Methods and compositions for the diagnosis of cancer

• US 20060160069A1
• Filed: 10/07/2004
• Published: 07/20/2006
• Est. Priority Date: 10/07/2003
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Dako Denmark A/S (Agilent Technologies, Inc.)
• Inventors: Lindberg, Martin, Wang, Zunde, Lin, Jone, Chau, Miu Fun, Lusk, Jason, Yoast, Sienna, Bisgaard-Franzen, Kirsten, Rasmussen, Ole Feldballe
• Granted Patent: US 8,221,970 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 33/574 for cancer

Abstract

In one embodiment, the invention relates to a method of detecting cervical cancer, and other types of cancer, using a combination of at least three genomic clones, or fragments thereof, of high risk Human Papilloma Virus. For example, the invention relates to a composition comprising at least three full length genomic clones, or fragments thereof, of high risk Human Papilloma Viruses.

Claims

1. A composition comprising a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 16 (SEQ ID NO:

1), or a fragment thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

1;

a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 18, (SEQ ID NO;

     2), or a fragment thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

2; and

a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 51 (SEQ ID NO;

     4), or a fragment thereof or a nucleic acid molecule substantially identical to SEQ ID NO;

4.

2. The composition according to claim 1, further comprising at least one of:

a) a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 11 (SEQ ID NO;

     3), or fragments thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

3;

b) a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 58 (SEQ ID NO;

     5), or fragments thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

5;

c) a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 66 (SEQ ID NO;

     7), or a fragment thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

7;

d) a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 73 (SEQ ID NO;

     8), or a fragment thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

8;

e) a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 70 (SEQ ID NO;

     9), or a fragment thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

9;

f) a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 56 (SEQ ID NO;

     6), or a fragment thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

6.

3. The composition according to claim 1 wherein the nucleic acid is DNA, RNA, LNA or PNA.

4. The composition according to claim 1, further comprising at least one additional molecule that binds to at least one protein marker for cancer or that binds to at least one additionalnucleic acid encoding a protein marker for cancer.

5. The composition according to claim 4, wherein the at least one protein marker for cancer is chosen from p16^INK4a, P63, c-Myc, Cox-2, HIF-1α

, a telomerase markers, a telomerase associated protein;

an extra-cellular matrix marker;

a proliferation marker;

a cell cycle marker;

or an apoptosis marker.

6. The composition according to claim 4, wherein the molecule that binds to the at least one protein marker for cancer is an antibody.

7. The composition according to claim 4, wherein the molecule that binds to the at least one additional nucleic acid encoding a protein marker for cancer is a nucleic acid.

8. The composition according to claim 7, wherein the nucleic acid encodes a protein chosen from p16^INK4a, P63, c-Myc, Cox-2, HIF-1α

, a telomerase markers, a telomerase associated protein;

an extra-cellular matrix marker;

a proliferation marker;

a cell cycle marker; and

an apoptosis marker.

9. The composition according to claim 2, wherein the nucleic acid molecules are labeled with a detectable substance.

10. The composition according to claim 9, wherein the detectable substance is covalently linked to the nucleic acid molecule.

11. The composition according to claim 9, wherein the detectable substance is linked to an intervening molecule.

12. The composition according to claim 11, wherein the intervening molecule is biotin.

13. The composition according to claim 11, wherein the intervening molecule is streptavidin.

14. The composition according to claim 9, wherein the detectable substance is chosen from a fluorescent material, a chemiluminescent material, a bioluminescent material, an enzyme, and a radioactive material.

15. The composition according to claim 1, further comprising a molecule, which hybridizes to at least one low risk HPV type.

16. The composition according to claim 15, wherein the molecule that hybridizes to said low risk HPV type is a nucleic acid molecule.

17. The composition according to claim 16, wherein the nucleic acid molecule is a nucleic acid molecule substantially identical to a full length genomic clone of a low risk HPV type, or fragment thereof.

