Multifactorial assay for cancer detection

• US 20050069963A1
• Filed: 08/13/2004
• Published: 03/31/2005
• Est. Priority Date: 08/15/2003
• Status: Abandoned Application

• Current Assignee: University of Pittsburgh of The Commonwealth System of Higher Education
• Original Assignee: University of Pittsburgh of The Commonwealth System of Higher Education
• Inventors: Gorelik, Elieser, Lokshin, Anna E.
• Application Number:
  US10/918,727
• Publication Number:
  US 20050069963A1
• Time in Patent Office: Days
• US Class Current: 435/7.230
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: G01N 33/57449 of ovaries
• CPC Class Codes: G01N 33/588 with semiconductor nanocrystal label, e.g. quantum dots

Abstract

Provided are methods for the rapid detection of ovarian cancer. The methods employ a multiplex immunoassay to detect levels of two or more of the markers EGF, G-CSF, IL-6, IL-8, CA-125, VEGF, MCP-1, anti-IL6, anti-IL8, anti CA-125, anti-c-myc, anti-p53, anti-CEA, anti-CA 15-3, anti-MUC-1, anti-survivin, anti-bHCG, anti-osteopontin, anti-PDGF, anti-Her2/neu, anti-Akt1, anti-cytokeratin 19, cytokeratin 19, EGFR, CEA, kallikrein-8, M-CSF, FasL, ErbB2 and Her2/neu in a sample of the patient'"'"'s blood, where the presence of abnormal levels of two or more of the markers indicates the presence of ovarian cancer in the patient. An array also is provided to quantitate levels of these markers in a patient'"'"'s blood. Also provided is a method of predicting onset of clinical ovarian cancer comprising determining the change in concentration over time of two or more of anti-Her2/neu, anti-MUC-1, anti-c-myc, anti-p53, anti-CA-125, anti-CEA, anti-CA 72-4, anti-PDGFRα, IFNγ, IL-6, IL-10, TNFα, MIP-1α, MIP-1β, EGFR and Her2/neu in a patient'"'"'s blood.

Claims

1. A method of determining the presence of ovarian cancer in a patient, comprising determining levels of markers in a blood marker panel comprising two or more of EGF, G-CSF, IL-6, IL-8, CA-125, VEGF, MCP-1, anti-IL6, anti-IL8, anti CA-125, anti-c-myc, anti-p53, anti-CEA, anti-CA 15-3, anti-MUC-1, anti-survivin, anti-bHCG, anti-osteopontin, anti-PDGF, anti-Her2/neu, anti-Akt1, anti-cytokeratin 19, cytokeratin 19, EGFR, CEA, kallikrein-8, M-CSF, FasL, ErbB2 and Her2/neu in a sample of the patient'"'"'s blood, where the presence of two or more of the following conditions indicates the presence of ovarian cancer in the patient:

EGF_LO, G-CSF_HI, IL-6_HI, IL-8_HI, VEGF_HI, MCP-1_LO, anti-IL-6_HI, anti-IL-8_HI, anti-CA-125_HI, anti-c-myc_HI, anti-p53_HI, anti-CEA_HI, anti-CA 15-3_HI, anti-MUC-1_HI, anti-survivin_HI, anti-bHCG_HI, anti-osteopontin_HI, anti-Her2/neu_HI, anti-Akt1_HI, anti-cytokeratin 19_HI and anti-PDGF_HI, CA-125_HI, cytokeratin 19_HI, EGFR_LO, Her2/neu_LO, CEA_HI, FasL_HI, kallikrein-8_LO, ErbB2_LO and M-CSF_LO.

2. The method of claim 1, wherein the panel comprises 3 to 5 of EGF, G-CSF, IL-6, IL-8, CA-125, VEGF, MCP-1, anti-c-myc, anti-p53, anti-CEA, anti-CA 15-3, anti-MUC-1, anti-survivin, anti-bHCG, anti-osteopontin, anti-PDGF, cytokeratin 19, EGFR, CEA, kallikrein-8, M-CSF and Her2/neu.

