WO2005040814A1

WO2005040814A1 - Methods and means of cancer detection by histone modification

Info

Publication number: WO2005040814A1
Application number: PCT/GB2004/004366
Authority: WO
Inventors: Carlos Caldas; Hilal Ozdag; Andrew Bannister; Tony Kouzarides; Alan Drummond
Original assignee: Cancer Research Technology Limited
Priority date: 2003-10-14
Filing date: 2004-10-13
Publication date: 2005-05-06

Abstract

Methods and Means of Cancer Detection by Histone Modification this invention relates to the detection and assessment of cancer conditions. In particular, methods of the invention relate to the determination of the presence or amount of post-translational modification of residues within histone sequences, for example, methylation of lysine residues, in order to assess a cancer condition. Methylated lysine residues which may be detected in these methods include, for example, H3 Lys 27, H3 Lys 36 and H4 Lys 20.

Description

Methods and Means of Cancer Detection by Histone Modification

This invention relates to the detection and assessment of cancer conditions using histone marks.

In eukaryotes, DNA is complexed with proteins to form nucleosomes, the basic sub-unit of chromatin. Nucleosomes consist of approximately 150 DNA base pairs wrapped around a histone core, which is a protein complex involving the four histones H4, H3, H2B and H2A. The amino-terminal tails of these proteins are among the most evolutionarily conserved proteins known. These tails are post-translationally modified by the addition of a range of chemical groups including methyl, acetyl and phosphoryl. These chemical modifications, or marks, play a key role in determining chromatin structure and hence access to the cells genomic DNA (Wu J and Grunstein M 2000 Trends Bioche . Sci. 25, 619-623; Berger SL 2001 Oncogene 20, 3007-3013) . It has also been shown that the marks are involved in the control mechanism for a wide range of cellular processes. For example, in general, deacetylation of marks and certain methylation marks are associated with gene silencing (Hu JF and Hoffman AR 2001 Methods Mol Biol 181, 285-296; Rice JC and Allis CD 2001 Curr Opin Cell Biol 13, 263-273; Carrozza MJ et al 2003 Trends Genet 19, 321-329; Nephew KP and Huang TH 2003 Cancer Lett 190, 125-133) and phosphoryl marks with apoptosis (Enomoto R et al 2001 Mol Cell Biol Res Commun 4, 276-281; Ajiro K 2000 J Biol Chem 275, 439- 443; Talasz H, et al 2002 Cell Death Differ 9, 27-39; Rogakou EP et al 2000 J Biol Chem 275, 9390-9395) and mitosis (Crosio et al 2002 Mol Cell Biol 22 874-885; Goto et al 2002 Genes

Cells 7, 11-17; Hans and Dimitrov 2001 Oncogene 20, 3021-3027;

Preuss et al 2003 Nucl Acids Res 31, 878-885) .

Methyltransferase enzymes such as EZH2 , ESET, SETDBl and G9a (Cao et al 2002 Science 298 1039-1043; Plath et al Science 2003 300 131-135; Yang et al Oncogene 2003 21 148-152; Schultz et al Genes Dev 2002 16 919-932; Tachibana et al J Biol Chem 2001 276 25309-25317) have been shown to introduce methyl marks on to specific lysine residues. Overexpression of EZH2 has been shown in certain prostate and breast cancer cells (Kleer et al (2003) Proc. Natl. Acad. Sci. USA 100 11607; Varambally et al . (2002) Na ture 419, 624-629)

The present inventors have discovered that histone modification in cancer cells is distinct from that in normal cells. In particular, specific histone lysine residues are methylated in cancer tissue but not in surrounding healthy tissue. Detection of histone modification, including, for example, methylation at specific lysine residues, may therefore be used to diagnose and assess cancer conditions.

One aspect of the invention provides a method of assessing a cancer condition in an individual comprising determining the presence or amount of a modified histone in a sample obtained from the individual.

The presence or amount of a modified histone in the sample is indicative of whether the individual has a cancer condition.