18. The composition according to claim 16, wherein the low risk HPV type is HPV 11 or HPV 70.

19. The composition according to claim 16, wherein the nucleic acid molecule is comprised of DNA, RNA, LNA or PNA.

20. The composition according to claim 16, wherein the nucleic acid which hybridizes to the low risk HPV types is not labeled with a detectable substance.

21. The composition according to claim 16, wherein the nucleic acid which hybridizes to the low risk HPV types is labeled with a detectable substance that is different from the detectable substance used to label the nucleic acid molecule which hybridizes to the HR-HPV.

22. The composition according to claim 21, wherein the detectable substance is chosen from a fluorescent material, a chemiluminescent material, a bioluminescent material, an enzyme and a radioactive material.

23. The composition according to claim 21, wherein the detectable substance is covalently linked to the nucleic acid which hybridizes to the low risk HPV types.

24. The composition according to claim 21, wherein the detectable substance is linked to an intervening molecule.

25. The composition according to claim 24, wherein the intervening molecule is biotin.

26. The composition according to claim 24, wherein the intervening molecule is streptavidin.

27. The composition according to claim 1, wherein the cancer markers detected are cervical cancer markers.

28. The composition according to claim 1, wherein the cancer markers detected are colon cancer markers.

29. The composition according to claim 1, wherein the cancer markers detected are anal cancer markers.

30. The composition according to claim 1, wherein the cancer markers detected are markers for HPV-related cancers.

31. The composition according to claim 1, further comprising a hybridization buffer comprising a low molecular weight dextran sulfate.

32. The composition according to claim 31, wherein the low molecular weight dextran sulfate has a molecular weight ranging from 25,000-75,000 daltons.

33. The composition according to claim 31, wherein the low molecular weight dextran sulfate has a molecular weight ranging from 35,000-50,000 daltons.

34. The composition according to claim 31, wherein the low molecular weight dextran sulfate is present in an amount ranging from 5-15 wt./vol. %.

35. The composition according to claim 31, wherein the low molecular weight dextran sulfate is present in an amount ranging from 10 wt./vol. %.

64. A method of detecting markers for cancer in a subject comprising

a) obtaining a sample comprising cells from the subject,

b) placing the sample on a solid support,

c) detecting HR-HPV in the sample from step b) by contacting the sample with the composition of claim 1,

d) performing a PAP stain on the same sample from step b) wherein the presence of HR-HPV and an abnormal PAP smear indicates the presence of cervical cancer or the risk of developing cancer.

65. The method according to claim 64, wherein the sample is a cervical cancer sample and the cells comprised in the sample cervical cells.

66. The method according to claim 64, wherein the sample is a colon cancer sample and the cells comprised in the sample colon cells.

67. The method according to claim 64, wherein the sample is a HPV-related cancer sample, and the cells comprised in the sample HPV-infected cells.

68. The method according to claim 64, wherein the detecting the HR-HPV in the sample is done by in situ hybridization of a nucleic acid probe specific to HR-HPV.

69. The method of claim 68, wherein the nucleic acid probe specific to HR-HPV detects at least 14 HR-HPV types.

71. A kit for detecting at least one marker associated with cancer comprising the following reagent:

a) the composition according to claim 1.

72. The kit according to claim 71, further comprising at least one of the following;

a) reagents for performing PAP stain

b) at least one reagent chosen from an antibody or nucleic acid probe, wherein the reagent is capable of detecting at least one of the following protein markers;

c-Myc, Cox-2, HIF-1α

, Histone H3, a telomerase marker, a telomerase associated protein;

an extra-cellular matrix marker;

a proliferation marker;

a cell cycle marker; and

an apoptosis marker.

c) at least one reagent for the detection of other cellular markers associated with the progression of cancer or risk of progression of cancer.

d) at least one reagent for the detection of antibodies or probes,

f) at least one sample for carrying out a positive control reaction for at least one of the above markers,

i) at least one container, and

j) instructions for performing an assay to detect cancer markers in a sample.

