Methods and Arrays for Use in the Same
20170192004 ยท 2017-07-06
Inventors
Cpc classification
G01N33/53
PHYSICS
International classification
Abstract
The present invention provides a method for diagnosing breast cancer comprising or consisting of the steps of (a) providing a sample to be tested; and (b) determining a biomarker signature of the test sample by measuring the presence and/or amount in the test sample of one or more biomarker selected from the group defined in Table A(i) and/or Table A(ii); wherein the presence and/or amount in the test sample of the one or more biomarker selected from the group defined in Table A(i) and/or Table A(ii) is indicative of the presence of breast cancer cells in the individual, corresponding uses, methods of treating breast cancer, together with arrays and kits for use in the same.
Claims
1. A method for diagnosing breast cancer comprising or consisting of the steps of: a) providing a sample to be tested; and b) determining a biomarker signature of the test sample by measuring the presence and/or amount in the test sample of one or more biomarker selected from the group defined in Table A(i) and/or Table A(ii); wherein the presence and/or amount in the test sample of the one or more biomarker selected from the group defined in Table A(i) and/or Table A(ii) is indicative of the presence of breast cancer cells in the individual.
2. The method according to claim 1 wherein the breast cancer is early breast cancer.
3. The method according to claim 1 or 2 wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table A, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113 or 114 of the biomarkers listed in Table A.
4. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table A(i), for example at least 2 or 3 of the biomarkers listed in Table A(i).
5. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table A(ii), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 of the biomarkers listed in Table A(ii).
6. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table A(iii), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 of the biomarkers listed in Table A(iii).
7. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table B(i), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 of the biomarkers listed in Table B(i).
8. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table B(ii), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 of the biomarkers listed in Table B(ii).
9. The method according to claim 8 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 0-52 weeks prior to diagnosis by conventional clinical methods.
10. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table B(iii), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43 of the biomarkers listed in Table B(iii).
11. The method according to claim 10 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 52-104 weeks prior to diagnosis by conventional clinical methods.
12. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table B(iv), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 of the biomarkers listed in Table B(iv).
13. The method according to claim 12 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 0-26 weeks prior to diagnosis by conventional clinical methods.
14. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table B(v), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 78 of the biomarkers listed in Table B(v).
15. The method according to claim 14 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 26-52 weeks prior to diagnosis by conventional clinical methods.
16. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table B(vi), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 of the biomarkers listed in Table B(vi).
17. The method according to claim 16 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 52-78 weeks prior to diagnosis by conventional clinical methods.
18. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table B(vii), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 of the biomarkers listed in Table B(vii).
19. The method according to claim 18 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 78-104 weeks prior to diagnosis by conventional clinical methods.
20. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table B(viii).
21. The method according to claim 20 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 0-35 weeks prior to diagnosis by conventional clinical methods of breast cancer consisting of tumours of less than or equal to 20 mm in any dimension.
22. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table B(ix), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38 of the biomarkers listed in Table B(ix).
23. The method according to claim 22 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 70-104 weeks prior to diagnosis by conventional clinical methods of breast cancer consisting of tumours of less than or equal to 20 mm in any dimension.
24. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table B(x), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or 63 of the biomarkers listed in Table B(x).
25. The method according to claim 24 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 20-26 or 26-52 weeks prior to diagnosis by conventional clinical methods.
26. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table B(xi), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 or 82 of the biomarkers listed in Table B(xi).
27. The method according to claim 26 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 26-52 or 52-78 weeks prior to diagnosis by conventional clinical methods.
28. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in Table B(xii), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 of the biomarkers listed in Table B(xii).
29. The method according to claim 28 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 52-78 or 78-104 weeks prior to diagnosis by conventional clinical methods.
30. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in
31. The method according to claim 30 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 0-26 or 26-52 weeks prior to diagnosis by conventional clinical methods.
32. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in
33. The method according to claim 32 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 26-52 or 52-78 weeks prior to diagnosis by conventional clinical methods.
34. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of 1 or more biomarker listed in
35. The method according to claim 34 wherein the method is indicative of whether or not the test sample is characteristic of a sample taken from an individual with breast cancer 52-78 or 78-104 weeks prior to diagnosis by conventional clinical methods.
36. The method according to any one of the preceding claims wherein step (b) comprises measuring the presence and/or amount of all of the biomarkers listed in Table A.
