotherapy; or one or more years following chemotherapy.
Even more preferably, the method for screening is undertaken one day following chemotherapy.
Preferably, the individual is a human individual.
Preferably, the individual is a non-human individual, such as a rodent
(for example a mouse, rat or hamster), a rabbit, a cat, a dog, a sheep, a pig, a goat, a horse, or cattle.
Preferably, the individual is an elderly individual, such as an elderly human individual. By "elderly human individual", we include an individual that is 50 or more years old - for example, 55 or more years old, 60 or more years old, 65 or more years old, 70 or more years old, 75 or more years old, 80 or more years old, 85 or more years old, or 90 or more years old. Preferably, the method further comprises step:
c) selecting a treatment for the individual.
Preferably, selecting the treatment in step c) is based on presence and/or quantity in the test sample of said biomarkers.
Preferably, the method further comprises step:
d) administering the selected treatment to the individual.
Preferably, the treatment is selected from the group consisting of: cancer treatment; thrombosis treatment; tumour treatment; and treatment for the adverse effects G-CSF.
Even more preferably, the treatment is cancer treatment.
Even more preferably, the treatment is thrombosis treatment.
Even more preferably, the treatment is tumour treatment.
Even more preferably, the treatment is for the adverse effects G-CSF. It will be appreciated that cancer treatment is often the same as tumour treatment.
Those skilled in the art of medicine, in particular an oncologist or pharmacist, will be aware of treatments suitable for cancer, thrombosis, tumours and the adverse effects of G-CSF.
Preferably, the cancer treatment or tumour treatment is one or more selected from the group consisting of: surgery; chemotherapy; and
radiotherapy.
Preferably, the treatment for thrombosis is selected from a list consisting of: heparin (such as unfractionated heparin); low molecular weight heparins (such as enoxaparin, dalteparin, tinzaparin, nadroparin, reviparin, ardeparin, and certoparin); pentasaccharide (such as fondaparinux, which is a synthetic pentasaccharide); antivitamin K agents (such as warfarin, dicoumarol, phenprocoumon, and acenocoumarol); direct acting oral coagulants (such as new oral anticoagulants (NOACs), dabigatran, rivaroxaban, apixaban, betrixaban, and edoxaban); antiplatelet agents (such as acetylsalicylic acid (aspirin)); clopidogrel; prasugrel; ticagrelor; and dipyridamole. Preferably, the treatment for a venous thrombotic disorder is selected from a list consisting of: heparin (such as unfractionated heparin); low molecular weight heparins (such as enoxaparin, dalteparin, tinzaparin, nadroparin, reviparin, ardeparin, and certoparin); pentasaccharide (such as fondaparinux, which is a synthetic pentasaccharide); antivitamin K agents (such as warfarin, dicoumarol, phenprocoumon, and acenocoumarol); and direct acting oral coagulants (such as new oral anticoagulants (NOACs), dabigatran, rivaroxaban, apixaban, betrixaban, and edoxaban).
Preferably, the treatment for an arterial thrombotic disorder is selected from a list consisting of: heparin (such as unfractionated heparin); low molecular weight heparins (such as, enoxaparin, dalteparin, tinzaparin, nadroparin, reviparin, ardeparin, and certoparin); pentasaccharide (such as fondaparinux, which is a synthetic pentasaccharide); antivitamin K agents (such as, warfarin, dicoumarol, phenprocoumon, and acenocoumarol); direct acting oral coagulants (such as new oral anticoagulants (NOACs),
dabigatran, rivaroxaban, apixaban, betrixaban, and edoxaban); antiplatelet agents (such as, acetylsalicylic acid (aspirin)); clopidogrel; prasugrel;
ticagrelor; and dipyridamole.
Preferably, the treatment for the adverse effects G-CSF is to stop G- CSF treatment or to decrease G-CSF treatment.
As described above, the present invention is also related to a diagnostic kit for use in any of the above described methods for diagnosing one or more of the described condition(s). In an embodiment of the invention, said diagnostic kit comprises at least one binding agent which could be chosen from a binding agent such as an anti-H3Cit-antibody or a fragment thereof capable of binding to the H3Cit. In an embodiment of the invention, the diagnosis of the one or more conditions is performed in a tailor made ELISA model, as described below, to measure the presence and quantity of said H3Cit.
Preferably, the kit further comprises an additional binding agent, such as an anti-G-CSF-antibody or a fragment thereof capable of binding to the G- CSF. Preferably, the kit further comprises an additional binding agent capable of binding to the anti-H3Cit-antibody or a fragment thereof and/or an anti-G-CSF-antibody or a fragment thereof (for example, an anti-lgG antibody).
Preferably, the additional binding agent comprises a detectable moiety, such as a fluorescent label, a fluorescein-type label, a rhodamine-type label, VIVOTAG 680 XL FLUOROCHROME™ (Perkin Elmer), phycoerythrin;
umbelliferone, Lissamine; a cyanine; a phycoerythrin, Texas Red, BODIPY FL-SE® (Invitrogen), a chemoluminescent label, an enzyme, a prosthetic group complex, a paramagnetic label, or a radioactive label.
Preferably, the fluorescent label is a rare earth chelate, such as europium chelate.
Preferably, the fluorescein-type label is selected from the group consisting of: fluorescein; fluorescein isothiocyanate; 5-carboxyfluorescein; 6- carboxy fluorescein; and dichlorotriazinylamine fluorescein.
Preferably, the rhodamine-type label is selected from the group consisting of: ALEXA FLUOR® 568 (Invitrogen); TAMRA®; or dansyl chloride.
Preferably, the chemoluminescent label is selected from the group consisting of: luminol; luciferase; luciferin; and aequorin.
Preferably, the enzyme is selected from the group consisting of:
horseradish peroxidase; alkaline phosphatase; beta-galactosidase; and or acetylcholinesterase.