3. The method of claim 1, wherein the panel comprises 4 of EGF, G-CSF, IL-6, IL-8, CA-125, VEGF, MCP-1, anti-c-myc, anti-p53, anti-CEA, anti-CA 15-3, anti-MUC-1, anti-survivin, anti-bHCG, anti-osteopontin, anti-PDGF, cytokeratin 19, EGFR, CEA, kallikrein-8, M-CSF and Her2/neu.

4. The method of claim 1, wherein the panel comprises 5 of EGF, G-CSF, IL-6, IL-8, CA-125, VEGF, MCP-1, anti-c-myc, anti-p53, anti-CEA, anti-CA 15-3, anti-MUC-1, anti-survivin, anti-bHCG, anti-osteopontin, anti-PDGF, cytokeratin 19, EGFR, CEA, kallikrein-8, M-CSF and Her2/neu.

5. The method of claim 1, wherein the panel comprises cytokeratin 19.

6. The method of claim 5, wherein the panel further comprises kallikrein-8.

7. The method of claim 6, wherein the panel further comprises CEA.

8. The method of claim 7, wherein the panel further comprises one or both of M-CSF and CA-125.

9. The method of claim 6, wherein the panel further comprises CA-125.

10. The method of claim 9, wherein the panel further comprises one or both of M-CSF and FasL.

11. The method of claim 1, wherein the panel comprises CA-125.

12. The method of claim 11, wherein the panel further comprises CK-19.

13. The method of claim 1, wherein the panel is one of:

a. CA-125, cytokeratin-19, Fas, M-CSF;

b. cytokeratin-19, CEA, Fas, EGFR, kallikrein-8;

c. CEA, Fas, M-CSF, EGFR, CA-125;

d. cytokeratin 19, kallikrein 8, CEA, CA 125, M-CSF;

e. kallikrein-8, EGFR, CA-125;

f. cytokeratin-19, CEA, CA-125, M-CSF, EGFR;

g. cytokeratin-19, kallikrein-8, CA-125, M-CSF, Fas;

h. cytokeratin-19, kallikrein-8, CEA, M-CSF;

i. cytokeratin-19, kallikrein-8, CEA, CA-125;

j. CA 125, cytokeratin 19, ErbB2; and

k. anti-CA 15-3, anti-IL-8, anti-survivin, anti-p53 and anti c-myc.

14. The method of claim 1, further comprising comparing the levels of the two or more markers in the patient'"'"'s blood with levels of the same markers in a control sample by applying a statistical method selected from the group consisting of linear regression analysis, classification tree analysis and heuristic naï

ve Bayes analysis.

15. The method of claim 14, wherein the statistical method is performed by a computer process.

16. The method of claim 14, wherein the statistical method is a classification tree analysis.

17. The method of claim 14, wherein the panel generates a sensitivity of at least about 80% and a specificity of at least about 80% using the statistical method.

18. The method of claim 17, wherein the panel generates a sensitivity of at least about 85% using the statistical method.

19. The method of claim 17, wherein the panel generates a specificity of at least about 85% using the statistical method.

20. The method of claim 17, wherein the panel generates a specificity of at least about 90% using the statistical method.

21. The method of claim 17, wherein the panel generates a specificity of at least about 99% using the statistical method.

22. The method of claim 1, wherein the panel comprises two or more of anti-c-myc, anti-p53, anti-CEA, anti-CA 15-3, anti-MUC-1, anti-survivin, anti-bHCG, anti-osteopontin and anti-PDGF.

23. The method of claim 1, wherein the panel comprises two or more of CA-125, cytokeratin 19, EGFR, kallikrein-8, M-CSF, FasL, CEA, and Her2/neu.

24. The method of claim 1, wherein the panel comprises Her2/neu, EGFR, CA-125 and cytokeratin 19.

25. The method of claim 1, wherein the panel comprises anti-CA15-3, IL-8, survivin, anti-p53 and anti-c-myc.

26. The method of claim 1, wherein the panel comprises anti-CA15-3, anti-CEA, anti-IL-6, anti-IL-8, anti-survivin, anti-p53, anti-bHGC and anti-c-myc.

