Focused microarray and methods of diagnosing cancer

• US 20070134687A1
• Filed: 09/12/2006
• Published: 06/14/2007
• Est. Priority Date: 09/12/2005
• Status: Abandoned Application

• Current Assignee: Aurelium BioPharma, Inc.
• Original Assignee: Aurelium BioPharma, Inc.
• Inventors: Georges, Elias, Bonneau, Anne-Marie, Boucher, Claudia
• Application Number:
  US11/520,476
• Publication Number:
  US 20070134687A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

Disclosed are methods for diagnosing cancer in a cell sample by detecting an increase in the levels of expression of marker genes in the cell sample as compared to the levels of expression of the same marker genes in a normal, nonneoplastic cell of the same tissue type. Also disclosed is a focused microarray device for diagnosis of cancer in a cell sample.

Claims

1. A method of diagnosing cancer in a subject, comprising:

(a) providing a focused microarray, the microarray having a plurality of nucleic acid capture probes, each capture probe being complementary to a marker gene selected from the group consisting of HDGF, cytokeratin 18, α

-enolase, GAPDH, GST-π

, TPI, 5C5-2, prohibitin, keratin 19, cytokeratin 7, HnRNP, pyrophosphatase inorganic, BIP, CBX3, ATP synthase δ

, PDI/ER-60 precursor, ATP synthase β

, prostasin, cathepsin β

, FAS, HSCP60, topoisomerase IIα

, PCNA, ezrin, PDI, cathepsin D, A-CRABP II, HSC70, rad 23 homolog β

, ETF3 subunit 2β

, proteosome B1 subunit proprotein, β

-tubulin, s1c9a3r1, prosolin, HSP60, HER-2, L-plastin, estrogen receptor α

, HSP27, thioredoxine peroxidase I, calumenin, 14-3-3 eta chain, Ki 67, MRP1, “

similar to stratifin,”

UCHL-1, mammaglobin 2, cellular RNA binding protein, p53, and annexin I;

(b) detecting a level of expression in a cell sample of the selected marker genes by contacting the nucleic acid capture probes with nucleic acids from the cell sample so as to allow for the hybridization of the nucleic acid capture probes with the nucleic acids from the cell sample; and

(c) comparing the level of expression of the selected marker genes in the cell sample to the level of expression of the same marker genes in a normal cell sample of the same tissue type,

wherein the presence of a cancer cell is indicated if the level of expression of five or more of the selected marker genes in the cell sample is greater than the level of expression of the same marker genes in the normal cell sample of the same tissue type.

2. The method of claim 1, wherein the microarray has a plurality of nucleic acid capture probes that are complementary to marker genes selected from the group consisting of cytokeratin 18, cytokeratin 7, keratin 19, α

-enolase, s1c9a3r1, TPI, and HER-2.

3. The method of claim 2, wherein the plurality of nucleic acid capture probes is at least six.

4. The method of claim 2, wherein the plurality of nucleic acid capture probes is at least seven.

5. The method of claim 1, wherein the presence of a cancer cell is indicated if the level of expression of six or more of the selected marker genes in the cell sample is greater than the level of expression of the same marker genes in the normal cell sample of the same tissue type.

6. The method of claim 1, wherein the presence of a cancer cell is detected if the level of expression of seven or more of the selected marker genes in the cell sample is greater than the level of expression of the same marker genes in the normal cell sample of the same tissue type.

7. The method of claim 1, wherein the plurality of marker genes selected is at least six, and an increased level of expression of at least five marker genes in the cell sample compared to the level of expression in the normal cell sample of the same tissue type indicates that the cell sample is neoplastic.

8. The method of claim 1, wherein the plurality of marker genes selected is at least seven, and an increased level of expression of at least five marker genes in the cell sample compared to the level of expression in the normal cell sample of the same tissue type indicates that the cell sample is neoplastic.

9. The method of claim 1, wherein the plurality of marker genes selected is at least eight, and an increased level of expression of at least six marker genes in the cell sample compared to the level of expression in the normal cell sample of the same tissue type indicates that the cell sample is neoplastic.