Preferably, the prosthetic group complex is selected from the group consisting of: streptavidin/biotin and avidin/biotin.
Preferably, the paramagnetic labels is selected from the list consisting of: paramagnetic ions of Aluminum (Al), Barium (Ba), Calcium (Ca), Cerium (Ce), Dysprosium (Dy), Erbium (Er), Europium (Eu), Gandolinium (Gd), Holmium (Ho), Iridium (Ir), Lithium (Li), Magnesi-um (Mg), Manganese (Mn), Molybdenum (M), Neodymium (Nd), Osmium (Os), Oxygen (O), Palladium (Pd), Platinum (Pt), Rhodium (Rh), Ruthenium (Ru), Samarium (Sm), Sodium (Na), Strontium (Sr), Terbium (Tb), Thulium (Tm), Tin (Sn), Titanium (Ti), Tungsten (W), or Zirconium (Zi), and particularly, Co+2, CR+2, Cr+3, Cu+2, Fe+2, Fe+3, Ga+3, Mn+3, Ni+2, Ti+3, V+3, and V+4, positron emitting metals using various positron emission tomogra-phies, and non-radioactive paramagnetic metal ions.
Preferably, the radioactive label is selected from the list consisting of: bismuth (213Bi); carbon (1 1 C, 13C, 14C); chromium (51 Cr); cobalt (57Co, 60Co); copper (64Cu); dysprosium (165Dy); erbium (169Er); fluorine (18F); gadolinium (153Gd, 159Gd); galli-um (68Ga, 67Ga); germanium (68Ge); gold (198Au); holmium (166Ho); hydrogen (3H); indium (1 1 1 In, 1 12ln, 1 13ln, 1 15ln); iodine (121 1, 123I, 125I, 131 1); iridium (1 92lr); iron (59Fe); krypton (81 mKr); lanthanium (140La); lutelium (177Lu); manganese (54Mn);
molybdenum (99Mo); nitrogen (13N, 15N); oxygen (150); palladium (103Pd); phosphorus (32P); potassium (42K); praseodymium (142Pr); promethium (149Pm); rhenium (186Re, 188Re); rhodium (105Rh); rubidium (81 Rb, 82Rb); ruthenium (82Ru, 97Ru); samarium (153Sm); scandium (47Sc);
selenium (75Se); sodium (24Na); strontium (85Sr, 89Sr, 92Sr); sulfur (35S); technetium (99Tc); thallium (201 Tl); tin (1 13Sn, 1 17Sn); xenon (133Xe); ytterbium (169Yb, 175Yb, 177Yb); yttrium (90Y); and zinc (65Zn).
Methods of detecting and using such detectable moieties will be known to those skilled in biology and chemistry, in particular a molecular biologist or a computational biologist.
Non-limiting examples of the invention are described to exemplify the invention below.
Experimental part
Example 1
In an ongoing study of stroke patients, it was sought to quantify H3Cit in plasma of patients with cerebral infarction and an underlying active cancer compared to patients with cerebral infarction without an underlying active cancer. H3Cit has previously not been quantified in plasma and there is no known or commercial method for doing so. It was therefore developed a tailor made method for the quantification of H3Cit in plasma using the following protocol:
H3Cit Roche ELISA
This H3Cit ELISA allows the specific detection of histone H3 citrullinated at arginines position 2, 8 and 17, which has been shown to be necessary for decondensation of the chromatin. The method uses the microplate modules and the coating anti-histone and incubation buffer of Cell death detection ELISA kit, Roche, cat nr 1 1774425001 . · Add 100 ul of coating solution (anti-histone 1 :10) to the pre-coated wells and incubate overnight at 4 degrees
• Remove the coating by tapping dry the wells
• Add 200 ul incubation buffer and incubate 1 h at room temp
• Remove solution, Wash 3X with PBS-tween 0.05%
· Add 40 ul incubation buffer + 10 ul plasma incubate 1 h30 room temp
• Remove solution, Wash 3X with PBS-tween 0.05%
• Add 100 ul of anti-H3Cit antibody in PBS-BSA 1 % incubate 1 h30 room temp
• Remove solution, Wash 3X with PBS-tween 0.05%
· Add 100 ul of anti-rabbit HRP in PBS-BSA 1 % incubate 1 h room temp
• Remove solution, Wash 3X with PBS-tween 0.05%
• Add 100 ul TMB and let develop the reaction
• Stop the reaction with 50 ul 2% sulfuric acid
• Read optical density at 450 nm with a reference correction wavelength at 570 nm
The results are expressed as the read optical density and are compared to the absorbance of plasma samples from healthy donors. A standard curve, using various concentration of in vitro citrullinated histone H3 diluted in pool plasma from healthy donors, is being developed to quantify the exact level of H3Cit in plasma samples. Results
The tailor made H3Cit Elisa was used to quantify the H3Cit levels in plasma of the patients with cerebral infarction and an underlying active cancer (n=8) compared to the patients with cerebral infarction without an underlying active cancer (n=23). It was found a three-fold increase in circulating H3Cit in plasma of patients with cerebral infarction and an underlying active cancer compared to patients with cerebral infarction without underlying cancer (Fig
1 )-
Surprisingly, the plasma levels of H3Cit in the patients without cancer were diminishingly small and it is believed that these patients had no H3Cit in plasma (ie background signal) as their levels were similar to 10 healthy controls (aged 62-93), and all of these had diminishingly low optical density suggesting no detectable H3Cit in plasma. This is highly unexpected as NETs have been described to underlie thrombosis and one would expect to identify H3Cit in plasma from these patients. Western Blot detection of H3Cit in the same patients revealed similar results, with positive signal of plasma H3Cit in the patients with underlying cancer (ie presence of H3Cit), and no signal (ie absence of H3Cit) in the patients without underlying cancer (Fig 2).
Interestingly, four out of eight patients with ischemic stroke and an underlying cancer did not have cancer diagnosed on stroke onset and inclusion in the study. In fact, these patients were diagnosed with cancer post mortem.
