Methods and compositions for the detection of cervical disease

• US 20050260566A1
• Filed: 03/23/2005
• Published: 11/24/2005
• Est. Priority Date: 03/24/2004
• Status: Active Grant

• Current Assignee: Tripath Imaging Incorporated (Becton, Dickinson & Co)
• Original Assignee: Tripath Imaging Incorporated (Becton, Dickinson & Co)
• Inventors: Malinowski, Douglas P., Fischer, Timothy J., Taylor, Adriann J., Parker, Margaret R.
• Granted Patent: US 7,157,233 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C07K 16/3069 Reproductive system, e.g. ovaria, uterus, testes, prostate
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/025 Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
• CPC Class Codes: G01N 33/57411 of cervix
• CPC Class Codes: G01N 33/6875 Nucleoproteins

Abstract

Methods and compositions for identifying high-grade cervical disease in a patient sample are provided. The methods of the invention comprise detecting overexpression of at least one biomarker in a body sample, wherein the biomarker is selectively overexpressed in high-grade cervical disease. In particular claims, the body sample is a cervical smear or monolayer of cervical cells. The biomarkers of the invention include genes and proteins that are involved in cell cycle regulation, signal transduction, and DNA replication and transcription. In particular claims, the biomarker is an S-phase gene. In some aspects of the invention, overexpression of a biomarker of interest is detected at the protein level using biomarker-specific antibodies or at the nucleic acid level using nucleic acid hybridization techniques. Kits for practicing the methods of the invention are further provided.

Claims

1. A method for diagnosing high-grade cervical disease in a patient, said method comprising detecting overexpression of at least one nuclear biomarker in a body sample from the patient, wherein the detection of overexpression of said nuclear biomarker specifically identifies samples that are indicative of high-grade cervical disease.

2. The method of claim 1, wherein the detection of overexpression of said biomarker distinguishes samples that are indicative of high-grade cervical disease from samples that are indicative of benign proliferation, early-stage HPV infection, or mild dysplasia.

3. The method of claim 1, wherein said method for diagnosing high-grade cervical disease is independent of morphological analysis or determination of HPV infection status.

4. The method of claim 1, wherein detecting overexpression comprises using at least one antibody to detect biomarker protein expression.

5. The method of claim 4, wherein the method comprises performing immunocytochemistry.

6. The method of claim 1, wherein overexpression of the nuclear biomarker is detected at the nucleic acid level.

7. The method of claim 6, wherein detecting overexpression comprises performing quantitative RT-PCR.

8. The method of claim 1, wherein the sample comprises cervical cells in suspension in a liquid-based preparation.

9. The method of claim 1, wherein the sample comprises a cervical tissue sample.

10. The method of claim 1, wherein the sensitivity of said method for diagnosing high-grade cervical disease is at least 90%.

11. The method of claim 10, wherein the positive predicative value of the method for diagnosing high-grade cervical disease is at least 40%.

12. The method of claim 1, wherein the specificity of said method for diagnosing high-grade cervical disease is at least 85%.

13. The method of claim 1, wherein the method is performed in response to the patient having an abnormal Pap smear result.

14. The method of claim 1, wherein the method is performed as a primary screen for high-grade cervical disease in a general patient population.

15. The method of claim 1, wherein the method is performed in conjunction with morphological analysis.

16. A method for diagnosing high-grade cervical disease in a patient, said method comprising detecting overexpression of at least two biomarkers in a body sample from the patient, wherein the detection of overexpression of said biomarkers specifically identifies samples that are indicative of high-grade cervical disease.

17. A method for diagnosing high-grade cervical disease in a patient, said method comprising:

a) obtaining a body sample from said patient;

b) contacting said sample with at least one antibody, wherein said antibody specifically binds to a nuclear biomarker protein that is selectively overexpressed in high-grade cervical disease; and

,

c) detecting binding of said antibody to said nuclear biomarker protein.

18. The method of claim 17, wherein the method comprises performing immunocytochemistry.

19. The method of claim 18, wherein the method is performed manually.

20. The method of claim 18, wherein the method is performed in an automated manner.

21. The method of claim 17, wherein the sample comprises cervical cells.

22. The method of claim 21, wherein the sample comprises a monolayer of cervical cells.

23. The method of claim 17, wherein the sensitivity of said method for diagnosing high-grade cervical disease is at least 90%.

24. The method of claim 17, wherein the specificity of said method for diagnosing high-grade cervical disease is at least 85%.

25. The method of claim 17, wherein the method is performed in response to the patient having an abnormal Pap smear result.