10. The method of claim 1, wherein the cell sample is from breast tissue.

11. The method of claim 10, wherein the plurality of marker genes is keratin 19, s1c9a3r1, FABP 4, and HER-2.

12. The method of claim 1, wherein the cell sample is from ovarian tissue and the plurality is at least three marker genes selected from the group consisting of cytokeratin 18, cytokeratin 7, α

-enolase, and TPI.

13. The method of claim 12, wherein the plurality is cytokeratin 18, cytokeratin 7, α

-enolase, and TPI.

14. The method of claim 1, wherein the step of comparing the level of expression of the selected marker genes further comprises using a class prediction algorithm to differentiate the level of expression of the selected marker genes in the cell sample from the level of expression of the same marker genes in the normal cell sample of the same tissue type.

15. The method of claim 14, wherein the one or more class prediction algorithms is selected from the group consisting of compound covariate predictor, diagonal linear discriminant analysis, nearest neighbor predictor, nearest centroid predictor, and support vector machine predictor.

16. The method of claim 1, wherein the presence of a cancer cell is indicated if the level of expression in the cell sample of at least one of the selected marker genes is at least two times the level of expression of the same marker gene in the normal cell sample of the same tissue type.

17. The method of claim 1, wherein the presence of a cancer cell is indicated if the level of expression in the cell sample of at least one of the selected marker genes is at least three times the level of expression of the same marker gene in the normal cell sample of the same tissue type.

18. The method of claim 1, wherein the presence of a cancer cell is indicated if the level of expression in the cell sample of at least one of the selected marker genes is at least four times the level of expression of the same marker gene in the normal cell sample of the same tissue type.

19. The method of claim 1, wherein the presence of a cancer cell is indicated if the level of expression in the cell sample of at least one of the selected marker genes is at least five times the level of expression of the same marker gene in the normal cell sample of the same tissue type.

20. The method of claim 1, wherein the presence of a cancer cell is indicated if the level of expression in the cell sample of at least one of the selected marker genes is at least six times the level of expression of the same marker gene in the normal cell sample of the same tissue type.

21. Any of the methods of claims 16-20, wherein the step of comparing the level of expression of the selected marker gene(s) comprises using one or more class prediction algorithms selected from the group consisting of compound covariate predictor, diagonal linear discriminant analysis, nearest neighbor predictor, nearest centroid predictor, and support vector machine predictor.

22. The method of claim 1, wherein the subject is a human.

23. The method of claim 1, wherein the presence of cancer being detected is selected from the group consisting of ovarian carcinoma, serous adenocarcinoma, clear cell adenocarcinoma, endometrioid carcinoma, mucinous adenocarcinoma, breast adenocarcinoma, and infiltrating ductal carcinoma.

24. A method of diagnosing breast cancer in a subject, comprising:

(a) selecting at least six marker genes from the group consisting of HDGF, cytokeratin 18, α

-enolase, GAPDH, GST-π

, TPI, 5C5-2, prohibitin, keratin 19, cytokeratin 7, HnRNP, pyrophosphatase inorganic, BIP, CBX3, PDI/ER-60 precursor, ATP synthase β

, prostasin, cathepsin β

, FAS, HSCP60, topoisomerase IIα

, PCNA, ezrin, PDI, cathepsin D, A-CRABP II, HSC70, β

-tubulin, s1c9a3r1, prosolin, HSP60, HER-2, L-plastin, estrogen receptor α

, HSP27, thioredoxine peroxidase I, calumenin, GAPDH, 14-3-3 eta chain, and annexin I;

(b) detecting a level of expression of the selected marker genes in a breast cell sample by contacting probes capable of binding or hybridizing with the marker genes isolated from the cell sample; and

(c) comparing the level of expression of the selected marker genes in the breast cell sample to a level of expression of the same marker genes detected in a normal breast cell sample,

wherein the presence of breast cancer is indicated if the level of expression of three or more marker genes in the breast cell sample is greater than the level of expression for in the normal breast cell sample.

25. The method of claim 24, wherein at least six marker genes are selected, and an increased level of expression of a plurality of at least five marker genes in the breast cell sample compared to the level of expression in the normal breast cell sample indicates the presence of breast cancer.

26. The method of claim 24, wherein at least seven marker genes are selected, and an increased level of expression of at least six marker genes in the breast cell sample compared to the level of expression in the normal breast cell sample indicates the presence of breast cancer.

