Gene Methylation as a Biomarker in Sputum

20080241842

11/995150

July 10, 2006

The present invention provides for a method to monitor the health of a subject. The method includes obtaining a test sample from the patient. A first probe specific for a CpG promoter region of a biomarker selected from p16, MGMT, PAX-α, PAX5-β, RASSF1A, HLHP, GATA4, GATA5, SFRP1, LAMC2, IGFBP3, H-cadherin, BETA 3, HLHP, and DAPK is provided to the sample. The probe contacts the test sample. The DNA of interest from the test sample is isolated. A second stage probe specific for a second CpG promoter region of a biomarker selected from p16, MGMT, PAX-α, PAX5-β, RASSF1A, HLHP, GATA4, GATA5, SFRP1, LAMC2, IGFBP3, H-cadherin, BETA 3, HLHP, and DAPK is provided to the sample to form a second stage PCR product. The DNA is analyzed for hypermethylation of the promoter region for at least one of p16, MGMT, PAX-α, PAX5-β, RASSF1A, HLHP, GATA4, GATA5, SFRP1, LAMC2, IGFBP3, H-cadherin, BETA 3, HLHP, and DAPK. Hypermethylation of the promoter region of at least one of p16, MGMT, PAX-α, PAX5-β, RASSF1A, HLHP, GATA4, GATA5, SFRP1, LAMC2, IGFBP3, H-cadherin, BETA 3, HLHP, and DAPK is an indication that the subject is at increased risk of developing cancer for example, non small cell lung cancer.

Belinsky, Steven A. (Albuquerque, NM, US)

LOVELACE RESPIRATORY RESEARCH INSTITUTE (Albuquerque, NM, US)

1. A method to monitor the health of a subject comprising: obtaining a test sample selected from tissue plasma, ejaculate, cerebrospinal fluid, serum, mammary duct fluid, urine, and fecal stool and sputum containing DNA from the subject; subjecting the DNA to bisulfite modification; expanding the number of copies of at least one gene selected from p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAB-2, DAL-1, RASSF2A, GATA5, and GATA4 by using a polymerase chain reaction with a first primer set to amplify a portion of the gene where the promoter methylation resides, thereby generating an amplification product; using an aliquot of the amplification product generated by the first polymerase chain reaction in a second, methylation-specific, polymerase chain reaction with a second set of primers having a temperature of annealing that exceeds the melting temperature; and detecting the presence of inactivation of the at least one specific gene selected from p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAB-2, DAL-1, RASSF2A, GATA5, and GATA4 to monitor the health of the subject.

2. The method of claim 1 further comprising applying an algorithm to the presence of inactivation of the at least one specific gene to produce an index value correlating to the health of the subject.

3. The method of claim 2 wherein the index value correlating to the health of the subject produced by the algorithm is further informed by clinical indicators selected from one or more of the following: the presence of chronic airway obstruction, family history of lung cancer, exposure to asbestos or number of cigarettes smoked over the subject's life time.

4. The method of claim 1 wherein the step of expanding the number of copies of at least one gene comprises amplifying the p16 gene.

5. The method of claim 4 wherein the step of amplifying the p16 gene comprises amplifying a 274 base pair fragment with a primer set comprising: [table included]

6. The method of claim 1 wherein the step of expanding the number of copies of at least one gene comprises amplifying the MGMT gene.

7. The method of claim 6 wherein the step of amplifying the MGMT gene comprises amplifying a 251 base pair fragment with a primer set comprising: [table included]

8. The method of claim 1 wherein the step of expanding the number of copies of at least one gene comprises amplifying the DAPK gene.

9. The method of claim 8 wherein the step of amplifying the DAPK gene comprises amplifying a 236 base pair fragment with a primer set comprising: [table included]

10. The method of claim 1 wherein the step of expanding the number of copies of at least one gene comprises amplifying the PAX5-alpha gene.

11. The method of claim 10 wherein the step of amplifying the PAX5-alpha gene comprises amplifying a 388 base pair fragment with a primer set comprising: [table included]

12. The method of claim 1 wherein the step of expanding the number of copies of at least one gene comprises amplifying the PAX5-beta gene.

13. The method of claim 12 wherein the step of amplifying the PAX5-beta gene comprises amplifying a 318 base pair fragment with a primer set comprising: [table included]

14. The method of claim 1 wherein the step of expanding the number of copies of at least one gene comprises amplifying the RASSF1A gene.

15. The method of claim 14 wherein the step of amplifying the RASSF1A gene comprises amplifying a 260 base pair fragment with a primer set comprising: [table included]

16. The method of claim 1 wherein the step of expanding the number of copies of at least one gene comprises amplifying the RASSF2A gene.

17. The method of claim 16 wherein the step of amplifying the RASSF2A gene comprises amplifying a 288 base pair fragment with a primer set comprising: [table included]

18. The method of claim 1 wherein the step of expanding the number of copies of at least one gene comprises amplifying the DAB2 gene.

19. The method of claim 18 wherein the step of amplifying the DAB2 gene comprises amplifying a 318 base pair fragment with a primer set comprising: [table included]

20. The method of claim 1 wherein the step of expanding the number of copies of at least one gene comprises amplifying the DAL-1 gene.

21. The method of claim 20 wherein the step of amplifying the DAL-1 gene comprises amplifying a 247 base pair fragment with a primer set comprising: [table included]

22. The method of claim 1 wherein the step of expanding the number of copies of at least one gene comprises amplifying the GATA5 gene.