37. The method according to any one of the preceding claims further comprising or consisting of the steps of: e) providing one or more control sample from: i. an individual not afflicted with breast cancer; and/or ii. an individual afflicted with breast cancer, wherein the sample was taken at a time period defined in claims 7-37 that differs from the time period that the test sample is characteristic of; f) determining a biomarker signature of the one or more control sample by measuring the presence and/or amount in the control sample of the one or more biomarkers measured in step (b); wherein the presence of breast cancer is identified in the event that the presence and/or amount in the test sample of the one or more biomarkers measured in step (b) is different from the presence and/or amount in the control sample of the one or more biomarkers measured in step (d). By the time period that the test sample is characteristic of we include the time period prior to breast cancer diagnosis by conventional clinical methods.
38. The method according to any one of the preceding claims further comprising or consisting of the steps of: g) providing one or more control sample from; i. an individual afflicted with breast cancer (i.e., a positive control); and/or ii. an individual afflicted with breast cancer, wherein the sample was taken at a time period defined in claims 7-35 that corresponds to the time period that the test sample is characteristic of; h) determining a biomarker signature of the control sample by measuring the presence and/or amount in the control sample of the one or more biomarkers measured in step (b); wherein the presence of breast cancer is identified in the event that the presence and/or amount in the test sample of the one or more biomarkers measured in step (b) corresponds to the presence and/or amount in the control sample of the one or more biomarkers measured in step (f).
39. The method according to claim 37 and/or 38 wherein the control samples comprise one or more sample taken from each of the time periods defined in claims 7-35 to be tested.
40. The method according to claim 37 and/or 38 wherein the control samples comprise one or more sample taken from each of the time periods defined in claims 7-35.
41. The method according to any of claims 37-40, wherein the individual from which the one or more control sample was obtained was not, at the time the sample was obtained, afflicted with breast abscess, breast fibroadenoma, fibroadenoma, fibrocystic breast disease, fibrocystic breasts, gynecomastia, mastalgia and/or mastitis.
42. The method according to any of claims 37-41, wherein the individual from which the one or more control sample was obtained was not, at the time the sample was obtained, afflicted with any disease or condition of the breast.
43. The method according to claim 37, wherein the individual not afflicted with breast cancer was not, at the time the sample was obtained, afflicted with any disease or condition.
44. The method according to claim 37 wherein the individual not afflicted with breast cancer is a healthy individual.
45. The method according to any one of claims 37-41 wherein the one or more individual afflicted with breast cancer is afflicted with a breast cancer selected from the group consisting of ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), invasive ductal breast cancer, invasive lobular breast cancer, Inflammatory breast cancer, medullary breast cancer, mucinous (mucoid or colloid) breast cancer, tubular breast cancer, adenoid cystic carcinoma of the breast (cribriform breast cancer), metaplastic breast cancer, angiosarcoma of the breast, lymphoma of the breast, basal type breast cancer, malignant phyllodes or cystosarcoma phyllodes and papillary breast cancer.
46. The method according to any one of the preceding claims wherein the breast cancer is invasive ductal breast cancer.
47. The method according to any one of the preceding claims wherein the method is repeated.
48. The method according to any one of the preceding claims wherein the method is repeated and wherein, in step (a), the sample to be tested is taken at different time to the previous method repetition.
49. The method according to claim 47 or 48 wherein the method is repeated using a test sample taken at a different time period to the previous test sample(s) used.
50. The method according to claim 48 or 49 wherein the method is repeated using a test sample taken between 1 day to 104 weeks to the previous test sample(s) used, for example, between 1 week to 100 weeks, 1 week to 90 weeks, 1 week to 80 weeks, 1 week to 70 weeks, 1 week to 60 weeks, 1 week to 50 weeks, 1 week to 40 weeks, 1 week to 30 weeks, 1 week to 20 weeks, 1 week to 10 weeks, 1 week to 9 weeks, 1 week to 8 weeks, 1 week to 7 weeks, 1 week to 6 weeks, 1 week to 5 weeks, 1 week to 4 weeks, 1 week to 3 weeks, or 1 week to 2 weeks.
51. The method according to claim 48 or 49 wherein the method is repeated using a test sample taken every period from the group consisting of: 1 day, 2 days, 3 day, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, 50 weeks, 55 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104, weeks, 105 weeks, 110 weeks, 115 weeks, 120 weeks, 125 weeks and 130 weeks.