27. The method of claim 24, wherein the plurality of marker genes comprises keratin 19, s1c9a3r1, and HER-2.

28. The method of claim 24, wherein the level of expression of marker genes is detected by nucleic acid capture probes attached to a solid support.

29. The method of claim 24, wherein the breast cancer is a breast adenocarcinoma or an infiltrating ductal carcinoma.

30. The method of claim 24, wherein the subject is a human.

31. The method of claim 24, wherein the presence of cancer is indicated if the level of expression in the breast cell sample of at least one of the selected marker genes is at least two times the level of expression of the same marker gene in the normal breast cell sample.

32. The method of claim 24, wherein the marker genes are selected from the group consisting of cathepsin D, ezrin, keratin 19, s1c9a3r1, A-CRABP II, HER-2, and estrogen receptor α

.

33. The method of claim 32, wherein the presence of cancer is indicated if the level of expression in the breast cell sample of at least one of the selected marker genes is at least three times the level of expression of the same marker genes in the normal breast cell sample.

34. The method of claim 33, wherein the average level of expression is determined in a breast cell sample and a normal breast cell sample for at least three marker genes selected from the group consisting of cathepsin D, ezrin, keratin 19, s1c9a3r1, A-CRABP II, HER-2, and estrogen receptor α

.

35. The method of claim 34, wherein the level of expression of the marker genes in the breast cell sample is compared to the level of expression of the same marker genes in the normal breast cell sample using one or more class prediction algorithms selected from the group consisting of compound covariate predictor, diagonal linear discriminant analysis, nearest neighbor predictor, nearest centroid predictor, and support vector machine predictor.

36. The method of claim 34, wherein the marker genes are keratin 19, s1c9a3r1, and HER-2.

37. The method of claim 36, wherein the presence of cancer is indicated if the level of expression in the breast cell sample of at least one of the plurality of selected marker genes is at least four times the level of expression of the same marker genes in the normal breast cell sample.

38. The method of claim 24, wherein the marker genes are selected from the group consisting of keratin 19, s1c9a3r1, HER-2, and FABP 4.

39. The method of claim 38, wherein the presence of cancer is indicated if the level of expression of keratin 19, s1c9a3r1, and HER-2 is at least four times greater in the breast cell sample than the level of expression for the same marker genes in the normal breast cell sample and the level of expression of FABP 4 is decreased in the breast cell sample as compared to the normal breast cell sample.

40. The method of claim 24, wherein comparing the level of expression of the selected marker genes further comprises using a class prediction algorithm to differentiate the level of expression of the selected marker genes in the cell sample from the level of expression of the same marker genes in the normal cell sample of the same tissue type.

41. The method of claim 40, wherein comparing the level of expression of the selected marker genes further comprises differentiating the level of expression of the selected marker genes in the cell sample from the level of expression of the same marker genes in the normal cell sample of the same tissue type using one or more class prediction algorithms selected from the group consisting of compound covariate predictor, diagonal linear discriminant analysis, nearest neighbor predictor, nearest centroid predictor, and support vector machine predictor.

42. The method of claim 24, wherein the level of expression of marker genes is determined using RT-PCR, PCR, nucleic acid blotting, dot blotting, or microarray.

43. A method of diagnosing ovarian cancer in a subject, comprising:

(a) selecting a plurality of marker genes from the group consisting of HDGF, cytokeratin 18, α

-enolase, GAPDH, GST-π

, TPI, 5C5-2, prohibitin, keratin 19, cytokeratin 7, HnRNP, pyrophosphatase inorganic, BIP, CBX3, PDI/ER-60 precursor, ATP synthase β

, prostasin, cathepsin β

, FAS, HSCP60, topoisomerase IIα

, PCNA, ezrin, PDI, cathepsin D, A-CRABP II, and annexin I;

(b) detecting a level of expression of the selected marker genes in an ovarian cell sample by contacting probes capable of binding or hybridizing with the marker genes isolated from the cell sample; and

(c) comparing the level of expression of the selected marker genes in the ovarian cell sample to a level of expression of the same marker genes detected in a normal ovarian cell sample,

wherein the presence of ovarian cancer is indicated if the level of expression of two or more marker genes in the ovarian cell sample is greater than the level of expression of the same marker genes in the normal ovarian cell sample.