A modified histone is a histone having one or more post- translational modifications. A modified histone may comprise a residue having a modification selected from the group consisting of acetylation, methylation, including mono-, di- or tri- ethylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and/or isomerisation. Residues having a modification may include Lys, Ser, Arg, His, Glu, Pro or Thr within the histone amino acid sequence. The positions of suitable residues within the sequence may be determined from the database histone sequences. For example, additional lysine residues may be methylated, acetylated or ubiquitinated, an arginine residue within the core histone sequence may be monomethylated, symmetrically or asymmetrically dimethylated or converted to citrulline, a serine, histidine or threonine residue within the core histone sequence may be phosphorylated and/or a proline residue within the core sequence may be isomerised.

The sample may be a tissue biopsy sample, for example from tissue suspected of malignancy, or may be a biological fluid sample, for example from blood, serum or plasma. A biological fluid sample may comprise cells, cell-free nucleosomes, free histones or fragments thereof, which may, optionally, be concentrated and/or isolated prior to contacting with the antibody.

Cancer conditions include, but are not limited to, lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carcinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma, lymphoma or leukaemia .

In some preferred embodiments, the cancer condition may be breast cancer. The presence of a modified histone may be determined using an antibody molecule that binds specifically to the modified histone .

For example, a method of assessing a cancer condition in an individual may comprise; contacting a sample obtained from the individual with an antibody which specifically binds to a modified histone; and, determining binding of said antibody.

Binding of the antibody is indicative of whether said individual has a cancer condition.

An antibody which specifically binds to an antigen such as a modified histone may not show any significant binding to molecules other than the antigen. In some cases, an antibody may specifically bind to a particular epitope which is carried by a number of antigens, in which case the antibody will be able to bind to the various antigens carrying the epitope.

An antibody specific for a modified histone is specific for a unique epitope formed by post-translational modification of a histone, for example a core histone such as H2A, H2B, H3, H4 (Luger,K. et al (1997) Nature 389, 251-260) or modifications or variants thereof (see for example (Ausio J (2001) Biochem Cell Bio 79, 693) . Known sequences of histones are described in the NHGRI/NCBI histone sequence database which is accessible on-line. The antibody may specifically bind to an epitope formed by a single modification or to an epitope formed by two or more modifications. An antibody specific for a modified histone does not specifically bind to the histone unless the histone contains one or more modified residues.

In preferred embodiments, a modified histone is a histone having a methylated lysine residue, for example a mono-, di- or tri-methylated lysine residue.

A method of assessing a cancer condition in an individual may comprise; determining the presence or amount of a histone having a methylated lysine residue in a sample obtained from the individual .

The presence or amount of a histone having a methylated lysine residue in the sample is indicative of whether the individual has a cancer condition.

A histone having a methylated lysine residue is preferably a core histone. In some embodiments, the methylated lysine residue within the core histone may be located outside the N- terminal 15 amino acid region. In some preferred embodiments, the methylated lysine residue may be a lysine residue other than H3 Lys 4 and/or H3 Lys 14.

The methylated lysine residue may be within the histone H3 sequence. In some preferred embodiments, the methylated lysine residue is H3 Lys 27 and/or H3 Lys 36.

The methylated lysine residue may be within the histone H4 sequence. In some preferred embodiments, the methylated lysine residue is H4 Lys 20. Examples of suitable histone methylations are shown in Table I.

The notation used to describe a particular histone modification indicates which histone has been modified, the particular amino acid(s) that have been modified and the type of modification that has occurred. For example H3 Lys 9 (Me) denotes the methylation of histone H3 at lysine 9.

As described above, methylation may include mono-, di- or tri- methylation.

The presence of histone having a methylated lysine residue may be conveniently determined using an antibody.

A method of assessing a cancer condition in an individual may comprise; contacting a sample obtained from the individual with an antibody which specifically binds to a histone having a methylated lysine residue; and, determining binding of said antibody.

In some embodiments, binding of the antibody to the sample may be determined relative to the binding of the antibody to a known cancerous or non-cancerous sample, for example, a sample from a healthy individual or a cancer patient. A method may comprise determining the binding of the antibody to a test sample obtained from the individual relative to a normal control sample, an increase or decrease in the binding of the antibody to the test sample relative to the normal control being indicative of whether the individual has a cancer condition.

For example, an increase in the binding of an antibody which specifically binds to H3 Lys 27, H3 Lys 36 and/or H4 Lys 20 to the test sample relative to a normal control may be indicative that the individual has a cancer condition.