A systemic induction of NETosis
Despite emerging research showing H3Cit (ie NETs) in thrombi and infarcted areas of myocardial and cerebral infarctions, the patients with large cerebral infarctions but no cancer had diminishing or no H3Cit in plasma. The patients with ischemic stroke and underlying active cancer had detectable and quantifiable levels of H3Cit in plasma.
The discrepancies between plasma H3Cit levels between patients with thrombosis only and patients with thrombosis and cancer leads us to believe that the NETs previously shown in thrombi and infarcted areas in patients without cancer may be the result of a different mechanism of NET induction than in thrombosis due to cancer. The strong inflammatory response, hypoxic milieu and oxidative stress surrounding an infarcted tissue could activate and prime neutrophils towards NETosis, inducing a local NET burden at the site of the infarction. Once released, NETs could subsequently contribute to thrombus growth and stabilization. In this setting, H3Cit may be found locally in thrombi and infarcted areas, but not in plasma. In cancer patients, cytokines (such as G-CSF) may be priming circulating neutrophils to
NETosis, resulting in circulating H3Cit in plasma, as shown in the patients. The circulating NETs may subsequently contribute to the prothrombotic state in cancer patients. Thereby, NETs in thrombus formation and infarction in patients without cancer may be regarded as locally induced NET burden, whereas NETs in cancer patients may be a systemically induced NET burden. H3Cit may therefore be detectable in plasma of cancer patients only. As the levels of NETs in plasma of cancer patients increase (it is
hypothesized that NET burden will increase with cancer progression), NETs may contribute to cancer-associated thrombosis.
Notably, the primary tumor sites in the patient group with ischemic stroke and cancer covered lung, pancreas, breast, prostate, urotheal and liver tumors. Comorbidity, age and infarct volume did not differ between the group with ischemic stroke without cancer and the group with ischemic stroke and cancer. In fact, all patients were old and had a high burden of comorbidity such as diabetes mellitus, renal insufficiency, hypertension, hearth failure and acute coronary artery disease.
Furthermore, in line with previous studies by Demers et al (Demers M, et al. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci USA
2012;109:13076-81 ), in which different types of murine cancers systemically released G-CSF priming neutrophils toward NETosis, the patients with cancer in this study had a seven-fold increase in plasma G-CSF compared to the patients without cancer (Fig 3). This increase correlated positively with high levels of H3Cit, supporting a cancer-released G-CSF-induced NET burden (Fig 4). These surprising data suggest a new model for NETs; infarction in patients without cancer have local NET burden, and no circulating H3Cit in plasma, whereas cancer patients have systemic high levels of H3Cit in plasma, which potentially could also relate to a future risk of infarction.
H3Cit in plasma as a biomarker for cancer
The cancer patients had detectable and quantifiable levels of H3Cit in plasma. To our surprise, the patients with ischemic stroke only (despite large infarctions, high age and a large burden of comorbidity), had negligible levels of H3Cit in plasma. Previous research discusses the possibility of NETs in screening for a variety of diseases. Our findings emphasize the fact that circulating H3Cit is not a general marker for high burden of comorbidity or age.
While NETs are probably crucial players in many disease settings, our results suggest that the NET burden in cancer is systemic, whereas the NET burden in many other conditions, such as thrombosis, is local. It is therefore believed that H3Cit in plasma could be used as a biomarker for cancer. Up to this date, the potential of circulating H3Cit as a biomarker for cancer has not been suggested. There are a number of clinically implemented biomarkers used for a variety of tumors, such as PSA (prostate cancer), CEA (colorectal cancer) and CA-125 (ovarian cancer). These biomarkers are hampered by low specificity and low sensitivity. On the contrary, H3Cit may have the potential to contribute in screening for cancer in general. H3Cit may also allow for a higher specificity and sensitivity than existing biomarkers. Cancer is one of the leading causes of death in the world. The poor prognosis is often due to late diagnosis. A biomarker, which could aid in diagnosing a wide variety of malignancies would allow for an earlier detection and thus a better prognosis for cancer patients in general. H3Cit in plasma may also be useful in the clinical follow up of tumor progression and screening for recurrence in patients with previously diagnosed cancer.
The present tailor made H3Cit ELISA is the first method described able to detect and quantify H3Cit in plasma Quantifying the levels of H3Cit in plasma could furthermore predict the susceptibility to cancer-associated thrombosis in cancer patients as well as cancer in patients with idiopathic thrombosis. A systemically induced NETosis may, however, have to reach a certain level to contribute to the prothrombotic state in cancer. A method to quantify H3Cit in plasma may therefore be crucial in the screening for cancer patients at highest risk for thrombosis. Low-molecular-weight heparin is currently the preferred treatment and prophylaxis of cancer-associated thrombosis. Anticoagulant therapy is, however, associated with a substantial risk of bleeding, especially in patients with active cancer, and the benefit-risk ratio is therefore too unfavourable to consider routine thrombo-prophylaxis. Quantification of H3Cit in plasma could reveal patients with the highest risk of thrombosis and allow for individualized thrombo-prophylaxis strategies.
Chemotherapy is known to increase the risk for thrombosis. Oxidative stress, such as in chemotherapy, induces NETosis. As NETs are highly procoagulant, it is conceivable to believe that quantifying plasma H3Cit before and after chemotherapy could aid in screening for patients at highest risk for chemotherapy-associated thrombosis.
Furthermore, Filgrastim, a human G-CSF manufactured by
recombinant DNA technology, is often administered after chemotherapy in order to reduce chemotherapy associated neutropenia and the incidence of febrile neutropenia. As tumor derived G-CSF is known to induce NETs, Filgrastim could hypothetically also induce NETs, which could be a
contributing factor in the development of adverse effects such as acute respiratory distress syndrome, a condition previoulsy linked to NETosis.