27. The method of claim 1, wherein the blood sample is a serum sample.

28. The method of claim 1, comprising performing an immunoassay to determine the quantities of the two or more of EGF, G-CSF, IL-6, IL-8, CA-125, VEGF, MCP-1, anti-c-myc, anti-p53, anti-CEA, anti-CA 15-3, anti-MUC-1, anti-survivin, anti-bHCG, anti-osteopontin, anti-PDGF, anti-Her2/neu, cytokeratin 19, EGFR, CEA, kallikrein-8, M-CSF, FasL, ErbB2 and Her2/neu in the patient'"'"'s blood.

29. The method of claim 28, wherein the immunoassay utilizes an array comprising binding reagents types specific to EGF, G-CSF, IL-6, IL-8, VEGF and MCP-1, wherein each binding reagent type is attached independently to a one or more discrete locations on one or more surfaces of one or more substrates.

30. The method of claim 29, wherein the substrates are beads comprising an identifiable marker, wherein each binding reagent type is attached to a bead comprising a different identifiable marker than beads to which a different binding reagent type is attached.

31. The method of claim 30, wherein the identifiable marker comprises a fluorescent compound.

32. The method of claim 30, wherein the identifiable marker comprises a quantum dot.

33. An array comprising binding reagent types specific to any two or more of EGF, G-CSF, IL-6, IL-8, CA-125, VEGF, MCP-1, anti-IL6, anti-IL8, anti CA-125, anti-c-myc, anti-p53, anti-CEA, anti-CA 15-3, anti-MUC-1, anti-survivin, anti-bHCG, anti-osteopontin, anti-PDGF, anti-Her2/neu, anti-Akt1, anti-cytokeratin 19, cytokeratin 19, EGFR, CEA, kallikrein-8, M-CSF, FasL, ErbB2 and Her2/neu, wherein each binding reagent type is attached independently to one or more discrete locations on one or more surfaces of one or more substrates.

34. The array of claim 33, wherein the substrates are beads comprising an identifiable marker, wherein each binding reagent type is attached to a bead comprising a different identifiable marker than beads to which a different binding reagent is attached.

35. The array of claim 34, wherein the identifiable marker comprises a fluorescent compound.

36. The array of claim 34, wherein the identifiable marker comprises a quantum dot.

37. The array of claim 33, consisting essentially of binding reagent types independently specific to any two or more of EGF, G-CSF, IL-6, IL-8, CA-125, VEGF, MCP-1, anti-c-myc, anti-p53, anti-CEA, anti-CA 15-3, anti-MUC-1, anti-survivin, anti-bHCG, anti-osteopontin, anti-PDGF, cytokeratin 19, EGFR and Her2/neu, each binding reagent type is attached independently to one or more discrete locations on one or more surfaces of one or more substrates.

38. The array of claim 33, wherein the panel comprises 3 to 5 of EGF, G-CSF, IL-6, IL-8, CA-125, VEGF, MCP-1, anti-c-myc, anti-p53, anti-CEA, anti-CA 15-3, anti-MUC-1, anti-survivin, anti-bHCG, anti-osteopontin, anti-PDGF, cytokeratin 19, EGFR, CEA, kallikrein-8, M-CSF and Her2/neu.

39. The array of claim 33, wherein the panel comprises 4 of EGF, G-CSF, IL-6, IL-8, CA-125, VEGF, MCP-1, anti-c-myc, anti-p53, anti-CEA, anti-CA 15-3, anti-MUC-1, anti-survivin, anti-bHCG, anti-osteopontin, anti-PDGF, cytokeratin 19, EGFR, CEA, kallikrein-8, M-CSF and Her2/neu.

40. The array of claim 33, wherein the panel comprises 5 of EGF, G-CSF, IL-6, IL-8, CA-125, VEGF, MCP-1, anti-c-myc, anti-p53, anti-CEA, anti-CA 15-3, anti-MUC-1, anti-survivin, anti-bHCG, anti-osteopontin, anti-PDGF, cytokeratin 19, EGFR, CEA, kallikrein-8, M-CSF and Her2/neu.