44. The method of claim 43, wherein the plurality of marker genes selected is at least six, and an increased level of expression of a plurality of at least four marker genes in the ovarian cell sample compared to the level of expression in the normal ovarian cell sample indicates the presence of ovarian cancer.

45. The method of claim 43, wherein the plurality of marker genes selected is at least seven, and an increased level of expression of a plurality of at least five marker genes in the ovarian cell sample compared to the level of expression in the normal ovarian cell sample indicates the presence of ovarian cancer.

46. The method of claim 43, wherein at least three marker genes selected from the group consisting of cytokeratin 18, cytokeratin 7, TPI, and α

-enolase.

47. The method of claim 43, wherein the marker genes are cytokeratin 18, cytokeratin 7, TPI, and α

-enolase.

48. The method of claim 43, wherein the step of comparing the level of expression of marker genes in the ovarian cell sample to the level of expression of the marker genes in the normal ovarian cell sample comprises using one or more class prediction algorithms selected from the group consisting of compound covariate predictor, diagonal linear discriminant analysis, nearest neighbor predictor, nearest centroid predictor, and support vector machine predictor.

49. The method of claim 43, wherein the level of expression of marker genes is detected by nucleic acid capture probes attached to a solid support.

50. The method of claim 43, wherein the ovarian cancer cell is an ovarian carcinoma, a serous adenocarcinoma, a clear cell adenocarcinoma, an endometrioid carcinoma, or a mucinous adenocarcinoma.

51. The method of claim 43, wherein the subject is a human.

52. The method of claim 43, wherein the presence of cancer is indicated if the level of expression in the ovarian cell sample of at least one of the selected marker genes is at least three times the level of expression of the same marker gene in the normal ovarian cell sample.

53. The method of claim 43, wherein the marker genes are selected from the group consisting of HDGF, cytokeratin 18, α

-enolase, GAPDH, GST-π

, TPI, keratin 19, cytokeratin 7, pyrophosphatase inorganic, ATP synthase β

, prostasin, cathepsin β

, cathepsin D, and A-CRABP II.

56. The method of claim 43, wherein at least five marker genes are selected from the group consisting of HDGF, cytokeratin 18, α

-enolase, GAPDH, GST-π

, TPI, keratin 19, cytokeratin 7, and A-CRABP II.

57. The method of claim 56, wherein the presence of cancer is indicated if the level of expression in the ovarian cell sample of at least one of the plurality of marker genes is at least five times the level of expression of the same marker genes in the normal ovarian cell sample.

58. The method of claim 56, wherein the presence of cancer is indicated if levels of expression are determined for all marker genes in the ovarian cell sample and the normal ovarian cell sample, and the levels of expression for all marker genes in the ovarian cell sample are at least five times the levels of expression for the same marker genes in the normal ovarian cell sample.

59. The method of claim 58, wherein the step of comparing the levels of expression of marker genes in the ovarian cell sample to the levels of expression of the marker genes in the normal ovarian cell sample comprises using a class prediction algorithm.

60. The method of claim 43, wherein the marker genes are selected from the group consisting of cytokeratin 18, α

-enolase, TPI, and cytokeratin 7.

61. The method of claim 60, wherein the presence of cancer is indicated if the level of expression in the ovarian cell sample of at least one of the plurality of selected marker genes is at least six times the level of expression of the same marker genes in the normal ovarian cell sample.

62. The method of claim 60, wherein the presence of cancer is indicated if levels of expression are determined for all marker genes in the ovarian cell sample and the normal ovarian cell sample, and the levels of expression for all marker genes in the ovarian cell sample are at least five times the levels of expression for the same marker genes in the normal ovarian cell sample.

63. The method of claim 62, wherein the step of comparing the levels of expression of marker genes in the ovarian cell sample to the levels of expression of the marker genes in the normal ovarian cell sample comprises using a class prediction algorithm.

64. The method of claim 43, wherein comparing the level of expression of the selected marker genes further comprises using a class prediction algorithm to differentiate the level of expression of the selected marker genes in the ovarian cell sample from the level of expression of the same marker genes in the normal ovarian cell sample.