26. The method of claim 17, wherein the method is performed as a primary screen for high-grade cervical disease in a general patient population.

27. The method of claim 17, wherein said nuclear biomarker is a gene involved in HPV-induced cell cycle disruption and activation of S-phase induction.

28. The method of claim 27, wherein said nuclear biomarker is selected from the group consisting of MCM2, MCM6, MCM7, p14ARF, p21^waf1, Topo2A, and Cyclin E.

29. The method of claim 28, wherein the nuclear biomarker is an MCM protein.

30. The method of claim 17, wherein said biomarker is a gene induced by the E2f transcription factor.

31. The method of claim 17, wherein said biomarker is a gene induced by the Sp-1 transcription factor.

32. The method of claim 17, wherein said biomarker is an S-phase gene.

33. The method of claim 17, wherein said biomarker is involved in cell cycle regulation.

34. The method of claim 17, wherein said biomarker is involved in DNA replication and transcription.

35. The method of claim 17 further comprising Papanicolaou (Pap) staining of the sample.

36. A method for diagnosing high-grade cervical disease in a patient, said method comprising:

a) obtaining a body sample from said patient;

b) contacting said sample with at least two antibodies, wherein each of said antibodies specifically binds to a distinct biomarker protein that is selectively overexpressed in high-grade cervical disease; and

,

c) detecting binding of said antibodies to said biomarker proteins.

37. The method of claim 36, wherein said biomarkers are selected from the group consisting of MCM2, MCM6, MCM7, p14ARF, p21^waf1, Topo2A, and Cyclin E.

38. The method of claim 37, wherein at least one of said biomarkers is an MCM protein.

39. The method of claim 37, wherein at least three antibodies are contacted with the sample, wherein a first and a second antibody specifically bind to MCM2, and wherein a third antibody specifically binds to Topo2A.

40. The method of claim 36, wherein said antibodies are contacted with said sample sequentially as individual antibody reagents.

41. The method of claim 37, wherein said antibodies are contacted with said sample simultaneously as an antibody cocktail.

42. The method of claim 36, wherein each of the antibodies is contacted with a separate microscope slide comprising a portion of the sample.

43. A method for diagnosing high-grade cervical disease in a patient, said method comprising:

a) obtaining a body sample from said patient;

b) contacting said sample with at least one antibody, wherein said antibody specifically binds to a biomarker protein that is selectively overexpressed in high-grade cervical disease;

c) detecting binding of said antibody to said biomarker protein; and

,

d) staining the sample with Papanicolaou (Pap) stain.

44. The method of claim 43, wherein the method is performed in response to the patient having an abnormal Pap smear result.

45. The method of claim 43, wherein the method is performed as a primary screen for high-grade cervical disease in a general patient population.

46. A method for diagnosing high-grade cervical disease, said method comprising detecting overexpression of at least one biomarker in a body sample from the patient, wherein overexpression of the biomarker is indicative of aberrant S-phase induction.

47. A method for diagnosing high-grade cervical disease, said method comprising detecting overexpression of at least one biomarker in a body sample from the patient, wherein overexpression of the biomarker is indicative of active transcription of oncogenic HPV E6 and HPV E7.

48. A method for diagnosing high-grade cervical disease, said method comprising detecting overexpression of at least one biomarker in a body sample from the patient, wherein overexpression of the biomarker is indicative of overexpression of oncogenic HPV E6 and HPV E7.

49. A kit comprising at least one antibody, wherein said antibody specifically binds to a nuclear biomarker protein that is selectively overexpressed in high-grade cervical disease.

50. The kit of claim 49, wherein said nuclear biomarker is selected from the group consisting of MCM2, MCM6, MCM7, p14ARF, p21^waf1, Topo2A, and Cyclin E.

51. The kit of claim 50, wherein the nuclear biomarker is an MCM protein.

52. The kit of claim 49, wherein said kit further comprises a peroxidase blocking reagent, a protein blocking reagent, chemicals for the detection of antibody binding to said biomarker proteins, a counterstain, a bluing agent, and instructions for use.

53. The kit of claim 52, wherein said chemicals for the detection of antibody binding comprise a chromogen and a secondary antibody conjugated to a labeled polymer, wherein the chromogen comprises 3′

,3′

-diaminobenzidine, and wherein the labeled polymer comprises horseradish peroxidase conjugated to a dextran polymer.

54. The kit of claim 52, wherein said counterstain comprises hematoxylin.

55. The kit of claim 52, wherein said bluing agent comprises a solution comprising Tris buffered saline, pH 7.4, Tween-20, and sodium azide.