36. A method for detecting markers for cancer in a subject comprising

a) obtaining a sample comprising cells from the subject;

b) contacting the sample with a composition comprising

a first nucleic acid molecule chosen from a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 16 (SEQ ID NO;

     1), or a fragment thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

1;

a second nucleic acid molecule chosen from a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 18, (SEQ ID NO;

     2), or a fragment thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

2; and

a third nucleic acid molecule chosen from a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 51(SEQ ID NO;

     4), or a fragment thereof or a nucleic acid molecule substantially identical to SEQ ID NO;

4,

under conditions such that at least one nucleic acid molecule chosen from the first, second and third nucleic acid molecules hybridizes to a human papilloma virus (HPV) nucleic acid contained in the sample thereby forming at least one nucleic acid—

HPV hybridization complex, and

c) detecting said nucleic acid—

HPV hybridization complex, wherein hybridization of the at least one nucleic acid molecule to the sample indicates the presence of cancer or the risk of developing cancer.

37. The method according to claim 36, further comprising contacting the sample with a probe comprising a nucleic acid molecule, comprising at least one of:

a) a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 56 (SEQ ID NO;

     6), or a fragment thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

6;

b) a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 58 (SEQ ID NO;

     5), or a fragment thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

5;

c) a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 66 (SEQ ID NO;

     7), or a fragment thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

7;

d) a nucleic acid molecule substantially identical to a full length genomic clone of human papilloma virus 73 (SEQ ID NO;

     8), or a fragment thereof, or a nucleic acid molecule substantially identical to SEQ ID NO;

8,

under conditions such that the probe hybridizes to a human papilloma virus nucleic acid contained in the sample;

and wherein hybridization of the probe to the sample indicates the presence of cervical cancer or the risk of developing cervical cancer.

38. The method according to claim 36, wherein the conditions such that the at least one nucleic acid molecule hybridizes to the human papilloma virus nucleic acid contained in the sample are high stringency conditions.

39. The method according to claim 36, wherein the conditions such that the at least one nucleic acid molecule hybridizes to the human papilloma virus nucleic acid contained in the sample are moderate stringency conditions.

40. The method according to claim 36, wherein the conditions such that the at least one nucleic acid molecule hybridizes to the human papilloma virus nucleic acid contained in the sample are low stringency conditions.

41. The method according to claim 36, wherein the conditions such that the at least one nucleic acid molecule hybridizes to the human papilloma virus nucleic acid contained in the sample include a hybridization buffer comprising 50% formamide, 0.3 M NaCl, and at least one non-specific DNA molecule.

42. The method according to claim 36, further comprising contacting the sample with at least one molecule that hybridizes to at least one low risk HPV type.

43. The method according to claim 42, wherein the at least one molecule is a nucleic acid molecule.

44. The method according to claim 43, wherein the nucleic acid molecule is a full length genomic clone of a low risk HPV type, or fragment thereof;

or a molecule which is substantially identical to a full length genomic clone of a low risk HPV type, or fragment thereof.

45. The method according to claim 44, wherein the low risk HPV type is HPV11 or HPV70.

46. The method according to claim 42, wherein the nucleic acid molecule is comprised of DNA, RNA, LNA or PNA.

47. The method according to claim 36, further comprising contacting the sample with at least one other agent that can detect cancer.

48. The method according to claim 47, wherein the cancer is cervical cancer.

49. The method according to claim 47, wherein the cancer is colon cancer.

50. The method according to claim 47, wherein the cancer is a HPV-related cancer.

51. The method according to claim 47, wherein the at least one other agent is a stain used in a PAP smear.

52. The method according to claim 51, wherein the stain is Papanicolaou. stain.

53. The method according to claim 47, wherein the at least one other agent is an agent which binds to a protein marker for cancer or a nucleic acid encoding a protein marker for cancer.

54. The method according to claim 53, wherein the agent that binds a protein marker is an antibody.

55. The method according to claim 47, wherein the agent is an agent that binds to a protein marker for cervical cancer or a nucleic acid encoding a protein marker for cervical cancer.