41. The array of claim 33, wherein the panel comprises cytokeratin 19.

42. The array of claim 41, wherein the panel further comprises kallikrein-8.

43. The array of claim 42, wherein the panel further comprises CEA.

44. The array of claim 43, wherein the panel further comprises one or both of M-CSF and CA-125.

45. The array of claim 42, wherein the panel further comprises CA-125.

46. The array of claim 45, wherein the panel further comprises one or both of M-CSF and FasL.

47. The array of claim 33, wherein the panel comprises CA-125.

48. The array of claim 47, wherein the panel further comprises CK-19.

49. The array of claim 33, wherein the panel is one of:

a. CA-125, cytokeratin-19, Fas, M-CSF;

b. cytokeratin-19, CEA, Fas, EGFR, kallikrein-8;

c. CEA, Fas, M-CSF, EGFR, CA-125;

d. cytokeratin 19, kallikrein 8, CEA, CA 125, M-CSF;

e. kallikrein-8, EGFR, CA-125;

f. cytokeratin-19, CEA, CA-125, M-CSF, EGFR;

g. cytokeratin-19, kallikrein-8, CA-125, M-CSF, Fas;

h. cytokeratin-19, kallikrein-8, CEA, M-CSF;

i. cytokeratin-19, kallikrein-8, CEA, CA-125;

j. CA 125, cytokeratin 19, ErbB2; and

k. anti-CA 15-3, anti-IL-8, anti-survivin, anti-p53 and anti c-myc.

50. A method of determining the presence of ovarian cancer in a patient, comprising determining levels of at least one of anti-Her2/neu, anti-IL-8, anti-osteopontin, anti-VEGF and anti-PDGF in a sample of the patient'"'"'s blood, where the presence of one or more of the following conditions indicates the presence of ovarian cancer in the patient:

anti-Her2/neu_HI, anti-IL-8_HI, anti-osteopontin_HI, anti-VEGF_HI, and anti-PDGF_HI.

51. A method of determining the presence of ovarian cancer in a patient, comprising determining levels of markers in a blood marker panel comprising anti-CA 15-3, anti-IL-8, anti-survivin, anti-p53 and anti c-myc in a sample of the patient'"'"'s blood, wherein the presence of the following conditions indicates the presence of ovarian cancer in the patient:

anti-CA 15-3_HI, anti-IL-8_HI, anti-survivin_HI, anti-p53_HI, and anti-c-myc_HI.

52. The method of claim 51, wherein the blood marker panel further comprises anti-CEA, anti-IL-6, anti-EGF and anti-bHCG.

53. The method of claim 51, further comprising comparing the levels of the markers in the patient'"'"'s blood with levels of the same markers in a control sample by applying a statistical method selected from the group consisting of linear regression analysis, classification tree analysis and heuristic naï

ve Bayes analysis.

54. The method of claim 53, wherein the statistical method is performed by a computer process.

55. The method of claim 53, wherein the statistical method is a classification tree analysis.

56. The method of claim 51, wherein the panel generates a sensitivity of at least about 90% and a specificity of at least about 99% using the statistical method.

57. The method of claim 51, wherein the panel generates a sensitivity of at least about 90% and a specificity of at least about 99% using the statistical method.

58. The method of claim 51, comprising performing an immunoassay to determine the quantities of anti-CA 15-3_HI, anti-IL-8_HI, anti-survivin_HI, anti-p53_HI and anti-c-myc_HI in the patient'"'"'s blood.

59. The method of claim 58, wherein the immunoassay utilizes an array comprising binding reagents types specific to EGF, G-CSF, IL-6, IL-8, VEGF and MCP-1, wherein each binding reagent type is attached independently to a one or more discrete locations on one or more surfaces of one or more substrates.

60. The method of claim 59, wherein the substrates are beads comprising an identifiable marker, wherein each binding reagent type is attached to a bead comprising a different identifiable marker than beads to which a different binding reagent type is attached.