56. The kit of claim 52 further comprising a positive control sample.

57. The kit of claim 56, wherein said positive control sample comprises SiHa cells.

58. The kit of claim 49 further comprising reagents for Pap staining.

59. The kit of claim 58, wherein the reagents for Pap staining comprise EA50 and Orange G.

67. A method of using the kit of claim 52 for the diagnosis of high-grade cervical disease.

60. A kit comprising at least two antibodies, wherein each of said antibodies specifically binds to a distinct biomarker protein that is selectively overexpressed in high-grade cervical disease.

61. The kit of claim 60, wherein said at least two antibodies are provided as separate reagents.

62. The kit of claim 60, wherein said at least two antibodies are provided as a cocktail.

63. The kit of claim 60, wherein at least one of said antibodies specifically binds to an MCM protein.

64. The kit of claim 60, wherein the kit comprises at least three antibodies, wherein a first and a second antibody in the kit specifically bind to MCM2, and wherein a third antibody specifically binds to Topo2A.

65. The kit of claim 64 further comprising a peroxidase blocking reagent, a protein blocking reagent, chemicals for the detection of antibody binding to said biomarker proteins, a counterstain, a bluing agent, and instructions for use.

68. A method of using the kit of claim 65 in the diagnosis of high-grade cervical disease.

66. A kit comprising at least one antibody and reagents for Pap staining, wherein the antibody specifically binds to a biomarker protein that is selectively overexpressed in high-grade cervical disease, and wherein the reagents for Pap staining comprise EA50 and Orange G.

69. A method for diagnosing high-grade cervical disease in a patient comprising detecting overexpression of at least one nucleic acid molecule that encodes a nuclear biomarker that is selectively overexpressed in high-grade cervical disease, wherein the nuclear biomarker is selected from the group consisting of MCM2, MCM6, MCM7, p14ARF, p21^waf1, Topo2A, and Cyclin E, the method comprising:

(a) obtaining a body sample from the patient;

(b) isolating nucleic acid material from the sample;

(c) mixing said nucleic acid material with at least one pair of oligonucleotide primers specific for the nuclear biomarker and a thermostable DNA polymerase under conditions that are suitable for amplification by polymerase chain reaction (PCR);

d) performing PCR; and

,

e) detecting PCR amplification products.

70. The method of claim 69, wherein performing PCR comprises performing RT-PCR.

71. The method of claim 70, wherein the sample comprises cervical cells in suspension in a liquid-based preparation.

72. The method of claim 70, wherein the sample comprises a cervical tissue sample.

73. The method of claim 69, wherein the nuclear biomarker is an MCM protein.

74. The method of claim 69, wherein the method is performed in response to the patient having an abnormal Pap smear result.

75. The method of claim 69, wherein the method is performed as a primary screen for high-grade cervical disease in a general patient population.

76. A method for diagnosing high-grade cervical disease, the method comprising detecting overexpression of at least two biomarkers in a body sample from the patient, wherein the detection of overexpression of the biomarkers specifically identifies samples that are indicative of high-grade cervical disease, and wherein overexpression of the biomarkers is detected at the nucleic acid level.

77. The method of claim 76, wherein detecting overexpression comprises performing RT-PCR.

78. A kit comprising at least one pair of oligonucleotide primers specific for a nuclear biomarker that is selectively overexpressed in high-grade cervical disease.

79. A kit comprising at least two pairs of oligonucleotide primers, wherein each pair of oligonucleotide primers is specific for a biomarker that is selectively overexpressed in high-grade cervical disease.

80. A method of using the kit of claim 79 for diagnosing high-grade cervical disease in a patient.

81. A slide pretreatment buffer comprising 0.05% to 5% sodium laureth-13-carboxylate.

82. The slide pretreatment buffer of claim 81 comprising 0.1% to 1% sodium laureth-13-carboxylate.

83. The slide pretreatment buffer of claim 82 comprising 0.5% sodium laureth-13-carboxylate.

84. A method for preparing a sample for use in an immunocytochemistry assay comprising applying a composition that comprises 0.05 to 5% sodium laureth-13-carboxylate to the sample.

85. The method of claim 84, wherein the composition comprises 0.1% to 1% sodium laureth-13-carboxylate.

86. The method of claim 85, wherein the composition comprises 0.5% sodium laureth-13-carboxylate.

87. The method of claim 84, wherein the composition is applied to the sample for less than one hour.

88. The method of claim 84 further comprising heating the sample.