Alternatively, the sample obtained from the individual may comprise one or more regions suspected of being cancerous (i.e. regions which show histological signs of being cancerous) and one or more normal regions (i.e. regions which are histologically normal or non-cancerous) . A method may comprise determining the binding of said antibody to a putative tumour region of said sample relative to a normal region of said sample, an increase or decrease in the binding of said antibody to the putative tumour region being indicative that the putative tumour region is cancerous and the individual has a cancer condition.

For example, an increase in the binding of an antibody which specifically binds to H3 Lys 27, H3 Lys 36 and/or H4 Lys 20 to putative tumour region relative to the normal region may be indicative that the putative tumour region is cancerous and the individual has a cancer condition.

In some embodiments, binding of the antibody to the sample may be determined relative to the binding of a second antibody to the same sample. The second antibody may specifically bind to a histone residue having a modification which is different from the methylated lysine residue which is bound by the first antibody (i.e. the second antibody may bind to a second modification) . The amount of binding to the sample of the first antibody relative to the binding to the sample of the second antibody may be indicative that the individual has a cancer condition.

The second antibody may bind specifically to a histone having a residue with a post-translational modification selected from the group consisting of acetylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and isomerisation or it may bind specifically to a histone having a residue which is methylated, where the methylated residue is not the lysine residue which is bound by the first antibody.

The first and second antibodies may bind to modified amino acid residues of the same histone; for example the ratio of H3 lys 9 (Me) to H3 lys 4 (Me) may be determined. Alternatively, the second antibody may bind to a modified residue on a different histone to the histone comprising the methylated lysine residue; for example, the ratio of H3 lys 9 (Me) to H4 arg 3 (Me) may be determined.

In other embodiments, the second antibody may bind to a different modification at the same lysine residue which, when methylated, is bound by the first antibody.

A cancer condition may thus be assessed by determining the ratio of different modifications of the same or different histone residues. In some embodiments, the amount of methylation relative to acetylation may be compared.

A method of assessing a cancer condition in an individual may comprise; determining the level or amount of histones having a methylated lysine residue relative to the level or amount of histones having an acetylated lysine residue in a sample obtained from the individual .

In some embodiments, acetylation and methylation may be determined at the same lysine residue, for example, the level or amount of H3 lys 9 (Me) may be compared to H3 lys 9 (Ac) . In other embodiments, acetylation and methylation may be

' determined at different lysine residues, for example the level or amount of H4 lys 20 (Me) may be compared to H4 lys 8 (Ac) .

Acetylation and methylation may be determined using modification-specific antibodies, as described above. A method for assessing a cancer condition in an individual may comprise; contacting a sample obtained from the individual with a first antibody which specifically binds to a histone having a methylated lysine residue and a second antibody which specifically binds to a histone having a acetylated lysine residue; and, determining the amount or level of binding of said first and second antibodies. The amount of binding of said first antibody relative to said second antibody is indicative of whether said individual has a cancer condition.

As described above, an antibody which specifically binds to a histone having a methylated lysine residue may recognise and specifically bind to an epitope comprising the methylated lysine residue and one or more additional modified residues.

For example such additional modifications may be selected from the group consisting of acetylation, methylation, including mono-, di- or tri-methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and/or isomerisation .

A histone residue having a modification may include a Lys, Ser, Arg, His, Glu, Pro or Thr residue within the histone amino acid sequence. The positions of suitable residues within the sequence may be determined from the database sequences of the core histones.

For example, additional lysine residues may be methylated, acetylated or ubiquitinated, an arginine residue within the core histone sequence may be monomethylated, symmetrically or asymmetrically dimethylated or converted to citrulline, a serine, histidine or threonine residue within the core histone sequence may be phosphorylated and/or a proline residue within the core sequence may be isomerised.

Binding of antibody molecules may be determined by any appropriate means . Tagging with individual reporter molecules is one possibility. The reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be direct or indirect, covalent, e.g. via a peptide bond, or non-covalent . Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion, encoding antibody and reporter molecule .

