Epigenetic modification of the loci for CAMTA1 and/or FOXP3 as a marker for cancer treatment
The present invention relates to a method, in particular an in vitro method, for pan-cancer diagnostics, comprising identifying the amount and/or proportion of stable regulatory T cells in a patient suspected of having cancer through analyzing the methylation status of at least one CpG position in the gene foxp3 and/or the gene camta1 or orthologous or paralogous genes thereof, wherein an increased amount and/or proportion of stable regulatory T cells in said patient is indicative for an unspecific cancerous disease. In a second aspect thereof, the present invention relates to a method for diagnosing the survival of a cancer patient, comprising identifying the amount and/or proportion of stable regulatory T cells in said cancer patient through analyzing the methylation status of at least one CpG position in the gene foxp3 and/or the gene camta1 or orthologous or paralogous genes thereof, wherein a demethylation in the gene foxp3 and/or the gene camta1 or orthologous or paralogous genes thereof, is indicative of a stable regulatory T cell, and wherein an increased amount and/or proportion of stable regulatory T cells in said cancer patient is indicative for a shorter survival for said cancer patient. Furthermore, the present invention relates to an improved treatment of cancers based on the inventive methods, and a kit for performing the above methods as well as respective uses.
1 . A method for pan-cancer diagnostics, comprising identifying the amount and/or proportion of stable regulatory T cells in a patient suspected of having cancer through analysing the methylation status of at least one CpG position in the gene foxp3 and/or camta1 or orthologous or paralogous genes thereof, wherein an increased amount and/or proportion of stable regulatory T cells in said patient is indicative for an unspecific cancerous disease.
2 . The method according to claim 1 , further comprising the analysis of at least one cancer- and/or tissue-specific marker.
3 . The method according to claim 1 , wherein said stable regulatory T cell is a CD25 + CD4 + regulatory T cell.
4 . The method according to claim 1 , further comprising the step of analysing the packaging of the chromatin structure in the region of the gene foxp3 and/or camta1 or orthologous or paralogous genes thereof, wherein an open chromatin structure in the region of the gene foxp3 and/or camta1 or orthologous or paralogous genes thereof is indicative of a stable regulatory T cell.
5 . The method according to claim 1 , wherein said analysis of the methylation status comprises analysing the methylation status of at least one CpG position in the 5′ region upstream from the transcription start, promoter regions, introns, and/or exon/intron borders of the gene foxp3 and/or camta1.
6 . The method according to claim 1 , wherein said analysis of the methylation status of foxp3 comprises amplification with at least one of the primer pairs selected from SEQ ID NO: 1 and 2; SEQ ID NO: 3 and 4, and orthologous or paralogous primer pairs thereof.
7 . The method according to claim 1 , wherein said analysis of the methylation status comprises a method selected from methylation specific enzymatic digests, bisulphite sequencing, MSP, HeavyMethyl, MethyLight, Ms-SNuPE or other methods relying on a detection of amplified DNA.
8 . The method according to claim 1 , wherein the analysis of the packaging of the chromatin structure comprises chromatin immunoprecipitation.
9 . The method according to claim 8 , wherein said immunoprecipitation comprises using antibodies against acylated histones
10 . The method according to claim 9 , wherein said histones are H3 and/or H4.
11 . The method according to claim 1 , wherein said patient is a human.
12 . The method according to claim 1 , wherein said cancer is selected from solid tumors, breast cancer, ovarian cancer, prostate cancer, and lung cancer.
13 . The method according to claim 1 , wherein the amount and/or proportion of stable regulatory T cells is identified in a blood or tissue sample obtained from said human.
14 . The method according to claim 1 , wherein the amount and/or proportion of stable regulatory T cells is identified through a comparison with the methylation status of at least one CpG position in the gene foxp3 and/or camta1 or orthologous or paralogous genes thereof in samples selected from all cells as present in said sample, all T-cells as present in said sample, and the number of stable regulatory T cells in a healthy patient.
15 . The method according to claim 1 , wherein an amount of regulatory T-cells corresponds to a demethylation of the CpG positions as analyzed to at least about 80%.
16 . The method according to claim 1 , wherein an amount of regulatory T-cells corresponds to a demethylation of the CpG positions as analyzed to at least about 90%.
17 . The method according to claim 1 , wherein an amount of regulatory T-cells corresponds to a demethylation of the CpG positions as analyzed to at least about 95%.
18 . The method according to claim 1 , wherein a demethylation and/or open chromatin structure in the region of the gene foxp3 and/or camta1 or orthologous or paralogous genes thereof corresponds to at least 10-fold of DNA relative to input for Me 3 K4.
19 . The method according to claim 1 , further comprising the step of providing a treatment for said cancer patient wherein said treatment reduces the amount and/or proportion of stable regulatory T cells in said cancer patient.
20 . The method according to claim 19 , wherein said treatment is selected from providing chemical and/or biological substances that selectively kill regulatory T-cells in said patient, or a treatment that reduces the expression of the gene foxp3 and/or camta1 or inhibits the biological activity of FoxP3 and/or Camta1 in said regulatory T-cells in said patient.
21 . The method according to claim 19 , wherein said substance is selected from an antibody that is selective for regulatory T-cells, such as an anti-CD25-antibody, a cytotoxic substance that is selective for regulatory T-cells, such as denileukin diftitox (Ontak®), antisense nucleic acids against the expression of the gene foxp3, and methylating agents.
22 . The method according to claim 20 , wherein said substance is selected from an antibody that is selective for regulatory T-cells, such as an anti-CD25-antibody, a cytotoxic substance that is selective for regulatory T-cells, such as denileukin diftitox (Ontak®), antisense nucleic acids against the expression of the gene foxp3, and methylating agents.