H3Cit may therefore also be useful in screening patients receiving Filgrastim.
Although there are probably heterogenous mechanisms behind a cancer-induced systemic NET burden, cancer-released G-CSF may play a central role. A combination of G-CSF and H3Cit quantification in plasma could thus further aid in the above diagnostic settings. Example 2
This Example describes a further characterisation of the assay discussed in Example 1 . This comprised the implementation of the standard curve generated from in vitro citrullinated H3Cit, mentioned in Example 1 , allowing for a more precise quantification of plasma H3Cit as well as a better reproducibility. Serial experiments to obtain the optimal concentrations of the standard curve, incubation times and dilutions of samples were performed, after which a methodological validation of the assay was performed.
Assay procedure
Reagents and equipment: Microplates with 96 streptavidin pre-coated wells, Monoclonal Anti-Histone-Biotin antibodies, and incubation buffer (all from Cell Death Detection ELISA PLUS kit, Roche, Cat. No. 1 1 774 425 001 ). Phosphate buffered saline (PBS; Life Technologies, Cat. No. 14190-250), tween 20 (Sigma-Aldrich, Cat. No. A9418), Rabbit Polyclonal Anti-Histone H3 (citrulline R2 + R8 + R17) antibody (Abeam, Cat. No. AB5103), Bovine serum albumin, BSA (Sigma-Aldrich, Cat. No. A9418), Goat Anti-Rabbit IgG Horseradish-peroxidase conjugate (BioRad, Cat. No. 170-6515), 3,3', 5,5'- tetramethylbenzidine (TMB) liquid substrate (Sigma-Aldrich, Cat. No. T0440), stop solution (Thermo Scientific, Cat. No. N600), Trizma Base (Sigma- Aldrich, Cat. No. T1503), CaCI2 (Sigma-Aldrich C1016), Phenylmethylsulfonyl fluoride (PMSF) protease inhibitor (Life Technologies, Cat. No. 36978), Dithiothreitol, DTT (Invitrogen, Cat. No. P2325), Human Recombinant PAD4 (Cayman Chemical, Cat. No. 10500), Human Recombinant Histone H3 (Cayman Chemical, Cat. No. 10263), ELISA reader (Tecan Sunrise).
A working stock solution of H3Cit was made as described previously (Lee P, et al. Mol Cell Biol 2008;28:4745-58). Briefly, human recombinant histone H3 and human recombinant PAD4 were incubated at 37 °C for 1 h in reaction buffer (50 imM Trizma base with 4 imM CaCI2, pH 7.6, 4 imM DTT and 1 imM PMSF). A final concentration of 10 000 ng/mL H3Cit was obtained by adding PBS-1 % BSA. The stock solution was aliquoted, frozen on dry ice, and stored at -80 °C until later use.
The microplate and diluents were kept at room temperature 30 min prior to starting the assay. Stock solution, antibodies, and samples were thawed on ice and kept on ice until loading of microplate. All incubations were at room temperature and washes were repeated 4 times with PBS-Tween (0.05%) with 20 sec soaking for each wash. The concentrations of the standard curve, incubation times and dilutions of samples were optimized in preliminary experiments.
The assay was performed as follows: 100 μΙ_ of anti-histone Biotin (1 :10 in incubation buffer) was added to Streptavidin pre-coated wells and incubated for 2 h. After washing, 50 μΙ_ of standard solutions or samples were added to each well and incubated for 1 .5 h, then washed again. 100 μΙ_ of anti-histone H3 (citrulline R2 + R8 + R17; Anti-H3Cit) antibody (1 :2000 in 1 % BSA in PBS) was applied to each well for 1 h incubation. After washing, the wells were incubated for another hour with 100 μΙ_ anti-rabbit HRP conjugate antibody (1 :5000 in 1 % BSA in PBS), followed by washing. For detection, 100 μΙ_ TMB was added to each well and incubated for 20 min in the dark. The reaction was stopped by adding 50 μΙ_ stop solution. The optical density (O.D.) was measured at a wavelength of 450 nm with a reference correction wavelength at 620 nm using an automatic plate reader. For validation of the assay, we assessed the following: linearity, stability, limit of detection, specificity, and precision. Trueness could not be determined as no reference analyte of known concentration is available, and there is no available assay for the "true" quantification of H3Cit in plasma for
comparison. The linear interval was defined as the linear section of the best- fit standard curve. Each standard curve was fitted using a four-parameter logistic (4PL) regression, and the 95% confidence interval (95 % CI) was considered. The limit of detection was approximated from the intersection of the lower asymptote of the upper 95% CI with the 4PL fit of the standards data. Specificity was assessed by the ability to detect citrullinated histone H3 but not non-citrullinated histone H3 in similar conditions by preparing a standard without PAD4, thus preventing the citrullination of histone H3.
Precision was expressed by the intra- and inter-assay coefficient of variation (%CV, defined as the ratio between standard deviation and mean value). The maximum accepted %CV for intra- and inter-assay variability were set to 15%. Stability was assessed by comparing the detector response obtained from freshly prepared and frozen aliquots of H3Cit standard, and comparing standard curves from frozen aliquots from three different batches of H3Cit that had been citrullinated on three different days. One vs. two freeze-thaw cycles of plasma samples were also compared.
Due to the role of NETs in infection, and data on the detection of H3Cit by western blotting in murine models of LPS-induced septic shock (Li Y et al. Surgery 201 1 ;150:442-51 ), we chose to conduct the assay validation on samples obtained from a human model of LPS-induced inflammation.
