Detection of loss of heterozygosity in tumor and serum of melanoma patients

• US 6,465,177 B1
• Filed: 10/26/1998
• Issued: 10/15/2002
• Est. Priority Date: 10/26/1998
• Status: Expired due to Term

• Current Assignee: John Wayne Cancer Institute
• Original Assignee: John Wayne Cancer Institute
• Inventors: Hoon, Dave S. B.
• Primary Examiner(s): Myers, Carla J.
• Application Number:
  US09/179,704
• Time in Patent Office: 1,450 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.31, 536/24.33
• US Class Current: 435/6.14
• 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 2600/156 Polymorphic or mutational markers

Abstract

A method is provided for assessing allelic losses on specific chromosomal regions in melanoma patents. The method relies on the evidence that free DNA may be released in the plasma/serum of cancer patients allowing the detection of DNA with LOH in the plasma/serum of cancer patients by analysis for microsatellite markers. The amount of and specific allelic loss allows a prognosis to be made regarding tumor diagnosis and progression, tumor metastasis, tumor recurrence, and mortality.

Claims

1. A method of detecting melanoma in a patient comprising:

a) performing a nucleic acid amplification reaction in a sample derived from acellular blood plasma or serum from the patient, wherein the amplification reaction is designed to amplify a loss of heterozygosity (LOH) marker from a locus selected from the group consisting of D1S214, D1S228, D3S1293, D6S264, D9S157, D9S161, S10S212, D10S216, and D11S925; and

b) detecting the presence or absence of the LOH marker;

wherein the LOH of the marker is indicative of melanoma in the patient.

2. The method of claim 1 wherein the sample is acellular blood plasma.

3. The method of claim 1 wherein the LOH marker is a CA-repeat microsatellite marker.

4. The method of claim 1 wherein the LOH marker is from the D3S1293 locus.

5. The method of claim 1, wherein step a) further comprises amplifying nucleic acid from more than one of the LOH markers, and step b) further comprises detecting the presence or absence of more than one of the LOH markers.

6. The method of claim 5, wherein the LOH markers are from the D9S157 and D3S1293 loci.

7. The method of claim 5 wherein the LOH markers are D9S157 and D1S228.

8. The method of claim 5 wherein the LOU markers are from the D11S925 and D3S1293 loci.

9. The method of claim 1 wherein the LOH markers are detected with a primer labeled with a chromophore.

41. The method of claim 1 wherein the sample is serum.

42. The method of claim 1 wherein the LOH marker is from the D1S214 locus.

43. The method of claim 1 wherein the LOH marker is from the D1S228 locus.

44. The method of claim 1 wherein the LOH marker is from the D6S264 locus.

45. The method of claim 1 wherein the LOH marker is from the D9S157 locus.

46. The method of claim 1 wherein the LOH marker is from the D9S161 locus.

47. The method of claim 1 wherein the LOH marker is from the D10S212 locus.

48. The method of claim 1 wherein the LOH marker is from the D10S216 locus.

49. The method of claim 1 wherein the LOH marker is from the D11S925 locus.

10. A method of monitoring treatment of melanoma in a patient comprising:

a) performing a nucleic acid amplification reaction in a sample derived from acellular blood plasma or serum from the patient, wherein the amplification reaction is designed to amplify a loss of heterozygosity (LOH) marker from a locus selected from the group consisting of D1S214, D1S228, D3S1293, D6S264, D9S157, D9S161, S10S212, D10S216, and D11S925; and

b) detecting the presence or absence of the LOH marker;

wherein the LOH of the marker is indicative of the efficacy of the treatment in the patient.

11. The method of claim 10 wherein the sample is acellular blood plasma.

12. The method of claim 10 wherein the LOH marker is a CA-repeat microsatellite.

13. The method of claim 10 wherein the LOH marker is from the D3S1293 locus.

14. The method of claim 10, wherein step a) further comprises amplifying nucleic acid from more than one of the LOH markers, and step b) further comprises detecting the presence or absence of more than one of the LOH markers.