23. The method of claim 22 wherein the step of amplifying the GATA5 gene comprises amplifying a 348 base pair fragment with a primer set comprising: [table included]

24. The method of claim 1 wherein the step of expanding the number of copies of at least one gene comprises amplifying the GATA4 gene.

25. The method of claim 24 wherein the step of amplifying the GATA4 gene comprises amplifying a 244 base pair fragment with a primer set comprising: [table included]

26. The method of claim 5 wherein a portion of a 274 base pair fragment of the p16 gene is interrogated with a methylation-specific primer set comprising: [table included]

27. The method of claim 7 wherein a portion of a 251 base pair fragment of the MGMT gene is interrogated with a methylation-specific primer set comprising: [table included]

28. The method of claim 9 wherein a portion of a 236 base pair fragment of the DAPK gene is interrogated with a methylation-specific primer set comprising: [table included]

29. The method of claim 11 wherein a portion of a 388 base pair fragment of the PAX5-alpha gene is interrogated with a methylation-specific primer set comprising: [table included]

30. The method of claim 13 wherein a portion of a 318 base pair fragment of the PAX5-beta gene is interrogated with a methylation-specific primer set comprising: [table included]

31. The method of claim 15 wherein a portion of a 260 base pair fragment of the RASSF1A gene is interrogated with a methylation-specific primer set comprising: [table included]

32. The method of claim 17 wherein a portion of a 288 base pair fragment of the RASSF2A gene is interrogated with a methylation-specific primer set comprising: [table included]

33. The method of claim 19 wherein a portion of a 318 base pair fragment of the DAB2 gene is interrogated with a methylation-specific primer set comprising: [table included]

34. The method of claim 21 wherein a portion of a 247 base pair fragment of the DAL-1 gene is interrogated with a methylation-specific primer set comprising: [table included]

35. The method of claim 23 wherein a portion of a 348 base pair fragment of the GATA5 gene is interrogated with a methylation-specific primer set comprising: [table included]

36. The method of claim 25 wherein a portion of a 244 base pair fragment of the GATA4 gene is interrogated with a methylation-specific primer set comprising: [table included]

37. A method of monitoring the efficacy of therapy for treating cancer in a subject comprising: obtaining a first test sample selected from tissue plasma, ejaculate, cerebrospinal fluid, serum, mammary duct fluid, urine, and fecal stool and sputum from the patient at a first time point; providing a primer specific for a CpG promoter region of a gene selected from p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAB-2, DAL-1, RASSF2A, GATA5, and GATA4; contacting the primer with the first test sample; analyzing DNA from the test sample for hypermethylation of the promoter region for at least one of p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAB-2, DAL-1, RASSF2A, and GATA5; providing the therapy for treating cancer to the subject; obtaining a second test sample selected from tissue plasma, ejaculate, cerebrospinal fluid, serum, mammary duct fluid, urine, and fecal stool and sputum from the patient at a second time point; providing a primer specific for the CpG promoter region of at least one of p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAB-2, DAL-1, RASSF2A, GATA5, and GATA4; contacting the primer with the second test sample; and analyzing DNA from the test sample for hypermethylation of the promoter region for at least one of p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAB-2, DAL-1, RASSF2A, GATA4, and GATA5.

38. The method of claim 37 wherein the cancer being treated is lung cancer.

39. The method of claim 37 wherein a decrease in the hypermethylation of the promoter region of at least one of p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAB-2, DAL-1, RASSF2A, GATA5, and GATA4 is an indication of the efficacy of the therapy.

40. A kit useful for the diagnosis, prognosis, monitoring and therapeutic treatment of a disease, comprising: a primer that is specific for detecting methylation of a CpG site in the promoter region of genes selected from p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAB-2, DAL-1, RASSF2A, GATA5, and GATA4; and a bisulfite reagent.

41. A computer program product for enabling a computer to produce and index value correlating to the health of a subject from one or more clinical indicators, the computer program product comprising: software instructions for enabling the computer to perform predetermined operations, and a computer readable medium embodying the software instructions; the pre-determined operations comprising: identifying from a sample selected from tissue plasma, ejaculate, cerebrospinal fluid, serum, mammary duct fluid, urine, and fecal stool and sputum, the methylation state of a promoter of one or more genes from a panel comprising p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAB-2, DAL-1, RASSF2A, GATA5, and GATA4 to determine a number of genes having hypermethylation of the promoter as a clinical indicator; applying an algorithm to the number of genes having hypermethylation of the promoter to produce an index value correlating to the health of a subject.

42. The method of claim 41 wherein clinical indicators further include one or more of the following: the presence of chronic airway obstruction, family history of lung cancer, exposure to asbestos or number of cigarettes smoked over the person's life time to produce an index value correlating to the health of the patient.

43. A computer system adopted to correlate the health of a subject from clinical indicators, the computer system comprising a processor and a memory including software instructions adapted to enable the computer system to perform operations comprising: identifying from a sample selected from tissue plasma, ejaculate, cerebrospinal fluid, serum, mammary duct fluid, urine, and fecal stool and sputum the methylation state of a promoter of one or more genes from a panel comprising p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAB-2, DAL-1, RASSF2A, GATA5, and GATA4 to determine the number of genes having hypermethylation of the promoter; applying an algorithm to the number of genes having hypermethylation of the promoter to produce an index value correlating to the health of a subject.

44. The method of claim 43 wherein clinical indicators further include on the following: the presence of chronic airway obstruction, family history of lung cancer, exposure to asbestos or number of cigarettes smoked over the person's life time to produce an index value correlating to the health of the patient.

435/6

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edspap.20080241842