METHOD FOR THE DETECTION OF HORMONE SENSITIVE DISEASE PROGRESSION

Abstract

The invention relates to the use of cell free nucleosome bound nuclear hormone receptor variant adducts for detecting the progression of hormone dependent disease to hormone therapy resistant disease or the effectiveness of a drug treatment in a patient. The invention also relates to methods for detecting said cell free nucleosome bound nuclear hormone receptor variant adducts.

Claims

1-4. (canceled)

5. A method of detecting the presence of a cell free nucleosome bound nuclear hormone receptor variant isolated from cancer tissue which comprises the steps of chromatin extraction followed by chromatin digestion.

6. A method of detecting circulating cell free nucleosome bound nuclear hormone receptor variant adduct in a blood, serum or plasma sample taken from a human or animal subject which comprises the steps of: (i) contacting the sample with a first binding agent which binds to nucleosomes or a component thereof; (ii) contacting the nucleosomes or sample with a second binding agent which binds specifically to said nuclear hormone receptor variant; (iii) detecting or quantifying the binding of said second binding agent which binds specifically to said nuclear hormone receptor variant; and (iv) using the presence or degree of such binding as a measure of the presence of circulating cell free nucleosome bound nuclear hormone receptor variant adducts in the sample.

7. A method of detecting circulating cell free nucleosome bound nuclear hormone receptor variant adduct in a blood, serum or plasma sample taken from a human or animal subject, which comprises the steps of: (i) contacting the sample with a first binding agent which binds specifically to said nuclear hormone receptor variant; (ii) contacting the nucleosomes or sample with a second binding agent which binds to nucleosomes or a component thereof; (iii) detecting or quantifying the binding of said second binding agent to nucleosomes or a component thereof; and (iv) using the presence or degree of such binding as a measure of the presence of circulating cell free nucleosome bound nuclear hormone receptor variant adducts in the sample.

8. The method as defined in any one of claims 5 to 7, wherein the hormone receptor variant is a variant of the androgen receptor, such as a truncated or short form variant.

9. The method of claim 8, wherein the truncated or short form variant of the androgen receptor lacks the ligand binding domain (LBD) of the androgen receptor.

10. The method of claim 8, wherein the truncated variant of the androgen receptor comprises androgen receptor variant 7 (AR-V7).

11. The method as defined in any one of claims 5 to 7, wherein the hormone receptor variant is a variant of the androgen receptor and the cancer is selected from castration resistant PCA.

12. The method as defined in any one of claims 5 to 7, wherein the hormone receptor variant is a variant of the estrogen receptor, such as a truncated or short form variant.

13. The method as defined in claim 12, wherein the truncated or short form variant of the estrogen receptor lacks the ligand binding domain (LBD) of the estrogen receptor.

14. The method as defined in claim 12, wherein the estrogen receptor variant is ERα-Δ5.

15. The method as defined in claim 12, wherein the estrogen receptor variant is the ERβ splice variant ERβ2 (or Eβcx).

16. The method as defined in any one of claims 5 to 7, wherein the hormone receptor variant is a variant of the estrogen receptor and the cancer is hormone therapy resistant breast cancer.

17. The method as defined in claim 10, wherein the AR-V7 variant adduct is measured in a blood sample taken from a prostate cancer patient and the adduct measurement is used to predict the efficacy of an androgen agonist, such as enzalutamide, or a CYP17 inhibitor, such as abiraterone, for the treatment of the subject from whom the sample was taken.

18. A method of detecting circulating cell free nucleosome bound AR-V7 adduct in a blood, serum or plasma sample taken from a human or animal subject which comprises the steps of: (i) contacting the sample with a first binding agent which binds to nucleosomes or a component thereof; (ii) contacting the nucleosomes or sample with a second binding agent which binds specifically to AR-V7; (iii) detecting or quantifying the binding of said second binding agent to AR-V7; and (iv) using the presence or degree of such binding as a measure of the presence of cell-free nucleosome bound AR-V7 adducts in the sample.