52. The method according to any one of claims 47-51 wherein the method is repeated at least once, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23, 24 times or 25 times.
53. The method according to any one of claims 47-51 wherein the method is repeated continuously.
54. The method according to any one of claims 47-51 wherein the method is repeated until diagnosis of breast cancer in the individual using conventional clinical methods.
55. The method according to any one of claims 47-54 wherein each repetition uses test sample taken from the same individual.
56. The method according to any one of claims 1 to 55 wherein step (b) comprises measuring the expression of the protein or polypeptide of the one or more biomarker(s)
57. The method according to any one of the preceding claims wherein step (b), (d) and/or step (f) is performed using one or more first binding agent capable of binding to a biomarker listed in Table A or Table B.
58. The method according to claim 57 wherein the first binding agent comprises or consists of an antibody or an antigen-binding fragment thereof.
59. The method according to claim 58 wherein the antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment thereof.
60. The method according to claim 58 or 59 wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: scFv; Fab; a binding domain of an immunoglobulin molecule.
61. The method according to any one of claims 58 to 60 wherein the first binding agent is immobilised on a surface.
62. The method according to any one of claims 57 to 61 wherein the one or more biomarkers in the test sample are labelled with a detectable moiety.
63. The method according to any one of claims 57 to 62 wherein the one or more biomarkers in the control sample(s) are labelled with a detectable moiety.
64. The method according to claim 62 or 63 wherein the detectable moiety is selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety; an enzymatic moiety.
65. The method according to claim 62 or 63 wherein the detectable moiety is biotin.
66. The method according to any one of claims 57 to 63 wherein step (b), (d) and/or step (f) is performed using an assay comprising a second binding agent capable of binding to the one or more biomarkers, the second binding agent comprising a detectable moiety.
67. The method according to any one of claim 66 wherein the second binding agent comprises or consists of an antibody or an antigen-binding fragment thereof.
68. The method according to claim 67 wherein the antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment thereof.
69. The method according to claim 67 or 68 wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: scFv; Fab; a binding domain of an immunoglobulin molecule.
70. The method according to any one of claims 68 to 71 wherein the detectable moiety is selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety; an enzymatic moiety.
71. The method according to claim 69 wherein the detectable moiety is fluorescent moiety (for example an Alexa Fluor dye, e.g. Alexa647).
72. The method according to any one of the preceding claims wherein the method comprises or consists of an ELISA (Enzyme Linked Immunosorbent Assay).
73. The method according to any one of the preceding claims wherein step (b), (d) and/or step (f) is performed using an array.
74. The method according to claim 73 wherein the array is a bead-based array.
75. The method according to claim 73 wherein the array is a surface-based array.
76. The method according to any one of claims 73 to 75 wherein the array is selected from the group consisting of: macroarray; microarray; nanoarray.
77. The method according to any one of the preceding claims wherein the method comprises: (v) labelling biomarkers present in the sample with biotin; (vi) contacting the biotin-labelled proteins with an array comprising a plurality of scFv immobilised at discrete locations on its surface, the scFv having specificity for one or more of the proteins in Table A or B; (vii) contacting the immobilised scFv with a streptavidin conjugate comprising a fluorescent dye; and (viii) detecting the presence of the dye at discrete locations on the array surface wherein the expression of the dye on the array surface is indicative of the expression of a biomarker from Table III in the sample.
78. The method according to any one of claims 1 to 55 wherein, step (b), (d) and/or (f) comprises measuring the expression of a nucleic acid molecule encoding the one or more biomarkers.
79. The method according to claim 78, wherein the nucleic acid molecule is a cDNA molecule or an mRNA molecule.
80. The method according to claim 78, wherein the nucleic acid molecule is an mRNA molecule.
81. The method according to claim 78, 79 or 80, wherein measuring the expression of the one or more biomarker(s) in step (b), (d) and/or (f) is performed using a method selected from the group consisting of Southern hybridisation, Northern hybridisation, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative real-time PCR (qRT-PCR), nanoarray, microarray, macroarray, autoradiography and in situ hybridisation.
82. The method according to any one of claims 78-81, wherein measuring the expression of the one or more biomarker(s) in step (b) is determined using a DNA microarray.
83. The method according to any one of claims 78-82, wherein measuring the expression of the one or more biomarker(s) in step (b), (d) and/or (f) is performed using one or more binding moieties, each individually capable of binding selectively to a nucleic acid molecule encoding one of the biomarkers identified in Table A or Table B.