61. The method of claim 60, wherein the identifiable marker comprises a fluorescent compound.

62. The method of claim 60, wherein the identifiable marker comprises a quantum dot.

63. An array comprising binding reagent types specific to anti-CA 15-3, anti-IL-8, anti-survivin, anti-p53 and anti c-myc, wherein each binding reagent type is attached independently to one or more discrete locations on one or more surfaces of one or more substrates.

64. The array of claim 63, further comprising binding reagent types specific to anti-CEA, anti-IL-6, anti-EGF and anti-bHCG, wherein each binding reagent type is attached independently to one or more discrete locations on one or more surfaces of one or more substrates.

65. A method of predicting onset of clinical ovarian cancer comprising determining the change in concentration at two or more time points of two or more of anti-Her2/neu, anti-MUC-1, anti-c-myc, anti-p53, anti-CA-125, anti-CEA, anti-CA 72-4, anti-PDGFRa, IFNγ

, IL-6, IL-10, TNFα

, MIP-1α

, MIP-1β

, EGFR and Her2/neu in a patient'"'"'s blood, wherein an increase in the concentration of anti-Her2/neu, anti-MUC-1, anti-c-myc, anti-p53, anti-CA-125, anti-CEA, anti-CA 72-4, anti-PDGFRα

, IFNγ

, IL-6 and IL-10 in the patient'"'"'s blood between the two time points and a decrease in the concentration of TNFα

, MIP-1α

, MIP-1, EGFR and Her2/neu in the patient'"'"'s blood between the two time points are predictive of the onset of clinical ovarian cancer.

66. A method of determining the presence of ovarian cancer in a patient, comprising determining levels of markers in a blood marker panel comprising three or more of EGF, G-CSF, IL-6, IL-8, VEGF and MCP-1 in as sample of the patient'"'"'s blood, where the presence of three or more of the following conditions indicates the presence of ovarian cancer in the patient:

EGF_LO, G-CSF_HI, IL-6_HI, IL-8_HI, VEGF_HI or MCP-1_LO.

67. The method of claim 66, wherein EGF_LO means less than about 224 pg/mL EGF, G-CSF_HI means greater than about 22 pg/mL G-CSF, IL-6_HI means greater than about 8.8 pg/mL IL-6, IL-8_HI means greater than about 10.2 pg/mL IL-8, CA-125_HI, means greater than about 10 pg/mL CA-125, VEGF_HI means greater than about 91 pg/mL VEGF or MCP-1_LO means less than about 342 pg/mL MCP-1.

68. The method of claim 66, comprising performing an immunoassay to determine the quantities of EGF, G-CSF, IL-6, IL-8, VEGF or MCP-1 in the patient'"'"'s blood.

69. The method of claim 68, wherein the immunoassay utilizes an array comprising binding reagents types specific to EGF, G-CSF, IL-6, IL-8, VEGF and MCP-1, wherein each binding reagent type is attached independently to a one or more discrete locations on one or more surfaces of one or more substrates.

70. The method of claim 69, wherein the substrates are beads comprising an identifiable marker, wherein each binding reagent type is attached to a bead comprising a different identifiable marker than beads to which a different binding reagent type is attached.

71. The method of claim 70, wherein the identifiable marker comprises a fluorescent compound.

72. The method of claim 70, wherein the identifiable marker comprises a quantum dot.

73. An array comprising binding reagent types specific to any three or more of EGF, G-CSF, IL-6, IL-8, VEGF, CA-125 and MCP-1, wherein each binding reagent type is attached independently to one or more discrete locations on one or more surfaces of one or more substrates.

74. The array of claim 73, wherein the substrates are beads comprising an identifiable marker, wherein each binding reagent type is attached to a bead comprising a different identifiable marker than beads to which a different binding reagent type is attached.

75. The array of claim 74, wherein the identifiable marker comprises a fluorescent compound.

76. The array of claim 74, wherein the identifiable marker comprises a quantum dot.

77. The array of claim 73, further comprising a binding reagent type specific to CA-125 attached independently to one or more discrete locations, as compared to the other binding reagents, on one or more surfaces of the one or more substrates.