19. A method of detecting circulating cell free nucleosome bound AR-V7 adduct in a blood, serum or plasma sample taken from a human or animal subject which comprises the steps of: (i) contacting the sample with a first binding agent which binds specifically to AR-V7; (ii) contacting the nucleosomes or sample with a second binding agent which binds to nucleosomes or a component thereof; (iii) detecting or quantifying the binding of said second binding agent to nucleosomes or a component thereof; and (iv) using the presence or degree of such binding as a measure of the presence of cell-free nucleosome bound AR-V7 adducts in the sample.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0032] FIG. 1: Circulating cell free nucleosome bound AR-V7 ELISA results for 9 cases of treatment naïve newly diagnosed prostate cancer, 1 case of treatment naïve PCA a few months post diagnosis, 1 case of advanced PCA treated by surgery and a negative control (−ve ctrl) sample.

DETAILED DESCRIPTION OF THE INVENTION

[0033] According to a first aspect of the invention there is provided the use of a cell free nucleosome bound nuclear hormone receptor variant adduct as a biomarker for the progression of hormone dependent cancer to hormone therapy resistant cancer.

[0034] According to a second aspect of the invention there is provided the use of a cell free nucleosome bound nuclear hormone receptor variant adduct as a biomarker for the effectiveness of a drug treatment in a patient.

[0035] In one embodiment, the cell free nucleosome bound nuclear hormone receptor variant adduct comprises a circulating cell free nucleosome bound nuclear hormone receptor variant adduct.

[0036] In one embodiment, the circulating cell free nucleosome bound nuclear hormone receptor variant is measured in a blood, serum or plasma sample.

[0037] According to a further aspect of the invention there is provided a method of detecting the presence of a cell free nucleosome bound nuclear hormone receptor variant as defined herein isolated from cancer tissue which comprises the steps of chromatin extraction followed by chromatin digestion.

[0038] According to a further aspect of the invention there is provided a method of detecting circulating cell free nucleosome bound nuclear hormone receptor variant adduct in a blood, serum or plasma sample taken from a human or animal subject which comprises the steps of:

[0039] (i) contacting the sample with a first binding agent which binds to nucleosomes or a component thereof;

[0040] (ii) contacting the nucleosomes or sample with a second binding agent which binds specifically to said nuclear hormone receptor variant;

[0041] (iii) detecting or quantifying the binding of said second binding agent which binds specifically to said nuclear hormone receptor variant; and

[0042] (iv) using the presence or degree of such binding as a measure of the presence of circulating cell free nucleosome bound nuclear hormone receptor variant adducts in the sample.

[0043] It will be appreciated that the method of this aspect of the invention is capable of detecting or predicting the progression of a hormone dependent cancer disease to a hormone therapy resistant cancer disease including castration resistant PCA and hormone therapy resistant breast cancer in the subject from whom the sample was taken.

[0044] It will also be appreciated that the method of this aspect of the invention is capable of predicting the efficacy of a drug in the subject from whom the sample was taken.

[0045] According to a further aspect of the invention there is provided a method of detecting circulating cell free nucleosome bound nuclear hormone receptor variant adduct in a blood, serum or plasma sample taken from a human or animal subject, which comprises the steps of:

[0046] (i) contacting the sample with a first binding agent which binds specifically to said nuclear hormone receptor variant;

[0047] (ii) contacting the nucleosomes or sample with a second binding agent which binds to nucleosomes or a component thereof;

[0048] (iii) detecting or quantifying the binding of said second binding agent to nucleosomes or a component thereof; and

[0049] (iv) using the presence or degree of such binding as a measure of the presence of circulating cell free nucleosome bound nuclear hormone receptor variant adducts in the sample.

[0050] It will be appreciated that the method of this aspect of the invention is capable of detecting or predicting the progression of a hormone dependent cancer disease to a hormone therapy resistant cancer disease including castration resistant PCA and hormone therapy resistant breast cancer in the subject from whom the sample was taken.

[0051] It will also be appreciated that the method of this aspect of the invention is capable of predicting the efficacy of a drug in the subject from whom the sample was taken.

[0052] In one embodiment, the hormone receptor variant is a variant of the androgen receptor. In a yet further embodiment, the androgen receptor variant comprises a truncated or short form variant. In a yet further embodiment, the truncated or short form variant of the androgen receptor lacks the ligand binding domain (LBD) of the androgen receptor. In a yet further embodiment, the truncated variant of the androgen receptor comprises androgen receptor variant 7 (AR-V7).

[0053] In a further embodiment, the hormone receptor variant is a variant of the androgen receptor and the cancer is selected from castration resistant PCA.