56. The method according to claim 47, wherein the agent is an agent that binds to a protein marker for HPV-related cancer or a nucleic acid encoding a protein marker for HPV-related cancer.

57. The method according to claim 47, wherein the protein marker for cancer is chosen from p16^INK4a, P63, c-Myc, Cox-2, HIF-1α

, a telomerase markers, a telomerase associated protein;

an extra-cellular matrix marker;

a proliferation marker;

a cell cycle marker; and

an apoptosis marker.

58. The method according to claim 36, wherein the sample is a cytology sample comprising cells.

59. The method according to claims 36, wherein the sample is a histology sample comprising cells.

60. The method according to claim 36, wherein the sample is provided on a solid support.

61. The method according to claim 60, wherein the solid support is chosen from a microscope slide, a bead, a micro-array and a chip.

62. The method according to claims 36, wherein the sample is placed in solution and the cells comprised in the sample are lysed before the sample is applied to the solid support.

63. The method according to claims 36, wherein the sample is screened for cancer by flow cytometry.

70. The methods according to claim 36, wherein the method is automated.

92. The method of claim 70 further comprising

a) creating a digital image of a sample

b) saving the digital image to a digital media,

c) analyzing the digital image using an algorithm which detects and quantifies molecules used to detect markers which indicate the presence of cancer or the risk of developing cancer, and

d) creating a report which contains information relating to the identification and quantification of markers for cancer.

93. The method according to claim 92, wherein the saved digital image is a high resolution image, and wherein step c) analyzing the digital image comprises

a) reducing the image resolution by sub sampling the high resolution digital image to create a second low resolution digital image;

b) analyzing the low resolution digital image to locate potential objects of interest within the low resolution image;

c) mapping potential objects of interest back onto the high resolution image;

d) analyzing each mapped object within the high resolution image to compile a list of descriptive statistics that describe each object; and

e) comparing the descriptive statistics for each object to an object definition to determine the likelihood that the described object is a nuclei.

94. The method according to claim 93, wherein analysing the low resolution digital image comprises

segmenting the low resolution digital image in HSI color space based on staining and counter staining colors;

detecting the edge of the cells to separate cells from background.

95. The method according to claim 94 further comprising smoothing the image.

96. The method according to claim 93 wherein analyzing each mapped object within the high resolution image comprises

classifying the objects (cells) based on chromatic, geometric, topological and biological information;

collecting statistics by using the original image to gather chromatic, geometric and topological information.

97. The method according to claim 96, wherein some objects are cells further comprising

filtering the cells based on chromatic, geometric, topological and biological information by comparing the object descriptive statistics to a pre-determined object definition to determine the probability that the cell fits the acceptance criteria and that the cell is captured by the filter;

storing the results for additional analysis.

73. A composition comprising at least one molecule that binds at least two protein markers for cancer, or at least two nucleic acids encoding protein markers for cancer, or a combination of at least one protein marker and at least one nucleic acid encoding a protein marker for cancer and optionally further comprising a PAP stain.

74. The composition according to claim 73, wherein the at least two protein markers for cancer are chosen from:

c-Myc, Cox-2, HIF-1α

, Histone H3, a telomerase marker, a telomerase associated protein;

an extra-cellular matrix marker;

a proliferation marker;

a cell cycle marker;

an apoptosis marker, and HR HPV.

75. The composition according to claim 73, wherein the markers for cancer are markers for HPV-related cancer.

76. The composition according to claim 73, wherein the markers for cancer are markers for cervical cancer.

77. A method for detection and quantitation of at least two markers for cancer comprising:

a) preparing a cytology sample on a solid support

b) contacting the sample of a) with the composition of claim 73,

c) detecting at least two markers for cancer,

d) quantifying the at least two markers for cancer

78. The method according to claim 77, wherein the cancer is HPV-related cancer.

79. The method according to claim 77, wherein the cancer is cervical cancer.

80. The method according to claim 77, wherein the cancer is colon cancer.

81. The method according to claim 77, wherein the markers for cancer are detected using at least one reagent chosen from an antibody, a nucleic acid molecule, and a PAP stain.