For example, the antibody may be labelled with a fluorophore such as FITC or rhodamine, a radioisotope, or a non-isotopic- labelling reagent such as biotin or digoxigenin; antibodies containing biotin may be detected using "detection reagents" such as avidin conjugated to any desirable label such as a fluorochrome . In some embodiments, an additional antibody may be used to detect the binding of the first antibody.

The mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.

Suitable approaches include immunohistochemical staining, immunocytochemical staining, Western Blotting, immunofluorescence, enzyme linked immunosorbent assays (ELISA) , radioimmunoassays (RIA) , immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA) , including sandwich assays using monoclonal and/or polyclonal antibodies. All of these approaches are well known in the art.

An antibody for use in a method described herein may be immobilised or non-immobilised i.e. free in solution.

An antibody may be immobilised, for example, by attachment to an insoluble support. The support may be in particulate or solid form and may include a plate, a test tube, beads, a ball, a filter or a membrane. An antibody may, for example, be fixed to an insoluble support that is suitable for use in affinity chro atography . Methods for fixing antibodies to insoluble supports are known to those skilled in the art. An immobilised antibody may be preferred, for example, for binding to antigens such as modified histones or nucleosomes, in solution, for example in a biological fluid sample.

The binding of the antibody to a methylated histone may be detected using a second antibody. The second antibody may bind to the first antibody, or may bind to a different region of the same histone or an associated antigen, for example in a sandwich assay. For example, the second antibody may bind to another nucleosome component, such as DNA or a different histone. Depending on the assay format employed, the second antibody may be immobilised or labelled with a detectable label .

In some embodiments, a labelled third antibody may be used to detect the binding of the second antibody.

Other aspects of the invention relate to methods for predicting the response of a cancer condition in an individual to an agent which reduces histone modification and methods of determining the effect of treatment with a agent which reduces histone modification on a cancer condition in an individual.

A method for predicting the response of a cancer condition in an individual to treatment with a histone modification inhibitor may comprise; contacting a sample obtained from the individual with an antibody which specifically binds to a histone having a modification; and, determining binding of said antibody

Suitable modifications include one or more methylated lysine residues. For example, a method for predicting the response of a cancer condition in an individual to treatment with a histone methylation inhibitor may comprise; contacting a sample obtained from the individual with an antibody which specifically binds to a histone having a methylated lysine residue; and, determining binding of said antibody.

Binding of said antibody is indicative of whether said cancer condition will respond to the treatment.

Methods may be used to identify individuals having a cancer condition that will respond to treatment with a histone methylation inhibitor. This allows treatment to be targeted to responsive individuals.

A suitable antibody may specifically bind to histone H3 which is methylated at Lys 27 and/or Lys 36 or may specifically bind to histone H4 which is methylated at Lys 20. A methylated histone which is specifically bound by an antibody may comprise one or more other modifications, as described above.

In some embodiments, a sample may comprise one or more cancerous regions (i.e. regions which show histological signs of being cancerous) and one or more normal regions (i.e. regions which are histologically normal or non-cancerous) . A method may comprise determining the binding of said antibody to a cancerous region of said sample relative to a normal region of said sample, an increase in the binding of said antibody to the cancerous region being indicative that said cancer condition will respond to treatment with a histone methylation inhibitor.

For example, an increase in the binding of an antibody which specifically binds to H3 Lys 27, H3 Lys 36 and/or H4 Lys 20 to putative tumour region relative to the normal region may be indicative that said cancer condition will respond to treatment with a histone methylation inhibitor.

In some embodiments, a method may comprise comparing the binding of said antibody to said sample with the binding of said antibody to a control non-cancerous sample, for example a sample obtained from an individual without a cancer condition or a non-cancerous sample from the patient, an increase in said binding relative to the control being indicative that said cancer condition will respond to said treatment.

For example, an increase in the binding of an antibody which specifically binds to H3 Lys 27, H3 Lys 36 and/or H4 Lys 20 to the sample relative to control may be indicative that said cancer condition will respond to said treatment.

In some embodiments, a method may comprise comparing the binding to the sample of a first antibody which specifically binds to a histone comprising a methylated lysine residue with the binding of a second antibody which specifically binds to a different histone modification. The amount of binding to the sample of the first antibody relative to the binding to the sample of the second antibody may be indicative that the cancer condition will respond to the treatment.