84. The method according to claim 83, wherein the one or more binding moieties each comprise or consist of a nucleic acid molecule.
85. The method according to claim 84 wherein, the one or more binding moieties each comprise or consist of DNA, RNA, PNA, LNA, GNA, TNA or PMO.
86. The method according to claim 84 or 85, wherein the one or more binding moieties each comprise or consist of DNA.
87. The method according to any one of claims 84-86 wherein the one or more binding moieties are 5 to 100 nucleotides in length.
88. The method according to any one of claims 84-86 wherein the one or more nucleic acid molecules are 15 to 35 nucleotides in length.
89. The method according to any one of claims 84-88 wherein the binding moiety comprises a detectable moiety.
90. The method according to claim 89 wherein the detectable moiety is selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety (for example, a radioactive atom); or an enzymatic moiety.
91. The method according to claim 90 wherein the detectable moiety comprises or consists of a radioactive atom.
92. The method according to claim 91 wherein the radioactive atom is selected from the group consisting of technetium-99m, iodine-123, iodine-125, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, phosphorus-32, sulphur-35, deuterium, tritium, rhenium-186, rhenium-188 and yttrium-90.
93. The method according to claim 90 wherein the detectable moiety of the binding moiety is a fluorescent moiety.
94. The method according to any one of the preceding claims wherein, the sample provided in step (a), (c) and/or (e) is selected from the group consisting of unfractionated blood, plasma, serum, tissue fluid, breast tissue, milk, bile and urine.
95. The method according to claim 94, wherein the sample provided in step (a), (c) and/or (e) is selected from the group consisting of unfractionated blood, plasma and serum.
96. The method according to claim 94 or 95, wherein the sample provided in step (a), (c) and/or (e) is serum.
97. The method according to any one of the preceding claims wherein the predicative accuracy of the method, as determined by an ROC AUC value, is at least 0.50, for example at least 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98 or at least 0.99.
98. The method according to claim 97 wherein the predicative accuracy of the method, as determined by an ROC AUC value, is at least 0.70.
99. The method according to any one of the preceding claims wherein, in the event that the individual is diagnosed with breast cancer, the method comprises the step of: (g) providing the individual with breast cancer therapy.
100. The method according to claim 99 wherein the breast cancer therapy is selected from the group consisting of surgery, chemotherapy, immunotherapy, chemoimmunotherapy and thermochemotherapy.
101. An array for determining the presence of breast cancer in an individual comprising one or more binding agent as defined in any one of claims 57-72 and 79-93.
102. The array according to claim 101 wherein the one or more binding agents is capable of binding to all of the proteins defined in Table A or Table B.
103. Use of one or more biomarkers selected from the group defined in Table A or Table B as a biomarker for determining the presence of breast cancer in an individual.
104. The use according to claim 103 wherein all of the proteins defined in Table A or Table B are used as a diagnostic marker for determining the presence of breast cancer in an individual.
105. The use of one or more binding moiety as defined in any one of claims 57-72 and 79-93 for determining the presence of breast cancer in an individual.
106. The use according to claim 105 wherein biomarkers for all of the proteins defined in Table A or Table B are used.
107. A kit for determining the presence of breast cancer comprising: C) one or more binding agent as defined in any one of claims 57-72 and 79-93 or an array according to claims 73-77 or claim 101-102; D) instructions for performing the method as defined in any one of claims 1-100.
108. A method of treating breast cancer in an individual comprising the steps of: (a) diagnosing breast cancer according to the method defined in any one of claims 1-100; and (b) providing the individual with breast cancer therapy.
109. The method according to claim 108 wherein the breast cancer therapy is selected from the group consisting of surgery, chemotherapy, immunotherapy, chemoimmunotherapy and thermochemotherapy.
110. A method or use for determining the presence of breast cancer in an individual substantially as described herein.
111. An array or kit for determining the presence of breast cancer in an individual substantially as described herein.
Description
[0195] Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:
[0196]
[0197] Classification of early (pre-diagnosed) breast cancer (BC) vs. healthy controls (N). (A) ROC curve for early BC vs. N. (B) Significantly differentially expressed analytes. A fold change >1 represents an up-regulation in BC vs. N, and vice versa. BC-1 represents a 9 serum biomarker signature reflecting BC at time of diagnosis, including patients taking inflammatory drugs and/or hormones, data adopted from (21). BC-2 represents a 8 serum biomarker signature reflecting BC at time of diagnosis, excluding patients taking inflammatory drugs and/or hormones, data adopted from (21).