Samples were taken from healthy individuals prior to and 3-4 h after receiving intravenous injection of lipopolysaccharide (LPS; 2 ng per kg of body weight Escherichia coli endotoxin, Lot H0K354 CAT number 1235503, United States Pharmacopeia, Rockville, MD, USA) or from healthy volunteers. Plasma samples were prepared from citrated whole blood following immediate centrifugation for 20 min at 2000 χ g after which they were stored at -80 °C until further analysis. At time of analysis, samples were thawed on ice and diluted 1 :2 in PBS unless otherwise indicated. All study individuals gave written informed consent for the use of their plasma, and the study complied with the Declaration of Helsinki. The study was approved by the ethics review board in Stockholm, Sweden (registration numbers 2014/1946-31 /1 and 2015/1533-31 /1 ). Results
Standard preparation and linearity. As no international standard preparation is available for H3Cit, we generated a standard curve using in vitro PAD4- citrullinated H3Cit, as described above. The stock was serially diluted 1 :2 in PBS-1 % BSA to obtain a standard curve and applied to a streptavidin-coated plate using an anti-histone biotin antibody as capture and an anti-H3Cit antibody for detection. To determine the suitable linear interval we interpolated the detected O.D. from the serial dilutions of H3Cit to different regressions. The best-fit curve was a sigmoidal 4PL curve rendering a linear interval of the curve between ~ 0.5 and 3.5 O.D., corresponding to concentrations between ~ 5 and ~ 300 ng/mL (Fig. 5).
Stability. The detector response when preparing standards from freshly citrullinated H3Cit was very similar to the detector response obtained from frozen aliquots of the same standards (Fig. 6). Moreover, the detector response when preparing standard curves from frozen aliquots from three different batches of H3Cit citrullinated on three different days were not significantly different (Fig. 5), allowing for a good reproducibility. Limit of detection. To determine the limit of detection, we approximated the lowest detectable concentration determined from the curve to ~ 5 ng/mL. This concentration corresponded to the intersection of the lower asymptote of the upper 95% CI with the 4PL fit of the standard curve. The limit of detection with stated probability was therefore set to approximately 5 ng/mL.
Specificity. To assess the specificity of the assay, we prepared a standard curve with histone H3 incubated under the same conditions as our standard preparation of H3Cit, but without PAD4, rendering non-citrullinated histones, and compared this to our standard curve with in vitro PAD4-citrullinated H3Cit. Although there was a low amount of antibody antigen detection when large amounts of non-citrullinated histone H3 were present, the antibody antigen detection was specific for citrullinated H3Cit in the linear interval of the assay (Fig. 7).
Concentrations of H3Cit in plasma in a human model of LPS-induced inflammation. Surrogate markers of NETs (cfDNA, nucleosomes and MPO- DNA complexes) have been identified in the plasma of septic patients and in murine models of lipopolysaccharide (LPS)-induced septic shock (Czaikoski PG et al. PLoS One 2016;doi: 10.1371 /journal. pone.0148142). Furthermore, H3Cit was detected by western blot in plasma of mice shortly after LPS injection (Li Y et al. Surgery 201 1 ;150:442-51 ). With the intention to perform the assay validation with samples containing H3Cit, and because NETs in septic models are established in the research field of NETs, we used previously collected and frozen plasma samples from healthy volunteers receiving intravenous LPS in an experimental model of inflammation. The samples were taken at baseline (before LPS injection) and after 3-4 h, with the hypothesis that LPS injection would induce a systemic NET formation resulting in elevated and detectable levels of H3Cit in plasma. Indeed, the levels of H3Cit in all samples taken at baseline were under the detection limit of approximately 5 ng/mL, and the levels of H3Cit in all samples taken from the same individuals 3-4 hours after LPS injection ranged from 28.7 ng/mL to 93.2 ng/mL (Fig. 8). These concentrations were all within the linear interval of the standard curve (Fig. 5). However, repeated freeze-thaw cycles of plasma samples with known concentrations of H3Cit rendered a mean reduction of 13.4 % ±2.3 % after a second freeze-thaw cycle. Freeze-thaw cycles of the plasma are therefore not recommended when applying this assay.
Precision and reproducibility. To assess the precision of the assay, we performed the assay on six replicates of eight samples (1 -8) within the same assay-run as well as duplicates of the same eight samples in four different assay runs performed on four different days. The CV were all <15 %, with the intra-assay ranging from 2.13-5.15 % and the inter-assay ranging from 5.80- 12.55 %, showing a high precision with good repeatability and reproducibility of the assay (Table 1 ).
Table 1 . Precision; intra-assay repeatability and four different days inter- assay reproducibility
Figure imgf000058_0001
This example establishes an assay allowing for the fast and reliable quantification of the NET specific biomarker H3Cit in human plasma. The validation of the assay revealed a high specificity for H3Cit as well as a high stability of the custom-made standard, rendering a good precision.
We show clear intra-individual elevations of H3Cit in healthy individuals receiving LPS injection. Surrogate markers of NETs have been implicated in septic conditions. This invention is related to H3Cit in cancer, but this
Example provides a proof-of-principle for the H3Cit assay. From this data, we consider that several measurements for plasma H3Cit over time, for example two measurements with one or a few weeks intervals, may identify those with stable elevations (ie induced by an active cancer) in contrast to those with temporary elevations (ie induced by a severe inflammatory reaction due to for example sepsis). Patients with sepsis will also present with symptoms of sepsis, alerting the clinician of a possible source of H3Cit other than cancer. All samples taken at baseline in the LPS-induced model of inflammation were below the limit of detection, suggesting that healthy individuals do not have a baseline systemic NET burden. Taken together, the optimization and validation of the tailor-made H3Cit ELISA-based assay presents a robust, precise and specific assay for the quantification of plasma H3Cit, which may be implicated, alone or in combination with existing screening protocols, in identifying cancer and cancer-associated conditions such as thrombosis.