23 . The method according to claim 19 , further comprising measuring and/or monitoring the amount of said regulatory T cells in response to chemical and/or biological substance.
24 . The method according to claim 20 , further comprising measuring and/or monitoring the amount of said regulatory T cells in response to chemical and/or biological substance.
25 . The method according to claim 21 , further comprising measuring and/or monitoring the amount of said regulatory T cells in response to chemical and/or biological substance.
26 . A kit for diagnosing the survival of a cancer patient, comprising materials for performing a method according to claim 1 .
27 . A method for diagnosing the survival of a cancer patient, comprising identifying the amount and/or proportion of stable regulatory T cells in said cancer patient through analysing the methylation status of at least one CpG position in the gene foxp3 and/or camta1 or orthologous or paralogous genes thereof,
wherein a demethylation in the gene foxp3 and/or camta1 or orthologous or paralogous genes thereof, is indicative of a stable regulatory T cell, and
wherein an increased amount and/or proportion of stable regulatory T cells in said cancer patient is indicative for a shorter survival for said cancer patient.
28 . The method according to claim 27 , wherein said stable regulatory T cell is a CD25 + CD4 + regulatory T cell.
29 . The method according to claim 27 , further comprising the step of analysing the packaging of the chromatin structure in the region of the gene foxp3 and/or camta1 or orthologous or paralogous genes thereof, wherein an open chromatin structure in the region of the gene foxp3 and/or camta1 or orthologous or paralogous genes thereof is indicative of a stable regulatory T cell.
30 . The method according to claim 27 , wherein said analysis of the methylation status comprises analysing the methylation status of at least one CpG position in the 5′ region upstream from the transcription start, promoter regions, introns, and/or exon/intron borders of the gene foxp3 and/or camta1.
31 . The method according to claim 27 , wherein said analysis of the methylation status of foxp3 comprises amplification with at least one of the primer pairs selected from SEQ ID NO: 1 and 2; SEQ ID NO: 3 and 4, and orthologous or paralogous primer pairs thereof.
32 . The method according to claim 27 , wherein said analysis of the methylation status comprises a method selected from methylation specific enzymatic digests, bisulphite sequencing, MSP, HeavyMethyl, MethyLight, Ms-SNuPE or other methods relying on a detection of amplified DNA.
33 . The method according to claim 27 , wherein the analysis of the packaging of the chromatin structure comprises chromatin immunoprecipitation.
34 . The method according to claim 33 , wherein said immunoprecipitation comprises using antibodies against acylated histones
35 . The method according to claim 34 , wherein said histones are H3 and/or H4.
36 . The method according to claim 27 , wherein said patient is a human.
37 . The method according to claim 27 wherein said cancer is selected from solid tumors, breast cancer, ovarian cancer, prostate cancer, and lung cancer.
38 . The method according to claim 27 , wherein the amount and/or proportion of stable regulatory T cells is identified in a blood or tissue sample obtained from said human.
39 . The method according to claim 27 , wherein the amount and/or proportion of stable regulatory T cells is identified through a comparison with the methylation status of at least one CpG position in the gene foxp3 and/or camta1 or orthologous or paralogous genes thereof in samples selected from all cells as present in said sample, all T-cells as present in said sample, and the number of stable regulatory T cells in a healthy patient.
40 . The method according to claim 27 , wherein an amount of regulatory T-cells corresponds to a demethylation of the CpG positions as analyzed to at least about 80%.
41 . The method according to claim 27 , wherein an amount of regulatory T-cells corresponds to a demethylation of the CpG positions as analyzed to at least about 90%.
42 . The method according to claim 27 , wherein an amount of regulatory T-cells corresponds to a demethylation of the CpG positions as analyzed to at least about 95%.
43 . The method according to claim 27 , wherein a demethylation and/or open chromatin structure in the region of the gene foxp3 and/or camta1 or orthologous or paralogous genes thereof corresponds to at least 10-fold of DNA relative to input for Me 3 K4.
44 . The method according to claim 27 , further comprising the step of providing a treatment for said cancer patient wherein said treatment reduces the amount and/or proportion of stable regulatory T cells in said cancer patient.
45 . The method according to claim 44 , wherein said treatment is selected from providing chemical and/or biological substances that selectively kill regulatory T-cells in said patient, or a treatment that reduces the expression of the gene foxp3 and/or camta1 or inhibits the biological activity of FoxP3 and/or Camta1 in said regulatory T-cells in said patient.
46 . The method according to claim 44 , wherein said substance is selected from an antibody that is selective for regulatory T-cells, such as an anti-CD25-antibody, a cytotoxic substance that is selective for regulatory T-cells, such as denileukin diftitox (Ontak®), antisense nucleic acids against the expression of the gene foxp3, and methylating agents.
47 . The method according to claim 45 , wherein said substance is selected from an antibody that is selective for regulatory T-cells, such as an anti-CD25-antibody, a cytotoxic substance that is selective for regulatory T-cells, such as denileukin diftitox (Ontak®), antisense nucleic acids against the expression of the gene foxp3, and methylating agents.
48 . The method according to claim 44 , further comprising measuring and/or monitoring the amount of said regulatory T cells in response to chemical and/or biological substance.
49 . The method according to claim 45 , further comprising measuring and/or monitoring the amount of said regulatory T cells in response to chemical and/or biological substance.
50 . The method according to claim 46 , further comprising measuring and/or monitoring the amount of said regulatory T cells in response to chemical and/or biological substance.
51 . A kit for diagnosing the survival of a cancer patient, comprising materials for performing a method according to claim 27.