[0198]
[0199] Immunoprofiling of early (pre-diagnosed) breast cancer (BC) vs. healthy controls (N), where the BC samples were divided into two cohorts based on the time of sample collection prior to diagnosis (0-52 and 52-104 weeks). (A) Significantly differentially expressed analytes for BC (weeks 0-52) vs. N. A fold change >1 represents an up-regulation in BC vs. N, and vice versa. (B) Significantly differentially expressed analytes for BC (weeks 52-104) vs. N.
[0200]
[0201] Immunoprofiling of early (pre-diagnosed) breast cancer (BC) vs. healthy controls (N), where the BC samples were divided into four cohorts based on the time of sample collection prior to diagnosis (0-26, 26-52, 52-78, and 78-104 weeks). (A) Significantly differentially expressed analytes for BC (weeks 0-26) vs. N. A fold change >1 represents an up-regulation in BC vs. N, and vice versa. (B) Significantly differentially expressed analytes for BC (weeks 26-52) vs. N. (C) Significantly differentially expressed analytes for BC (weeks 52-78) vs. N. (D) Significantly differentially expressed analytes for BC (weeks 78-104) vs. N.
[0202]
[0203] Immunoprofiling of early (pre-diagnosed) breast cancer (BC), where the BC samples were divided into four cohorts based on the time of sample collection prior to diagnosis (0-26, 26-52, 52-78, and 78-104 weeks). (A) Classification of BC (weeks 0-26) vs. BC (weeks 26-52) vs. BC (weeks 52-78) vs. BC (weeks 78-104). The ROC AUC values (LOO cross-validation) are stated. (B) The number of differentially expressed analytes for BC (weeks 0-26) vs. BC (weeks 26-52) vs. BC (weeks 52-78) vs. BC (weeks 78-104). In each comparison, a majority of the de-regulated analytes were either up- or down-regulated, as indicated by the direction of the arrows. (C) The top 15 differentially expressed analytes for BC (weeks 0-26) vs. BC (weeks (26-52). (D) The top 15 differentially expressed analytes for BC (weeks 26-52) vs. BC (weeks (52-78). (E) The top 15 differentially expressed analytes for BC (weeks 52-78) vs. BC (weeks (78-104).
[0204]
[0205] Immunoprofiling of early (pre-diagnosed) breast cancer (BC), where the BC samples were divided into four cohorts based on the time of sample collection prior to diagnosis (0-26, 26-52, 52-78, and 78-104 weeks). (A) Expression pattern for ten selected key analytes. Significantly differentially up- or down-regulated analytes are indicated by an arrow. (B) Expression pattern for three selected key analytes, in terms of the observed antibody microarray signal intensities. The intensities are normalized and logged.
[0206] Supplementary
[0207] Correlation between tumor size at time of diagnosis and time (weeks) for sample collection prior to diagnosis.
[0208] Supplementary
[0209] Mapping of clinical parameters onto the early BC samples, and stratification using PCA analysis. (A) ER positive vs. ER negative. (B) PgR positive vs. PgR negative. (C) Histological grade 1 vs. grade 2 vs. grade 3. (D) Pre-menopausal vs. post-menopausal. (E) BMI group 1 (<18.5) vs. group 2 (18.5-24.9) vs. group 3 (25.0-29.9) vs. group 4 (30.0-34.9) vs. group 5 (35.0-39.9) vs. group 6 (>6). The grouping was based on the guidelines from World Health Organization.
[0210] Supplementary
[0211] Immunoprofiling of early (pre-diagnosed) breast cancer (BC), where the BC samples were filtered for tumor size 20 mm to be included) and divided into three cohorts based on the time of sample collection prior to diagnosis (0-35, 35-69, and 70-104 weeks). (A) Significantly differentially expressed analytes for BC (weeks 0-35) vs. N. A fold change >1 represents an up-regulation in BC vs. N, and vice versa. (B) Significantly differentially expressed analytes for BC (weeks 70-104) vs. N.