[0054] In an alternative embodiment, the hormone receptor variant is a variant of the estrogen receptor. In a yet further embodiment, the estrogen receptor variant comprises a truncated or short form variant. In a yet further embodiment, the truncated or short form variant of the estrogen receptor lacks the ligand binding domain (LBD) of the estrogen receptor. In one embodiment the estrogen receptor variant is ERα-Δ5. In a further embodiment, the estrogen receptor variant is the ERβ splice variant ERβ2 (or ERβcx).

[0055] It will be appreciated that the method of this aspect of the invention is capable of detecting or predicting the progression of an estrogen dependent breast cancer disease to a hormone therapy resistant breast cancer disease in the subject from whom the sample was taken.

[0056] It will also be appreciated that the method of this aspect of the invention is capable of predicting the efficacy of a drug in the subject from whom the sample was taken.

[0057] In a further embodiment, the hormone receptor variant is a variant of the estrogen receptor and the cancer is hormone therapy resistant breast cancer.

[0058] In preferred embodiments of the invention a circulating cell free nucleosome bound short form or truncated ER variant adduct is measured in a blood sample taken from a patient. In another preferred embodiment a circulating cell free nucleosome bound short form or truncated AR variant adduct is measured. In both these preferred embodiments the adduct measurements are used to predict the efficacy of a drug for the treatment of the subject from whom the sample as taken and/or to predict or detect the progression of a hormone dependent cancer disease to a hormone therapy resistant cancer disease including castration resistant PCA and hormone therapy resistant breast cancer in the subject from whom the sample was taken.

[0059] In particularly preferred embodiments of the invention a circulating cell free nucleosome bound short form or truncated AR-V7 variant adduct is measured in a blood sample taken from a prostate cancer patient and the adduct measurements are used to detect the progression of a hormone dependent prostate cancer disease to a castration resistant prostate cancer.

[0060] In further particularly preferred embodiments of the invention a circulating cell free nucleosome bound short form or truncated AR-V7 variant adduct is measured in a blood sample taken from a prostate cancer patient and the adduct measurement is used to predict the efficacy of an androgen agonist or a CYP17 inhibitor for the treatment of the subject from whom the sample was taken. In preferred embodiments of the invention the androgen agonist is enzalutamide. In another preferred embodiment the CYP17 inhibitor is abiraterone.

[0061] In a particularly preferred embodiment of the invention, there is provided a method of detecting circulating cell free nucleosome bound AR-V7 adduct in a blood, serum or plasma sample taken from a human or animal subject which comprises the steps of:

[0062] (i) contacting the sample with a first binding agent which binds to nucleosomes or a component thereof;

[0063] (ii) contacting the nucleosomes or sample with a second binding agent which binds specifically to AR-V7;

[0064] (iii) detecting or quantifying the binding of said second binding agent to AR-V7; and

[0065] (iv) using the presence or degree of such binding as a measure of the presence of cell-free nucleosome bound AR-V7 adducts in the sample.

[0066] It will be appreciated that the method of this embodiment is capable of predicting or detecting the progression of a hormone dependent PCA disease to a castration resistant PCA disease in the subject from whom the sample was taken.

[0067] It will also be appreciated that the method of this embodiment is capable of predicting the efficacy of a drug in the subject from whom the sample was taken.

[0068] In an alternative particularly preferred embodiment of the invention, there is provided a method of detecting circulating cell free nucleosome bound AR-V7 adduct in a blood, serum or plasma sample taken from a human or animal subject which comprises the steps of:

[0069] (i) contacting the sample with a first binding agent which binds specifically to AR-V7;

[0070] (ii) contacting the nucleosomes or sample with a second binding agent which binds to nucleosomes or a component thereof;

[0071] (iii) detecting or quantifying the binding of said second binding agent to nucleosomes or a component thereof; and

[0072] (iv) using the presence or degree of such binding as a measure of the presence of cell-free nucleosome bound AR-V7 adducts in the sample.

[0073] It will be appreciated that the method of this embodiment is capable of predicting or detecting the progression of a hormone dependent PCA disease to a castration resistant PCA disease in the subject from whom the sample was taken.

[0074] It will also be appreciated that the method of this embodiment is capable of predicting the efficacy of a drug in the subject from whom the sample was taken.

[0075] In further embodiments of the invention multiple measurements of different circulating cell free nucleosome bound nuclear hormone receptor variant adducts are performed to ascertain the relative abundance of different variant adducts in a sample, as well as the relative abundance of circulating cell free nucleosome bound wild type nuclear hormone receptor adducts.