Example 3
To further validate H3Cit as a biomarker in screening for cancer, a study was conducted with a larger cohort of cancer patients with the intention to cover a normal biological variation as well as a broader variation in primary malignant tumor types. In a prospective case-control design, patients with active cancer admitted to Stockholms Sjukhem were recruited as case patients. To explore the specificity of H3Cit in cancer, 51 age- and gender matched hospitalized patients with other diseases but without known cancer, admitted to
Danderyds Hospital were included as control group. These diagnoses were determined by previous diagnoses in medical journals (according to the ICD system) and/or indication for the current hospitalization. The hospitalized patients without known cancer had multiple comorbidities, but the indications for the current hospitalizations were acute infection (n=7), severe venous thrombotic events (n=4), acute myocardial infarction (n=1 ), acute ischemic stroke (n=14), acute liver failure (n=5), acute renal failure (n=1 ), acute pulmonary disease (n=2), severe hyperglycemia (n=2), severe inflammatory bowel disease (n=1 ), confusion of unknown cause (n=6), severe anemia (n=3), bone fracture with complications (n=2), severe hyponatremia (n=1 ), epileptic seizure (n=1 ) and alcohole intoxication (n=1 ). Age- and gender- matched healthy individuals were recruited (through advertisements) as reference. Active cancer was defined as diagnosis of, or treatment for, cancer within the prior six months, or known recurrent or metastatic disease.
All patients and healthy individuals were included in the study between September 2015 and March 2016. Written informed consent was obtained from each study participant, and the study complied with the Declaration of Helsinki. The study was approved by the ethics review board in Stockholm, Sweden (registration number 2015/1533-31 /3). In the cancer group, there were 6 different types of primary malignant tumors (both epithelial, other solid malignant tumors and hematological cancers), including the most common cancer types (Table 2): colon (n=1 1 ), breast (n=10), gastrointestinal (n=8), lung (n=6), prostate (n=5),
gynecological (n=4), malignant melanoma (n=3), glioblastoma (n=3), pancreas (n=2), lymphoma (n=1 ), acute myeloid leukemia (n=1 ), gingival (n=1 ), liposarcoma (n=1 ), sarcoma (n=1 ), neuroendocrine (n=1 ). One patient had primary tumors of both lung and prostate, and one patient had primary tumor of unknown origin. Table 2
Figure imgf000060_0001
SSH27 70 M Prostate adenocarcinoma
SSH28 78 F Sarcoma Soft tissue sarcoma
SSH29 53 M Lung adenocarcinoma
SSH30 91 F Breast adenocarcinoma
SSH31 54 F Breast adenocarcinoma
SSH32 73 M Colorectal adenocarcinoma rectum
SSH33 74 M Neuroendocrine Multifokal NET ileum
SSH34 67 F Breast adenocarcinoma
SSH35 67 M Glioblastoma Glioblastoma WHO grade IV
SSH36 71 M Prostate adenocarcinoma
SSH37 66 M Liposarcoma liposarcoma grade II
SSH38 70 M Colon adenocarcinoma
SSH39 69 M AML AML
SSH40 61 M Prostate adenocarcinoma
SSH42 78 M Prostate adenocarcinoma
SSH43 69 M Colon adenocarcinoma
SSH44 55 F Lung squamous cell
SSH45 83 F Colon adenocarcinoma
SSH46 34 F Melanoma malignant melanoma
SSH47 89 F Lymphoma B-cell non-Hodgkin lymphoma
SSH48 68 F Gastrointestinal adenocarcinoma peritoneum
SSH49 85 F Gastrointestinal adenocarcinoma gall bladder
SSH50 79 F Gastrointestinal adenocarcinoma peritoneum
SSH51 63 F Melanoma malignant melanoma
SSH52 69 F Gynecological adenocarcinoma corpus uteri
SSH53 50 F Breast adenocarcinoma
SSH54 69 F Melanoma malignant melanoma
SSH55 59 F Breast adenocarcinoma
SSH56 70 M Glioblastoma Glioblastoma WHO grade IV
SSH57 65 M Glioblastoma Glioblastoma WHO grade IV
SSH58 37 F Gingival squamous cell
adenocarcinoma unknown
SSH59 50 M Gastrointestinal origin Gl
adenocarcinoma (both
SSH60 68 M Lung/Prostate primaries)
SSH61 73 M Colorectal adenocarcinoma rectum
Patient sample SSH41 is omitted from the above table, as the patient was inadvertently included in the study twice.
There were no significant differences in age or sex distribution between the patients with active cancer (median age 70.5, 41 .7% men), hospitalized patients with other diseases but no known cancer (median age 77, 40% men), and the healthy individuals (median age 70.4, 42% men) (Figure 9). Venous blood sampling was performed once. Plasma samples were prepared from citrated whole blood following immediate centrifugation for 20 min at 2000 x g after which they were stored at -80 °C until further analysis. At time of analysis, samples were thawed on ice and diluted 1 :2 in PBS.
Plasma samples were analyzed for H3Cit using our tailor made H3Cit assay presented and validated in Example 1 and 2. H3Cit was detectable in 44/60 cancer patients (73%), in 17/55 hospitalized patients with other diseases but no known cancer (33%), and in 9/50 healthy individuals (18%) (Fig 10).
Cancer patients had a significant 5-fold increase in plasma H3Cit levels compared to healthy individuals (mean 36.1 ng/mL vs. 6.6 ng/mL, p<0.001 ) and a significant 3-fold increase compared to hospitalized patients with other diseases but no known cancer (mean 36.1 ng/mL vs. 1 1 .5 ng/mL, p<0.001 ). The difference in plasma H3Cit levels between healthy individuals and hospitalized patients with other diseases but no known cancer was not statistically significant (p=0.510). The mean levels of plasma H3Cit in patients with the different malignant tumor types are depicted in Figure 1 1 and in Table 3. Table 3.