[0212] Supplementary
[0213] Immunoprofiling of early (pre-diagnosed) breast cancer (BC), where the BC samples were divided into four cohorts based on the time of sample collection prior to diagnosis (0-26, 26-52, 52-78, and 78-104 weeks). (A) Significantly differentially expressed analytes for BC (weeks 0-26) vs. BC (weeks 26-52). A fold change >1 represents an up-regulation in BC vs. N, and vice versa. (B) Significantly differentially expressed analytes for BC (weeks 26-52) vs. BC (weeks 52-78). (C) Significantly differentially expressed analytes for BC (weeks 52-78) vs. BC (weeks 78-104).
[0214] Supplementary
[0215] The smallest panel of antibodies required to achieve the best classification (minimized error) of the various early BC cohorts vs. N, using a backward elimination strategy was implemented. (A) Early BC (all samples) vs. N. (B) Early BC (0-52 weeks) vs. N. (C) Early BC (52-104 weeks) vs. N. (D) Early BC (0-26 weeks) vs N. (E) Early BC (26-52 weeks) vs. N. (F) Early BC (52-78 weeks) vs. N. (G) Early BC (78-104 weeks) vs. N.
EXAMPLES
Introduction
[0216] There is a significant need for deciphering disease-associated biomarkers in early breast cancer (BC), which could pave the way for early and improved diagnosis, as well as provide a deeper understanding of the underlying disease biology and processes involved in early BC. In this study, we have for the first time performed immunoprofiling of early human BC by targeting human serum samples collected up to two years before clinical diagnosis. To this end, we utilized the immune system as an early and specific sensor for disease, by profiling predominantly immunoregulatory and cancer-associated analytes using affinity proteomics. The data showed that several disease progression associated serum biomarkers could be delineated in early human BC. The observed serological profiles shed light on biological processes involved in early BC, such as cancer immunoediting, and provide novel opportunities for early BC diagnosis and classification. Taken together, this study demonstrated that a minimally invasive blood sample harbored cancer-specific information, reflecting early human BC and key associated biological processes thereof (at least) up to two years before diagnosis.
[0217] Material and Methods
[0218] Clinical Samples
[0219] The Malm Diet and Cancer study is a population-based, prospective study in which a total of 17,035 women (born between 1923 and 1950) were enrolled and followed (between 1991 and 1996) (24, 25). After informed consent, serum samples were collected at time of enrollment and stored at 80 until use. A total of 255 patients were selected, including 85 patients clinically diagnosed with breast cancer (BC)<2 years after enrollment (i.e. sample collection), and 170 patients (healthy controls, N) matched with age and weight (body mass index, BMI).
[0220] The serum samples were biotinylated, using a previously optimized protocol (18, 19). Briefly, crude samples were diluted 1:45 in PBS, resulting in an approximate protein concentration of 2 mg/mL, and labeled with a 15:1 molar excess of biotin to protein, using 0.6 mM EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill., USA). Unbound biotin was removed by dialysis against PBS for 72 hours. Labeled samples were aliquoted and stored at 20 C. until further use.
[0221] Antibodies
[0222] In total, 293 human recombinant single-chain Fv (scFv) antibodies directed against 98 antigens and 31 peptide motifs, denoted CIMS (contex-independent motif specific) antibody clones 1-31 (26), were used as microarray content (Table 2). A majority of the antibodies were selected against immunoregulatory analytes and cancer-associated proteins in order to exploit the immune system as an early and specific sensor for disease (23). The specificity, affinity (normally in the nM range), and on-chip functionality of these phage display derived scFv antibodies (27) (Sall et al, 2014 manuscript in preparation) was ensured by using i) stringent phage-display selection and screening protocols, ii) multiple clones (1-4) per target, and iii) a molecular design, adapted for microarray applications (28). In addition, the specificity of several of the antibodies has previously also been validated using well-characterized, standardized serum samples (with known levels of the targeted analytes), and/or orthogonal methods, such as mass spectrometry (affinity pull-down experiments), ELISA, MesoScaleDiscovery (MSD) assay, cytometric bead assay, and MS, as well as using spiking and blocking experiments (29-37). Notably, the reactivity of some antibodies might be lost since the label (biotin) used to enable detection could block the affinity binding to the antibodies (epitope masking), but we have bypassed this problem, as in this study, by frequently including more than one antibody against the same protein, but directed against different epitopes (28).