[0076] A binding agent which binds specifically to a nuclear hormone receptor variant, as used in the present invention, refers to a binding agent which binds strongly and preferentially to a particular variant or isoform, or a particular group of variants or isoforms, but binds less strongly to many, most, or all other variants or isoforms of the nuclear hormone receptor. It will be clear to those skilled in the art that the binding agent used need not bind uniquely to the variant concerned but that this variant should be preferentially bound over other variants commonly present in high levels in samples containing cell free nucleosome nuclear hormone variant adducts. Binding agents which bind specifically to short or truncated variant forms of nuclear hormone receptors are known in the art (Antonarakis).

[0077] The binding agents which bind to nucleosomes, as referred to herein, refer to any binding agent which selectively binds to any part of a cell free nucleosome-nuclear hormone receptor variant adduct or complex appropriate to the embodiment. In preferred embodiments, without limitation, the binding agents used are antibodies (including antibody fragments such as light chains or antibody variable regions), aptamers and naturally occurring binding proteins. The binding agent referred to in any of the previous embodiments which binds to nucleosomes or a component thereof, may bind to any component of a nucleosome including without limitation a histone, modified histone, histone variant, DNA or modified DNA component or it may bind to a composite epitope present in intact nucleosomes but not in individual disassociated nucleosome components. In a preferred embodiment of the invention the binding agent referred to in all of the previous embodiments which binds to nucleosomes or a component thereof, is an antibody directed to bind to the histone variant H3.1, H3.2 or H3t.

[0078] In further embodiments of the invention any of the methods of the previous embodiments are used but the sample to be tested is a cell free nucleosome preparation derived from cancer tissue removed from a patient (for example by biopsy or surgery). A solution of cell free nucleosomes may be prepared from the tissue by any method known in the art. In a preferred embodiment chromatin is extracted from the cancer tissue and the extract is digested using a nuclease to produce a solution of mononucleosomes. Commercial kits, for example the Pierce Chromatin Prep Module, are available for this purpose.

[0079] The invention will now be illustrated with reference to the following non-limiting examples.

EXAMPLE 1

[0080] Serum samples were taken from 9 treatment naïve subjects with newly diagnosed prostate cancer and 2 subjects who had been diagnosed within the previous six months. One of these two subjects was also treatment naïve and the other had advanced disease for which surgery was the selected treatment. Blood was drawn from this subject on the day of surgery. A horse serum sample was used as a negative control. The samples were tested for circulating cell free nucleosome bound AR-V7 by an ELISA method using a solid phase anti-histone capture antibody and a biotinylated anti-AR-V7 detection antibody as follows: Serum sample (10 μL/well) and assay buffer (50 μL/well), were added to microtitre plate wells and incubated overnight at 4° C. The serum and assay buffer mixture was decanted and the wells were washed three times with wash buffer. A solution of biotinylated anti-AR-V7 detection antibody was added (50 μL/well) and incubated for 90 minutes at room temperature with mild agitation. Excess detection antibody was decanted and the wells were again washed three times with wash buffer. A solution containing a streptavidin-horse radish peroxidase conjugate was added (50 μL/well) and incubated for 30 minutes at room temperature with mild agitation. Excess conjugate was decanted and the wells were again washed three times with wash buffer. A coloured substrate solution (100 μL/well 2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) was added and incubated for 20 minutes at room temperature with mild agitation. The optical density (OD) of the wells was measured at a wavelength of 405 nm using a standard microtitre plate reader. A low ELISA signal was observed for all of the 9 treatment naïve newly diagnosed prostate cancer cases as well as the treatment naïve subject that had been diagnosed a few months prior to sampling. These results indicated that no AR variant mediated disease progression, and no development of enzalutamide or abiraterone therapy resistance, had yet occurred in these treatment naïve subjects. However, a high signal was observed for the subject treated with surgery indicating that the disease in this subject had progressed to a castration resistant prostate cancer disease which is also enzalutamide and abiraterone therapy resistant. This data additionally demonstrates that circulating cell free nucleosome nuclear hormone variant adduct levels, measured using the method of the invention, correlate with clinical treatment decisions made by prostate cancer oncologists using current methods for detection of disease progression. The results are shown in FIG. 1.