15. The method of claim 14 wherein the LOH markers are from D9S157 and D3S1293 loci.

16. The method of claim 14 wherein the LOH markers are D9S157 and D1S228.

17. The method of claim 14 wherein the LOH markers are from the D11S925 and D3S1293 loci.

18. The method of claim 10 wherein the LOH markers are detected with a primer labeled with a chromophore.

50. The method of claim 10 wherein the sample is serum.

51. The method of claim 10 wherein the LOH marker is from the D1S214 locus.

52. The method of claim 10 wherein the LOH marker is from the D1S228 locus.

53. The method of claim 10 wherein the LOH marker is from the D6S264 locus.

54. The method of claim 10 wherein the LOH marker is from the D9S157 locus.

55. The method of claim 10 wherein the LOH marker is from the D9S161 locus.

56. The method of claim 10 wherein the LOH marker is from the D10S212 locus.

57. The method of claim 10 wherein the LOH marker is from the D10S216 locus.

58. The method of claim 10 wherein the LOH marker is from the D11S925 locus.

19. A method of detecting subclinical melanoma in a patient comprising:

a) performing a nucleic acid amplification reaction in a sample derived from acellular blood plasma or serum from the patient, wherein the amplification reaction is designed to amplify a loss of heterozygosity (LOH) marker from a locus selected from the group consisting of D1S214, D1S228, D3S1293, D6S264, D9S157, D9S161, S10S212, D10S216, and D11S925; and

b) detecting the presence or absence of the LOH marker;

wherein the LOH of the marker is indicative of subclinical melanoma in the patient.

20. The method of claim 19 wherein the sample is acellular blood plasma.

21. The method of claim 19 wherein the LOH marker is a CA-repeat microsatellite marker.

22. The method of claim 19 wherein the LOH marker is from the D3S1293 locus.

23. The method of claim 19, wherein step a) further comprises amplifying nucleic acid from more than one of the LOH markers, and step b) further comprises detecting the presence or absence of more than one of the LOH markers.

24. The method of claim 23 wherein the LOH markers are from D9S157 and D3S1293 loci.

25. The method of claim 23 wherein the LOH markers are D9S157 and D1S228.

26. The method of claim 23 wherein the LOH markers are from the D11S925 and D3S1293 loci.

27. The method of claim 19 wherein the LOH markers are detected with a primer are labeled with a chromophore.

59. The method of claim 19 wherein the sample is serum.

60. The method of claim 19 wherein the LOH marker is from the D1S214 locus.

61. The method of claim 19 wherein the LOH marker is from the D1S228 locus.

62. The method of claim 19 wherein the LOH marker is from the D6S264 locus.

63. The method of claim 19 wherein the LOH marker is from the D9S157 locus.

64. The method of claim 19 wherein the LOH marker is from the D9S161 locus.

65. The method of claim 19 wherein the LOH marker is from the D10S212 locus.

66. The method of claim 19 wherein the LOH marker is from the D10S216 locus.

67. The method of claim 19 wherein the LOH marker is from the D11S925 locus.

28. A method of prognosing melanoma in a melanoma patient comprising:

a) performing a nucleic acid amplification reaction in a sample derived from acellular blood plasma or serum from the patient, wherein the amplification reaction is designed to amplify a loss of heterozygosity (LOH) marker from a locus selected from the group consisting of D1S214, D1S228, D3S1293, D6S264, D9S157, D9S161, S10S212, D10S216, and D11S925; and

b) detecting the presence or absence of the LOH marker;

wherein an incease in the LOH of the marker is indicative of a poorer prognosis in the patient.

29. The method of claim 28 wherein the biological fluid is acellular blood plasma.

30. The method of claim 28 wherein the LOH marker is a CA-repeat microsatellite marker.

31. The method of claim 28 wherein the LOH marker is from the D3S1293 locus.

32. The method of claim 28, wherein step a) further comprises amplifying nucleic acid from more than one of the LOH markers, and step b) further comprises detecting the presence or absence of more than one LOH of the markers.

33. The method of claim 32 wherein the LOH markers are from the D9S157 and D3S1293 loci.

34. The method of claim 32 wherein the LOH markers are D9S157 and D1S228.

35. The method of claim 32 wherein the LOH markers are from the D1S925 and D3S1293 loci.

36. The method of claim 28 wherein the LOH markers are detected with a primer labeled with a chromophore.

68. The method of claim 28 wherein the sample is serum.

69. The method of claim 28 wherein the LOH marker is from the D1S214 locus.

70. The method of claim 28 wherein the LOH marker is from the D1S228 locus.

71. The method of claim 28 wherein the LOH marker is from the D6S264 locus.

72. The method of claim 28 wherein the LOH marker is from the D9S157 locus.

73. The method of claim 28 wherein the LOH marker is from the D9S161 locus.

74. The method of claim 28 wherein the LOH marker is from the D10S212 locus.

75. The method of claim 28 wherein the LOH marker is from the D10S216 locus.

76. The method of claim 28 wherein the LOH marker is from the D11S925 locus.

37. A method of detecting melanoma in a patient comprising:

a) performing a nucleic acid amplification reaction in a sample derived from acellular blood plasma or serum from the patient, wherein the amplification reaction is designed to amplify a loss of heterozygosity (LOH) marker from a locus selected from the group consisting of D1S214, D1S228, D3S1293, D6S264, D9S157, D9S161, S10S212, D10S216, and D11S925; and

b) detecting the presence or absence of two or more of the LOH markers;

wherein the LOH of the markers is indicative of melanoma in the patient.