Figure imgf000062_0001
Figure imgf000063_0001
The data clearly shows that H3Cit is elevated in cancer patients, and that this elevation can be seen in many different cancer types. As a biomarker H3Cit gives an approximate 75% sensitivity, as depicted in Figure 10 and 1 1 , and therefore it exceeds the sensitivity of the to-date clinically available screening biomarkers, ranging from 20-80% in various studies. Furthermore, H3Cit seems to be elevated in a large variety of cancers, suggesting that it may be applicable as a screening biomarker for cancer in general. There are as of today no clinically available biomarkers for screening for cancer in general. H3Cit may also, due to its potential as a biomarker in screening for several malignant tumor types, be implemented in combination with existing biomarkers, such as PSA, CA-125 and CEA, allowing for an increased sensitivity and specificity.
Plasma H3Cit was detectable in some hospitalized patients with other diseases but no known cancer (33%) and in some healthy individuals (18%). Although these study participants had no known cancer diagnosis at the time of blood sampling, they were of old age (median age 70.4 years), and there may be a possibility that some of these individuals have an unknown cancer. Furthermore, the hospitalized patients with other diseases but no known cancer had a large amount of comorbidities, with a median comorbidity index score of 6 using the Charlson Comorbidity Index Scoring System (Charlson ME, et al. A new method of classifying prognostic comorbidity in longitudinal studies: Development and validation. 1987 Journal of Chronic Diseases. 1987;40:373-83).
The cancer patients had a significantly lower median comorbidity index score of 3 (p<0.001 ), suggesting that plasma H3Cit is not a biomarker for disease burden in general (Fig 12). Indeed, there was no significant difference in the levels of plasma H3Cit between healthy individuals and hospitalized patients with other diseases but no known cancer, and there was no correlation between the plasma H3Cit levels and comorbidity index score in any of the groups (r=0.16, p=0.224 in the cancer group and r=0.08, p=0.589 in the group with hospitalized patients with other diseases but no known cancer). The levels of plasma H3Cit in healthy individuals as well as in hospitalized patients with other diseases but no known cancer with detectable plasma H3Cit were also lower than in cancer patients with detectable levels of plasma H3Cit, suggesting that there may be a cut-off level for when plasma H3Cit is indicative of cancer.
Further and larger studies of plasma H3Cit in patients with known cancer diagnosis (with a variety of malignant tumors and stages) as well as individuals without known cancer will be warranted to explore the sensitivity, specificity and cut-off levels of plasma H3Cit as a screening biomarker for cancer. Based on these data, however, plasma H3Cit, alone or in
combination with existing screening biomarkers and protocols, may increase the sensitivity and specificity in screening for cancer, cancer recurrence and cancer progression in many different types of cancer.

Claims

1 . A method for screening for the presence of cancer in an individual
comprising the steps:
a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or quantity in the plasma sample of a
biomarker chosen from citrullinated histone H3 (H3Cit), wherein said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating cancer in said individual.
2. A method for screening for cancer-associated thrombosis or an elevated risk for cancer-associated thrombosis in cancer individuals or for screening for cancer in individuals with idiopathic thrombosis, comprising the steps:
a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or the quantity in the plasma sample of a biomarker chosen from citrullinated histone H3 (H3Cit), wherein said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating cancer-associated thrombosis or an elevated risk for cancer-associated thrombosis in said cancer individuals or cancer in said individuals with idiopathic thrombosis.
3. A method for screening for thrombosis or an elevated risk for
thrombosis in cancer individuals following chemotherapy, comprising the steps:
a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or the quantity in the plasma sample of a biomarker chosen from citrullinated histone H3 (H3Cit), wherein said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating thrombosis or an elevated risk for thrombosis in said cancer individuals following chemotherapy.
4. A method for screening for tumour progression in individuals or
screening for recurrence of cancer in individuals with previously diagnosed cancer, comprising the steps:
a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or the quantity in the plasma sample of a biomarker chosen from citrullinated histone H3 (H3Cit), wherein said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating tumour progression in said individuals or cancer in said individuals with previously diagnosed cancer.
5. A method for screening for cancer, an elevated risk for cancer- associated thrombosis and/or cancer-associated thrombosis in individuals, comprising the steps: a) providing a plasma sample to be tested from the individual; and b) measuring the presence and/or the quantity in the plasma sample of biomarkers chosen from citrullinated histone H3 (H3Cit) and granulocyte colony stimulating factor (G-CSF), wherein said presence and/or quantity in the test sample of said biomarkers chosen from citrullinated histone H3 (H3Cit) and granulocyte colony stimulating factor (G-CSF) is indicating cancer, an elevated risk for cancer-associated thrombosis and/or cancer-associated thrombosis in said individuals. A method for screening for adverse effects in individuals receiving granulocyte colony stimulating factor (G-CSF) following chemotherapy, comprising the steps:
providing a plasma sample to be tested from the individual; and measuring the presence and/or the quantity in the plasma sample of a biomarker chosen from citrullinated histone H3 (H3Cit), wherein said presence and/or quantity in the test sample of said biomarker chosen from citrullinated histone H3 (H3Cit) is indicating adverse effects in individuals receiving granulocyte colony stimulating factor (G-CSF) following chemotherapy.
A method according to any one of claims 1 -6, wherein step b)
comprises comparing the presence and/or the quantity of the H3Cit biomarker in the plasma sample with the presence and/or the quantity of the H3Cit biomarker in one or more plasma samples from healthy individual(s) having known concentrations of citrullinated histone H3 (H3Cit) in said plasma samples.
A method according to claim 7, wherein the presence and/or the quantity of the H3Cit biomarker in the plasma sample is higher than the presence and/or the quantity of the H3Cit biomarker in one or more plasma samples from healthy individual(s).
A method according to claim 8, wherein the presence and/or the quantity of the H3Cit biomarker in the plasma sample is two-fold higher or more than the presence and/or the mean or median quantity of the H3Cit biomarker in one or more plasma samples from healthy
individual(s).
10. A method according to any one of claims 1 -6, wherein step b)
comprises measuring the optical density of the plasma sample.