65. The method of claim 57, wherein comparing the level of expression of the selected marker genes further comprises differentiating the level of expression of the selected marker genes in the ovarian cell sample from the level of expression of the same marker genes in the normal ovarian cell sample using one or more class prediction algorithms selected from the group consisting of compound covariate predictor, diagonal linear discriminant analysis, nearest neighbor predictor, nearest centroid predictor, and support vector machine predictor.

66. The method of claim 43, wherein the level of expression of marker genes is determined using RT-PCR, PCR, nucleic acid blotting, dot blotting, or microarray.

54. The method of claim 54, wherein the presence of cancer is indicated if the level of expression in the ovarian cell sample of at least one of the selected marker genes is at least four times the level of expression of the same marker genes in the normal ovarian cell sample.

55. The method of claim 54, wherein the presence of cancer is indicated if a level of expression in the ovarian cell sample of at least two marker genes is at least four times the level of expression for the same marker genes in the normal ovarian cell sample.

67. A focused microarray for diagnosing a neoplasm, comprising:

a) a first set of nucleic acid capture probes comprising a plurality of nucleic acid capture probes, each capture probe being complementary to a marker gene selected from the group consisting of HDGF, cytokeratin 18, α

-enolase, GAPDH, GST-π

, TPI, 5C5-2, prohibitin, keratin 19, cytokeratin 7, HnRNP, pyrophosphatase inorganic, BIP, CBX3, ATP synthase δ

, PDI/ER-60 precursor, ATP synthase β

, prostasin, cathepsin β

, FAS, HSCP60, topoisomerase IIα

, PCNA, ezrin, PDI, cathepsin D, A-CRABP II, HSC70, rad 23 homolog β

, ETF3 subunit 2β

, proteosome BI subunit proprotein, β

-tubulin, s1c9a3r1, prosolin, HSP60, HER-2, L-plastin, estrogen receptor α

, HSP27, thioredoxine peroxidase I, calumenin, 14-3-3 eta chain, Ki 67, MRP1, “

similar to stratifin,”

UCHL-1, mammaglobin 2, cellular RNA binding protein, p53, and annexin I;

b) a second set of nucleic acid capture probes comprising a plurality of nucleic acid capture probes, each capture probe being complementary to a marker gene encoding an endogenous housekeeping gene;

c) a solid support to which the first and second set of nucleic acid capture probes are attached at predetermined positions.

68. The focused microarray of claim 67, wherein the first set of capture probes is complementary to at least three of the marker genes selected from the group consisting of cytokeratin 7, cytokeratin 18, TPI, α

-enolase, keratin 19, s1c9a3r1, and HER-2.

69. The focused microarray of claim 67, wherein the first set of capture probes is complementary to at least four of the marker genes selected from the group consisting of cytokeratin 7, cytokeratin 18, TPI, α

-enolase, keratin 19, s1c9a3r1, and HER-2.

70. The focused microarray of claim 67, wherein the first set of capture probes is complementary to at least five marker genes selected from the group consisting of cytokeratin 7, cytokeratin 18, TPI, α

-enolase, keratin 19, s1c9a3r1, and HER-2.

71. The focused microarray of claim 67, wherein the first set of capture probes is complementary to at least six of the marker genes selected from the group consisting of cytokeratin 7, cytokeratin 18, TPI, α

-enolase, keratin 19, s1c9a3r1, and HER-2.

72. The focused microarray of claim 67, wherein at least one capture probe of the second set is complementary to a marker gene selected from the group consisting of EF-2 and EIF-4B.

73. The focused microarray of claim 67, wherein the second set of capture probes consists of the marker genes EF-2 and EIF-4B.

74. The focused microarray of claim 67, wherein the first set of capture probes is complementary to marker genes selected from the group consisting of HDGF, cytokeratin 18, α

-enolase, GAPDH, GST-π

, TPI, 5C5-2, prohibitin, keratin 19, cytokeratin 7, HnRNP, pyrophosphatase inorganic, BIP, CBX3, PDI/ER-60 precursor, ATP synthase β

, prostasin, cathepsin β

, FAS, HSCP60, topoisomerase IIα

, PCNA, ezrin, PDI, cathepsin D, A-CRABP II, HSC70, β

-tubulin, s1c9a3r1, prosolin, HSP60, HER-2, L-plastin, estrogen receptor α

, HSP27, thioredoxine peroxidase I, calumenin, GAPDH, 14-3-3 eta chain, and annexin I.