As described above, a different modification may include methylation at a residue other than the lysine residue which is recognised by the first antibody, or a post-translational modification selected from the group consisting of acetylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and isomerisation.

The first and second antibodies may bind to modified amino acid residues of the same histone; for example the ratio of H3 lys 9 (Me) to H3 lys 4 (Me) in the sample may be determined. Alternatively, the second antibody may bind to a modified residue on a different histone to the histone comprising the methylated lysine residue; for example, the ratio of H3 lys 9 (Me) to H4 arg 3 (Me) may be determined.

The response of a cancer condition in an individual to treatment with a histone methylation inhibitor may thus be predicted by determining the ratio of different modifications of the same or different histone residues.

In some embodiments, the amount of methylation relative to acetylation of the same histone residue may be compared. A method for predicting the response of a cancer condition in an individual to treatment with a histone methylation inhibitor may comprise; determining the level or amount of histones having a methylated lysine residue relative to the level or amount of histones having an acetylated lysine residue in a sample obtained from the individual.

In some embodiments, acetylation and methylation may be determined at the same lysine residue, for example, the level or amount of H3 lys 9 (Me) may be compared to H3 lys 9 (Ac) .

In other embodiments, acetylation and methylation may be determined at different lysine residues, for example the level or amount of H4 lys 20 (Me) may be compared to H4 lys 8 (Ac) .

Acetylation and methylation may be determined using antibodies . A method for predicting the response of a cancer condition in an individual to treatment with a histone methylation inhibitor may comprise; contacting a sample obtained from the individual with a first antibody which specifically binds to a histone having a methylated lysine residue and a second antibody which specifically binds to a histone having a acetylated lysine residue; and, determining the amount or level of binding of said first and second antibodies.

The amount of binding of said first antibody relative to said second antibody is indicative of whether said cancer condition will respond to the treatment. A histone modification inhibitor may inhibit one or more enzymes which post-translationally modify histone residues. For example, a histone methylation inhibitor may inhibit histone methylases, in particular methylases which methylate H3 Lys 27, H3 Lys 36 or H4 Lys 20. Such an inhibitor may, for example, be a small organic molecule, a polypeptide, or a nucleic acid molecule (e.g. a sense, anti-sense or RNAi molecule) .

Suitable samples, antibodies and detection methods for use in such methods are described above.

In some embodiments, a sandwich assay format may be used to determine the presence of modified histones in a biological fluid sample from said individual. A nucleosome containing a modified histone may be comprised within a cell in said fluid sample or may be a cell-free nucleosome within the sample.

Binding of the first antibody to a methylated histone as described above may be determined using a second antibody. The second antibody may bind to the first antibody, to the same antigen as the first antibody but at a different site, or to an antigen which is associated with such an antigen, for example another component of the nucleosome.

The binding of said specific first antibody to said sample may be compared with the binding of said first antibody with a control sample obtained from a individual without a cancer condition. An increase in binding relative to the control being indicative that said cancer condition will respond to said treatment. Other aspects of the invention relate to the analysis of histone modification, for example histone methylation, to determine the effect of treatment with a histone modification inhibitor on a cancer condition.

A method for determining the effect on a cancer condition in an individual of treatment with a histone modification inhibitor may comprise; contacting samples obtained from the individual at first and second time points in said treatment with an antibody which specifically binds to a histone having a modification; and, determining binding of said antibody to said samples.

Suitable histone modifications include one or more methylated lysine residues. For example, a method for determining the effect on a cancer condition in an individual of treatment with a histone methylation inhibitor may comprise; contacting samples obtained from the individual at first and second time points in said treatment with an antibody which specifically binds to a histone having a methylated lysine residue; and, determining binding of said antibody to said samples.

A change in the binding of said antibody at said second time point relative to the first may be indicative that the treatment has a beneficial effect on said condition.

A suitable antibody may specifically bind to histone H3 which is methylated at Lys 27 and/or Lys 36 or may specifically bind to histone H4 which is methylated at Lys 20. A decrease in the binding of an antibody which specifically binds to H3 Lys 27, H3 Lys 36 and/or H4 Lys 20 to the sample at the second time point relative to the first may be indicative that the treatment has a beneficial effect on said cancer condition.