82. The method according to claim 77, wherein the composition is chosen from a PAP stain, c-Myc, Cox-2, HIF-1α

, Histone H3, a telomerase marker, a telomerase associated protein, an extra-cellular matrix marker, a proliferation marker, a cell cycle marker, an apoptosis marker, and HR-HPV.

83. The method according to claim 77, wherein the method is an automated method.

84. A method of detecting markers for cancer in a subject comprising

a) obtaining a sample comprising cells from the subject;

b) contacting the sample with a composition according to claim 73, comprising at least two molecules that bind to at least two protein markers for cancer under conditions such that the at least two molecules bind to the at least two protein markers for cancer in the sample;

wherein binding of the two molecules to the sample indicates the presence of cancer or the risk of developing cancer.

85. The method according to claim 84, wherein cancer is HPV-related cancer.

86. The method according to claim 84, wherein the cancer is cervical cancer.

87. The method according to claim 84, where the protein markers for cancer are chosen from c-Myc, Cox-2, HIF-1α

, Histone H3, a telomerase marker, a telomerase associated protein;

an extra-cellular matrix marker;

a proliferation marker;

a cell cycle marker;

HR HPV, and an apoptosis marker.

88. The method according to claim 84, wherein the sample is contained on a single solid support.

89. The method according to claim 88, where the single solid support is a microscope slide.

90. The method according to claim 84, further comprising contacting the sample with PAP stain, and wherein an abnormal PAP stain and binding of the at least two molecules to the sample indicates the presence of cancer or the risk of developing cancer.

91. The method of claim 84, wherein the method is automated.

98. A computer controlled software algorithm for performing image analysis of high resolution microscopic digital images of cells containing nuclei comprising the following steps:

a) analyzing a saved high resolution digital image, said digital image saved as multiple gigabytes;

b) sub sampling the high resolution digital image to create a second low resolution digital image;

c) analyzing the low resolution digital image to locate potential objects of interest within the low resolution image;

d) mapping potential objects of interest back onto the high resolution image;

e) analyzing each mapped object within the high resolution image to generate a list of descriptive statistics that describe each object; and

f) comparing the descriptive statistics for each object to an object definition to determine the likelihood that the described object is a nuclei.

99. The image analysis algorithm according to claim 98, wherein step c), the analysis of the low resolution digital image to locate potential objects of interest in the low resolution image may comprise the following steps:

a) reducing image resolution;

b) smoothing the image;

c) segmenting the image in HSI color space;

d) detecting the edges of objects.

100. The image analysis algorithm according to claim 98, wherein step e), the analyzing of each mapped object, comprises

classifying the object based on chromatic, geometric, topological and biological information;

and

collecting statistics by using the original image to gather chromatic, geometric and topological information.

101. An image analysis algorithm according to claim 100, where the collected statistics for a mapped object within the high resolution image include:

a) contour length;

b) size;

c) symmetry;

d) compactness;

e) topology;

f) color;

g) saturation; and

h) intensity

102. The image analysis algorithm according to claim 100, where the collected statistics for the whole image or the regions of interest within the high resolution image include:

a) total number of objects in each categories;

b) average and mean intensity;

c) average size; and

d) topology.

103. A nucleic acid hybridization buffer for in situ hybridization comprising a low molecular weight dextran sulfate.

104. The hybridization buffer according to claim 103, wherein dextran sulfate has a molecular weight range of about 25,000-75,000.

105. The hybridization buffer according to claim 103, wherein the dextran sulfate has a molecular weight of about 35,000-50,000.

106. The hybridization buffer according to any of claim 103, wherein the low molecular weight dextran sulfate is present in an amount ranging from 5-15 wt./vol. %.

107. The hybridization buffer according to any of claim 103, wherein the low molecular weight dextran sulfate is present in an amount ranging from 10 wt./vol. %.