EXAMPLE 2

[0081] Serum samples were taken from treatment naïve subjects with newly diagnosed breast cancer and from subjects with treated breast cancer. The samples were tested for circulating cell free nucleosome bound ERα-Δ5 by an ELISA method similar to that described in EXAMPLE 1 above but instead using a biotinylated anti-ERα-Δ5 detection antibody. Low ELISA signals were observed in the treatment naïve newly diagnosed breast cancer cases. The results indicated that there had been no development of ERα-Δ5 mediated hormone therapy resistance in these treatment naïve subjects. However, a high signal was observed for some of the subjects with treated breast cancer disease indicating elevated levels of circulating cell free nucleosome bound ERα-Δ5 variant adducts in the serum samples from those subjects and disease progression to a hormone therapy resistant breast cancer disease in those subjects.

EXAMPLE 3

[0082] Serum samples were taken from treatment naïve subjects with newly diagnosed breast cancer and from subjects with treated breast cancer. The samples were tested for circulating cell free nucleosome bound ERβ2 by an ELISA method similar to that described in EXAMPLE 1 above but instead using a biotinylated anti-ERβ2 detection antibody. Low ELISA signals were observed in the treatment naïve newly diagnosed breast cancer cases. The results indicated that there had been no development of ERβ2 mediated hormone therapy resistance in these treatment naïve subjects. However, a high signal was observed for some of the subjects with treated breast cancer disease indicating elevated levels of circulating cell free nucleosome bound ERβ2 variant adducts in the serum samples from those subjects and disease progression to a hormone therapy resistant breast cancer disease in those subjects.

EXAMPLE 4

[0083] A cancer tissue biopsy sample was taken from treatment naïve subjects with newly diagnosed prostate cancer and from subjects with treated prostate cancer. A solution of cell free mononucleosomes and/or oligonucleosomes was prepared from the tissue samples using the Pierce Chromatin Prep Module according to the manufacturer's instructions. The samples were tested for cell free nucleosome bound AR-V2 by an ELISA method similar to that described in EXAMPLE 1 above. Low ELISA signals were observed in the treatment naïve newly diagnosed prostate cancer cases. The results indicated that there had been no development of AR-V7 mediated hormone therapy resistance in these treatment naïve subjects. However, a high signal was observed for some of the subjects with treated prostate cancer disease indicating elevated levels of cell free nucleosome bound AR-V7 variant adducts in the samples from those subjects and disease progression to a hormone therapy resistant prostate cancer disease in those subjects which is also enzalutamide and abiraterone therapy resistant.

EXAMPLE 5

[0084] A cancer tissue biopsy sample was taken from treatment naïve subjects with newly diagnosed breast cancer and from subjects with treated breast cancer. A solution of cell free mononucleosomes and/or oligonucleosomes was prepared from the tissue samples using the Pierce Chromatin Prep Module according to the manufacturer's instructions. The samples were tested for cell free nucleosome bound ERα-Δ5 by an ELISA method similar to that described in EXAMPLE 2 above. Low ELISA signals were observed in the treatment naïve newly diagnosed breast cancer cases. The results indicated that there had been no development of ERα-Δ5 mediated hormone therapy resistance in these treatment naïve subjects. However, a high signal was observed for some of the subjects with treated breast cancer disease indicating elevated levels of cell free nucleosome bound ERα-Δ5 variant adducts in the samples from those subjects and disease progression to a hormone therapy resistant breast cancer disease in those subjects.

EXAMPLE 6

[0085] A cancer tissue biopsy sample was taken from treatment naïve subjects with newly diagnosed breast cancer and from subjects with treated breast cancer. A solution of cell free mononucleosomes and/or oligonucleosomes was prepared from the tissue samples using the Pierce Chromatin Prep Module according to the manufacturer's instructions. The samples were tested for cell free nucleosome bound ERβ2 by an ELISA method similar to that described in EXAMPLE 3 above. Low ELISA signals were observed in the treatment naïve newly diagnosed breast cancer cases. The results indicated that there had been no development of ERβ2 mediated hormone therapy resistance in these treatment naïve subjects. However, a high signal was observed for some of the subjects with treated breast cancer disease indicating elevated levels of cell free nucleosome bound ERβ2 variant adducts in the samples from those subjects and disease progression to a hormone therapy resistant breast cancer disease in those subjects.

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