As described above, a different modification may be methylation at a residue other than the lysine residue which is recognised by the first antibody, or a post-translational modification selected from the group consisting of acetylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and isomerisation .

As described above, the first and second antibodies may bind to modified amino acid residues of the same histone or the second antibody may bind to a modified residue on a different histone to the histone comprising the methylated lysine residue .

In other embodiments, the second antibody may bind to a different modification at the same lysine residue which, when methylated, is bound by the first antibody. The effect on a cancer condition in an individual of treatment with a histone methylation inhibitor may be determined from a change in the ratio of the different modifications of the same or different histone residues at said first and second time points.

In some embodiments, the amount of methylation relative to acetylation may be compared.

A method for determining the effect on a cancer condition in an individual of treatment with a histone methylation inhibitor may comprise; determining the level or amount of histones having a methylated lysine residue relative to the level or amount of histones having an acetylated lysine residue in samples obtained from the individual at first and second time points in said treatment .

A change in ratio of histones having a methylated lysine residue relative to histones having an acetylated lysine residue at said second time point relative to the first may be indicative that the treatment has a beneficial effect on said condition.

Acetylation and methylation may be determined at the same or different lysine residues, as described above.

Antibodies may be used to determine acetylation and methylation. A method for determining the effect on a cancer condition in an individual of treatment with a histone methylation inhibitor may comprise; contacting samples obtained from the individual at first and second time points in said treatment with a first antibody which specifically binds to a histone having a methylated lysine residue and a second antibody which specifically binds to a histone having a acetylated lysine residue; and, determining the amount or level of binding of said first and second antibodies at the first and second time points.

A change in ratio of binding of said first antibody relative to said second antibody at the second time point relative to the first time point may be indicative that the treatment has a beneficial effect on said condition.

Suitable samples, antibodies and detection methods for use in such methods are described above. Any standard technique may be used to perform such methods, including immunohistochemistry and immunocytochemistry . In some embodiments, a sandwich assay format may be used.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents referenced in this specification are incorporated herein by reference.

The skilled person will understand that the invention may be carried out with various combinations and sub-combinations of the features described above, and all these combinations and sub-combinations, whether or not specifically described or exemplified, are encompassed by the invention. Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the tables below.

Examples

Materials and Methods

Frozen slices of normal and breast cancer tissue were obtained from the Addenbrooke's Tumour Bank with appropriate Ethical Committee approval .

The frozen slides were left to dry overnight at room temperature, fixed in acetone for ten minutes and washed twice for 5 minutes with phosphate buffered saline (PBS) . They were then treated with rabbit polyclonal antibodies that targeted specific methyl modifications, diluted appropriately in PBS. The slides were then washed twice for 5 minutes in PBS and treated with a second, fluorescein isothiocyanate-coupled swine anti-rabbit antibody (1/50 diluted in PBS) for 30 minutes in the dark. They were then washed twice for 5 minutes in PBS. The slides were mounted on Vectashield PI-H1300 and analysed using a confocal microscope. Nuclei were identified by 4 ' 6- diamidino-2-phenylindole-2HCl (DAPI) stain and the antibodies against the methyl mark identified by green, fluorescein fluorescence .

Results For the antibodies raised against the methyl modifications shown in table I, more intense staining of cancerous breast tissue was observed compared to normal breast tissue. For the antibodies raised against the methyl marks shown in table II, no such differences were observed. Methyl marks that distinguish cancer tissue from normal tissue as described herein have potential as both tumour markers and therapeutic targets.

Mark

H4 Lys 20(monoMel

H4 Lys 20 (triMe)

H3 Lys 27 (monoMe;

H3 Lys 27 (diMe)

H3 Lys 27 (triMe)

H3 Lys 36(monoMe)

Table I

Mark

H3 Lys 4 (monoMe)

H3 Lys 4 (diMe)

H3 Lys 4 (triMe)

H3 Lys 14 (diMe)

Table II

Claims

Claims :

1. A method of assessing a cancer condition in an individual comprising; determining the presence or amount of a histone having a modification in a sample obtained from the individual.