77. The method of claim 37 wherein the sample is acellular blood plasma.

78. The method of claim 37 wherein the sample is serum.

79. The method of claim 37 wherein one of the two markers is D3S1293.

80. The method of claim 37 wherein one of the two markers is D1S228.

81. The method of claim 37 wherein the LOH markers are from the D9S157 and D3S1293 loci.

82. The method of claim 37 wherein the LOH markers are D9S157 and D1S228.

83. The method of claim 37 wherein the LOH markers are from the D11S925 and D3S1293 loci.

84. The method of claim 37 wherein the LOH markers are detected with a primer labeled with a chromophore.

38. A method of monitoring treatment of melanoma in a patient comprising:

a) performing a nucleic acid amplification reaction in a sample derived from acellular blood plasma or serum from the patient, wherein the amplification reaction is designed to amplify a loss of heterozygosity (LOH) marker from a locus selected from the group consisting of D1S214, D1S228, D3S1293, D6S264, D9S157, D9S161, S10S212, D10S216, and D11S295; and

b) detecting the presence or absence of two or more of the LOH markers;

wherein the LOH of the markers is indicative of the efficacy of the treatment in the patient.

85. The method of claim 38 wherein the sample is acellular blood plasma.

86. The method of claim 38 wherein the sample is serum.

87. The method of claim 38 wherein one of the two markers is D3S1293.

88. The method of claim 38 wherein one of the two markers is D1S228.

89. The method of claim 38 wherein the LOH markers are from the D9S157 and D3S1293 loci.

90. The method of claim 38 wherein the LOH markers are D9S157 and D1S228.

91. The method of claim 38 wherein the LOH markers arc from the D11S925 and D3S1293 loci.

92. The method of claim 38 wherein the LOH markers are detected with a primer labeled with a chromophore.

39. A method of detecting subclinical melanoma in a patient comprising:

a) performing a nucleic acid amplification reaction in a sample derived from acellular blood plasma or serum from the patient, wherein the amplification reaction is designed to amplify a loss of heterozygosity (LOH) marker from a locus selected from the group consisting of D1S214, D1S228, D3S1293, D6S264, D9S157, D9S161, S10S212, D10S216, and D11S925; and

b) detecting the presence or absence of two or more of the LOH markers;

wherein the LOH of the markers is indicative of subclinical melanoma in the patient.

93. The method of claim 39 wherein the sample is acellular blood plasma.

94. The method of claim 39 wherein the sample is serum.

95. The method of claim 39 wherein one of the two markers is D3S1293.

96. The method of claim 39 wherein one of the two markers is D1S228.

97. The method of claim 39 wherein the LOH markers are from the D9S157 and D3S1293 loci.

98. The method of claim 39 wherein the LOH markers are D9S157 and D1S228.

99. The method of claim 39 wherein the LOH markers are from the D11S925 and D3S1293 loci.

100. The method of claim 39 wherein the LOH markers are detected with a primer labeled with a chromophore.

40. A method of prognosing melanoma in a melanoma patient comprising:

a) performing a nucleic acid amplification reaction in a sample derived from acellular blood plasma or serum from the patient, wherein the amplification reaction is designed to amplify a loss of heterozygosity (LOH) marker from a locus selected from the group consisting of D1S214, D1S228, D3S1293, D6S264, D9S157, D9S161, S10S212, D10S216, and D11S925; and

b) detecting the presence or absence of two or more of the LOH markers;

wherein an increase in the LOH of the markers is indicative of a poorer prognosis in the patient.

101. The method of claim 40 wherein the sample is acellular blood plasma.

102. The method of claim 40 wherein the sample is serum.

103. The method of claim 40 wherein one of the two markers is D3S1293.

104. The method of claim 40 wherein one of the two markers is D1S228.

105. The method of claim 40 wherein the LOH markers are from the D9S157 and D3S1293 loci.

106. The method of claim 40 wherein the LOH markers are D9S157 and D1S228.

107. The method of claim 40 wherein the LOH markers are from the D11S925 and D3S1293 loci.

108. The method of claim 40 wherein the LOH markers are detected with a primer labeled with a chromophore.