1 1 . A method according to claim 10, wherein step b) comprises measuring the optical density of the plasma sample and comparing to the optical density of one or more plasma samples from healthy individual(s) having known concentrations of citrullinated histone H3 (H3Cit) in said plasma samples.
12. A method according to claim 1 1 , wherein the optical density of the
plasma sample is two-fold higher or more than the mean or median optical density of one of more plasma samples from healthy
individual(s).
13. A method according to any one of claims 1 -12, wherein step b)
comprises using a binding agent capable of binding to the biomarker citrullinated histone H3 (H3Cit) .
14. A method according to claim 13, wherein said binding agent is an anti- H3Cit-antibody or a fragment thereof.
15. A method according to any one of claims 1 -14, wherein the cancer is selected from a group consisting of: leukemia; carcinoma; AIDS-related cancers; lymphoma; anal cancer; appendix cancer; astrocytoma; bile duct cancer, extrahepatic cancer; bladder cancer; bone tumor, osteosarcoma fibrous histiocytoma; malignant fibrous histiocytoma; glioma; brain cancer; brain tumor; breast cancer; bronchial adenomas; bronchial carcinoids; cervical cancer; childhood cancers; chronic myeloproliferative disorders; colon cancer; desmoplastic small round cell tumor; endometrial cancer; ependymoma; epitheliod
hemangioendothelioma (EHE); esophageal cancer; extrahepatic bile duct cancer; eye cancer; gallbladder cancer; gastric (i.e. stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor
(GIST); gestational trophoblastic tumor; head and neck cancer; heart cancer; hepatocellular (i.e. liver) cancer; primary hepatocellular (i.e. liver) cancer; hypopharyngeal cancer; kidney cancer; renal cell cancer; laryngeal cancer; lip and oral cavity cancer; Waldenstrom macroglobulinemia; male breast cancer; childhood medulloblastoma; melanoma; merkel cell cancer; metastatic squamous neck cancer with occult primary; mouth cancer; childhood multiple endocrine neoplasia syndrome; multiple myeloma; plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myelodysplastic diseases;
myeloproliferative diseases; chronic myeloproliferative disorders;
myxoma; nasal cavity and paranasal sinus cancer; neuroblastoma;
oligodendroglioma; oral cancer; oropharyngeal cancer; ovarian cancer; pancreatic cancer; islet cell pancreatic cancer; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal germinoma; pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; pituitary adenoma; pleuropulmonary blastoma; prostate cancer; rectal cancer; renal pelvis and ureter cancer; salivary gland cancer; Sezary syndrome; skin cancer; small intestine cancer; metastatic squamous neck cancer with occult primary; stomach cancer; childhood supratentorial primitive neuroectodermal tumor; fungoides and Sezary syndrome; testicular cancer; throat cancer; childhood thymoma; thyroid cancer; childhood thyroid cancer; transitional cell cancer of the renal pelvis and ureter; gestational trophoblastic tumor; urethral cancer; endometrial uterine cancer; vaginal cancer; vulvar cancer; and childhood Wilms tumor.
6. A method according to any one of claims 1 -14, wherein the cancer is selected from a group consisting of: lung cancer; pancreatic cancer; breast cancer; prostate cancer; urothelial cancer; liver cancer; colon cancer; colorectal cancer; gastrointestinal cancer; gynecological cancer; glioblastoma; lymphoma; acute myeloid leukemia; gingival cancer;
liposarcoma; sarcoma; neuroendocrine cancer and melanoma (such as malignant melanoma).
7. A method according to any one of claims 1 -14, wherein the cancer is selected from a group consisting of: lung cancer; pancreatic cancer; breast cancer; prostate cancer; urotheal cancer; and liver cancer.
18. A method according to any one of claims 1 -17, further comprising step: c) selecting a treatment for the individual.
19. A method according to claim 18, further comprising step: d) administering the selected treatment to the individual.
20. A diagnostic kit for use in a method according to any one of claims 1 - 19 comprising; a) a binding agent capable of binding to the biomarker citrullinated histone
H3 (H3Cit); and
instructions for performing the method according to any one of claims 1 9.
A diagnostic kit for use according to claim 20, wherein said binding agent is an anti-H3Cit-antibody or a fragment thereof.
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US10287353B2 (en) 2016-05-11 2019-05-14 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-1 inhibitors
US10385131B2 (en) 2016-05-11 2019-08-20 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-L1 inhibitors
US10385130B2 (en) 2016-05-11 2019-08-20 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-1 inhibitors
US11535670B2 (en) 2016-05-11 2022-12-27 Huyabio International, Llc Combination therapies of HDAC inhibitors and PD-L1 inhibitors
US11872241B2 (en) 2018-11-30 2024-01-16 Beth Israel Deaconess Medical Center, Inc. Compositions and methods for reducing major thrombotic events in cancer patients
WO2020221921A1 (en) * 2019-05-02 2020-11-05 Belgian Volition Sprl Method for the detection of cancer
CN112773805A (en) * 2019-11-11 2021-05-11 中国科学院脑科学与智能技术卓越创新中心 Novel therapeutic agent for ischemic brain injury
WO2021119189A1 (en) * 2019-12-09 2021-06-17 Beth Israel Deaconess Medical Center, Inc., Compositions and methods for identifying and modulating thrombotic conditions in a cancer patient
TWI799799B (en) * 2019-12-09 2023-04-21 美商貝斯以色列女執事醫療中心有限公司 Compositions and methods for identifying and modulating thrombotic conditions in a cancer patient
CN112881704A (en) * 2020-12-30 2021-06-01 中山大学 Application of detection reagent of CIT-H3 protein in preparation of liver cancer prognosis and/or recurrence prediction reagent
CN113358872A (en) * 2021-06-03 2021-09-07 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） Marker group and system for evaluating curative effect of tumor immunotherapy
WO2023170724A1 (en) * 2022-03-08 2023-09-14 The University Of Jordan Use of substituted quinolone derivatives for treating cancer

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