2. A method according to claim 1 wherein the modification is a methylated lysine residue.

3. A method according to claim 2 wherein the methylated lysine residue is selected from H3 Lys 27, H3 Lys 36 and H4 Lys 20.

4. A method according to claim 2 or claim 3 wherein the lysine residue is mono-, di- or tri- methylated.

5. A method according to any one of claims 2 to 4 wherein the histone further comprises one or more additional modifications .

6. A method according to claim 5 wherein each additional modification is selected from the group consisting of acetylation, mono-, di- or tri-methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and isomerisation.

7. A method according to any one of the preceding claims wherein the presence or amount of said histone is indicative of said individual having a cancer condition.

8. A method according to any one of the preceding claims wherein the amount of histones in said sample having said modification is determined relative to the amount of histones having a different modified residue.

9. A method according to claim 8 wherein the amount of histones having a methylated lysine residue is determined relative to the amount of histones having an acetylated lysine residue .

10. A method according to any one of the preceding claims comprising; contacting a sample obtained from the individual with an antibody molecule which specifically binds to said histone having a methylated lysine residue; and, determining binding of said antibody.

11. A method according to claim 10 wherein the antibody molecule comprises a detectable label.

12. A method according to claim 10 comprising detecting the binding of a labelled secondary antibody which binds to said antibody molecule.

13. A method according to any one of the preceding claims wherein said sample is a biopsy sample.

14. A method according to any one of claims 1 to 12 wherein said sample is a biological fluid sample.

15. A method according to claim 14 wherein said biological fluid is blood, serum or plasma.

16. A method according to claim 14 or claim 15 wherein the histone is not nucleosome associated.

17. A method according to claim 14 or claim 15 wherein said histone is comprised in a cell in said biological fluid sample .

18. A method according to claim 14 or claim 15 wherein said histone is comprised in a cell-free nucleosome in said biological fluid sample.

19. A method according to claim 17 or claim 18 wherein binding to said antibody is determined using a nucleosome specific antibody.

20. A method according to claim 19 wherein the nucleosome specific antibody comprises a detectable label.

21. A method according to claim 19 wherein binding of said nucleosome specific antibody is determined by detected the binding of a labelled secondary antibody.

22. A method according to any one of claims 17 to 21 wherein said sample is contacted with a nucleosome specific antibody and the binding to said nucleosome specific antibody determined using said antibody which specifically binds to a histone having a methylated lysine residue.

23. A method according to any^' one of claims 14 to 22 wherein the antibody is immobilised.

24 A method according to any one of claims 1 to 23 wherein said cancer is breast cancer.

25. A method for predicting the response of a cancer condition in an individual to treatment with a histone methylation inhibitor comprising; determining the presence or amount of a histone having a methylated lysine residue in a sample obtained from the individual .

26. A method according to claim 25 wherein the methylated lysine residue is selected from H3 Lys 27, H3 Lys 36 and H4 Lys 20.

27. A method according to claim 25 or claim 26 wherein the histone having a methylated lysine residue further comprises one or more other modified residues.

28. A method according to claim 27 wherein each said modified residue has a modification selected from the group consisting of acetylation, methylation, which may be mono-, di- or tri- methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and/or isomerisation.

29. A method according to any one of claims 25 to 28 wherein the presence or amount of said histone is indicative that said cancer condition will respond to treatment with a histone methylation inhibitor.

30. A method according to any one of claims 25 to 29 wherein the amount of histones having said methylated lysine residue in said sample is determined relative to the amount of histones having a different modification.

31. A method according to claim 30 wherein the different modification is an acetylated lysine residue.

32. A method according to any one of claims 25 to 31 comprising; contacting a sample obtained from the individual with an antibody molecule which specifically binds to said histone having a methylated lysine residue; and, determining binding of said antibody.

33. A method according to claim 32 comprising; comparing the binding of said antibody to a cancerous region of said sample with the binding of said antibody with a non-cancerous region of said sample, an change in said binding relative to the non-cancerous region being indicative of whether said cancer condition will respond to said treatment.

34. A method according to claim 32 comprising; comparing the binding of said antibody to said sample to the binding of said antibody with a control sample obtained from a individual without a cancer condition, a change in said binding relative to the control being indicative that of whether said cancer condition will respond to said treatment.