75. The focused microarray of claim 67, wherein the first set of capture probes is complementary to marker genes selected from the group consisting of keratin 19, s1c9a3r1, and HER-2.

76. The focused microarray of claim 67, wherein the first set of capture probes is complementary to marker genes selected from the group consisting of HDGF, cytokeratin 18, α

-enolase, GAPDH, GST-π

, TPI, 5C5-2, prohibitin, keratin 19, cytokeratin 7, HnRNP, pyrophosphatase inorganic, BIP, CBX3, PDI/ER-60 precursor, ATP synthase β

, prostasin, cathepsin β

, FAS, HSCP60, topoisomerase IIα

, PCNA, ezrin, PDI, cathepsin D, A-CRABP II, and annexin I.

77. The focused microarray of claim 67, wherein the first set of capture probes is complementary to marker genes selected from the group consisting of cytokeratin 18, cytokeratin 7, TPI, and cytokeratin 7.

78. A kit for diagnosing cancer in a subject, comprising:

a) a first set of probes for the detection of one or more marker genes selected from the group consisting of HDGF, cytokeratin 18, α

-enolase, GAPDH, GST-π

, TPI, 5C5-2, prohibitin, keratin 19, cytokeratin 7, HnRNP, pyrophosphatase inorganic, BIP, CBX3, ATP synthase δ

, PDI/ER-60 precursor, ATP synthase β

, prostasin, cathepsin β

, FAS, HSCP60, topoisomerase IIα

, PCNA, ezrin, PDI, cathepsin D, A-CRABP II, HSC70, rad 23 homolog β

, ETF3 subunit 2β

, proteosome B1 subunit proprotein, β

-tubulin, s1c9a3r1, prosolin, HSP60, HER-2, L-plastin, estrogen receptor α

, HSP27, thioredoxine peroxidase I, calumenin, 14-3-3 eta chain, Ki 67, MRP1, “

similar to stratifin,”

UCHL-1, mammaglobin 2, cellular RNA binding protein, p53, and annexin I;

b) a second set of probes for the detection of one or more marker genes selected from endogenous housekeeping genes; and

c) a detection means for identifying a probe hybridizing to a target marker gene.

79. The kit of claim 78, wherein the first set of probes is a plurality of nucleic acids complementary to mRNA encoding the selected marker genes.

80. The kit of claim 79, wherein the nucleic acid is selected from the group consisting of single-stranded RNA, double-stranded RNA, double-stranded DNA, single-stranded DNA, and RNA-DNA hybrids.

81. The kit of claim 78, wherein the second set of probes is a plurality of nucleic acids complementary to an mRNA encoding marker genes that do not vary statistically significantly in level of expression between cancer cell samples and normal cell samples.

82. The kit of claim 78, wherein the second set of probes is a plurality of nucleic acids complementary to an mRNA encoding EF-2 and EIF-4B.

83. The kit of claim 82, wherein the nucleic acid is selected from the group consisting of single-stranded RNA, double-stranded RNA, double-stranded DNA, single-stranded DNA, RNA-DNA hybrids, and siRNA.

84. The kit of claim 78, wherein the detection means is selected from the group consisting of fluorophores, chemical dyes, radiolabels, chemiluminescent compounds, colorimetric enzymatic reactions, chemiluminescent enzymatic reactions, magnetic compounds, and paramagnetic compounds.

85. The kit of claim 78, wherein the first set and second set of probes are attached to a solid support at predetermined positions.

86. The kit of claim 78, wherein the cancer being detected is selected from the group consisting of breast adenocarcinoma, infiltrating ductal carcinoma, ovarian carcinoma, serous adenocarcinoma, clear cell adenocarcinoma, endometrioid carcinoma, and mucinous adenocarcinoma.

87. The kit of claim 78, wherein a training is provided that comprises a pamphlet supplying information on the levels of expression of the marker genes in a normal cell sample and a neoplastic cell sample detected by the first set of probes.