35. A method according to any one of claims 25 to 34 wherein said sample is a biopsy sample.

36. A method according to any one of claims 25 to 35 wherein the antibody comprises a detectable label.

37. A method according to any one of claims 25 to 35 wherein binding of said antibody is determined by detected the binding of a labelled secondary antibody.

38. A method according to any one of claims 25 to 34 wherein said sample is a biological fluid sample.

39. A method according to claim 38 wherein said biological fluid is blood, serum or plasma.

40. A method according to claim 38 or claim 39 wherein the histone is not nucleosome associated.

41. A method according to claim 38 or claim 39 wherein said histone is comprised in a cell in said biological fluid sample .

42. A method according to claim 38 or claim 39 wherein said histone is comprised in a cell-free nucleosome in said biological fluid sample.

43. A method according to any one of claims 38 to 42 wherein the antibody is immobilised.

44. A method according to any one of claims 41 to 43 wherein binding to said antibody is determined us.ing a nucleosome specific antibody.

45. A method according to claim 44 wherein the nucleosome specific antibody comprises a detectable label.

46. A method according to claim 45 wherein binding of said nucleosome specific antibody is determined by detected the binding of a labelled secondary antibody.

47. A method according to any one of claims 41 to 46 wherein said sample is contacted with a nucleosome specific antibody and the binding to said nucleosome specific antibody determined using said antibody which specifically binds to a histone having a methylated lysine residue.

48. A method for determining the effect of treatment with a histone methylation inhibitor on a cancer condition in an individual comprising; determining the level of histones having a methylated lysine residue in samples obtained from the individual first and second time points in said treatment.

49. A method according to claim 48 wherein the methylated lysine residue is selected from H3 Lys 27, H3 Lys 36 and H4 Lys 20.

50. A method according to claim 48 or claim 49 wherein the histone having a methylated lysine residue further comprises one or more other modifications.

51. A method according to claim 50 wherein said one or more other modifications are selected from the group consisting of acetylation, methylation, which may be mono-, di- or tri- methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and isomerisation.

52. A method according to any one of claims 48 to 51 wherein a decrease in level of said histones at said second time point relative to the first is indicative of the treatment having a beneficial effect on said condition

53. A method any one of claims 48 to 52 wherein wherein the level of histones having a methylated lysine residue is determined relative to the level of histones having a different modification.

54. A method according to claim 53 wherein the different modification is an acetylated lysine residue.

55. A method according to any one of claims 48 to 55 comprising; contacting samples obtained from the individual at first and second time points in said treatment with an antibody which specifically binds to a histone having a methylated lysine residue; determining binding of said antibody to said samples; a decrease in the binding of said antibody to said second sample relative to the first being indicative of the effect of said treatment.

56. A method according to any one of claims 48 to 55 wherein said sample is a biopsy sample.

57. A method according to claim 56 wherein the antibody comprises a detectable label.

58. A method according to claim 56 wherein binding of said antibody is determined by detecting the binding of a labelled secondary antibody which binds to said antibody molecule.

59. A method according to any one of claims 48 to 55 wherein said sample is a biological fluid sample.

60. A method according to claim 59 wherein said biological fluid is blood, serum or plasma.

61. A method according to claim 59 or claim 60 wherein the histone is not nucleosome associated.

62. A method according to claim 59 or claim 60 wherein said histone is comprised in a cell in said biological fluid sample .

63. A method according to claim 59 or claim 60 wherein said histone is comprised in a cell-free nucleosome in said biological fluid sample.

64. A method according to any one of claims 59 to 63 wherein the antibody is immobilised.

65. A method according to any one of claims 62 to 64 wherein binding to said antibody is determined using a nucleosome specific antibody.

66. A method according to claim 65 wherein the nucleosome specific antibody comprises a detectable label.

67. A method according to claim 65 wherein binding of said nucleosome specific antibody is determined by detecting the binding of a labelled secondary antibody.

68. A method according to any one of claims 62 to 67 wherein said sample is contacted with a nucleosome specific antibody and the binding to said nucleosome specific antibody determined using said antibody which specifically binds to a histone having a methylated lysine residue.

69. A method of assessing a cancer condition, said method being substantially as described herein.