[0223] The antibodies were produced in 15 mL E. coli cultures and purified from the periplasm in 300 L, using a MagneHis Protein Purification system (Promega, Madison, Wis., USA) and a KingFisher96 robot (Thermo Fisher Scientific, Waltham, Mass., USA). The elution buffer was exchanged for PBS, using Zeba 96-well desalt spin plates (Pierce). The protein concentration was measured, using NanoDrop (Thermo Scientific, Wilmington, Del., USA) and the purity was checked, using 10% SDS-PAGE (Invitrogen, Carlsbad, Calif., USA).
[0224] Antibody Microarrays
[0225] The antibody microarray analysis was performed using a previously optimized protocol (17, 38) (Delfani et al, manuscript in preparation). The antibody microarrays were produced on black MaxiSorp slides (Nunc, Roskilde, Denmark) using a non-contact printer (SciFlexarrayer S11, Scienon, Berlin, Germany). Thirteen identical subarrays were printed on each slide, consisting of 3331 spots, with a spot diameter of 130 m, and a spot center-to-center distance of 200 m. Each subarray was divided into three segments, separated by printed rows of labeled BSA. Each antibody was spotted in triplicates, one replicate in each segment. 10 slides were printed each day, resulting in a total of 130 subarrays per day, for three days. The slides were produced over night, and the arrays were subsequently used for array analysis the following day.
[0226] Each slide was mounted in a hybridization gasket (Schott, Jena, Germany) and blocked with 1% (w/v) milk, 1% (w/v) Tween-20 in PBS (MTPBS) for one hour. In the meantime, samples were thawed on ice, and diluted 1:10 in MTPBS in Costar non-treated 96-well plates (Corning, N.Y., USA). The slides were washed 4 times with 0.05% (v/v) Tween-20 in PBS (PBST) before 120 L of the samples were added. Samples were incubated for 2 hours on a rocking table, slides washed 4 times with PBST, incubated with 1 g/mL Streptavidin-Alexa in PBSMT for 1 hour on a rocking table, and again washed 4 times with PBST. Finally, the slides were dismounted from the hybridization chambers, directly immersed in dH.sub.2O, and dried under a stream of N2. The slides were immediately scanned in a confocal microarray scanner (PerkinElmer Life and Analytical Sciences, Wellesley, Mass., USA) at 10 m resolution, using 60% PMT gain and 90% laser power. Signal intensities were quantified using the ScanArray Express software version 4.0 (Perkin Elmer Life and Analytical Sciences), and the fixed circle option. Signal intensity values with local background subtraction were used for data analysis.
[0227] Microarray Data Pre-Processing
[0228] The antibody microarray data was pre-processed using a previously designed strategy (17, 38) (Delfani et al, manuscript in preparation). An average value of three replicate spots, spread out over the array, was used unless any replicate coefficient of variation (CV) exceeded 15% from the mean value, in which case it was dismissed, and the average value of the two remaining replicates was used instead. The average CV of replication was 7%. Applying a cut-off CV of 15%, 88% of the data values were calculated from all three replicate spots, and the remaining 12% from two replicates.
[0229] The limit of detection (LOD) was defined as the average blank signal (PBS) plus 2 standard deviations. Any antibodies from which signal intensities were found to be below LOD in >30% of samples were removed, resulting in the removal of four antibody clones against GLP-1R, MCP-4, IL-3, and STAT1. Hence, the subsequent microarray data analysis was performed based on 289 antibodies.
[0230] For evaluation of normalization strategies and initial analysis on variance, the data was visualized using principal component analysis (PCA) and hierarchical clustering (QIucore, Lund, Sweden). PCA on log 2 raw data showed some systematic differences between days of analysis, and minor systematic differences between arrays within in the same day of analysis. These differences were effectively neutralized by normalization, which was carried out in two steps. First, the differences between days of analysis were eliminated using a subtract by group mean strategy (39). Briefly, the average intensity for each antibody over all the samples within one day was calculated, and subtracted from the single values, thus zero centering the data. To avoid negative values, the global mean signal of each antibody was then added to each respective data point. In a next step, the array to array differences observed within the same day of analysis were removed by using a semi-global normalization approach reported earlier (17, 38) (Delfani et al, manuscript in preparation), with a minor modification. Thus, a scaling factor was calculated for each subarray, based on the 15% of antibodies with the lowest standard deviation (previously CV) over all samples. This scaling factor was then applied to the data from each sample.
[0231] Microarray Data Analysis
[0232] The antibody microarray data was analysed using a previously designed strategy (17, 38) (Delfani et al, manuscript in preparation). The BC patients were analysed as one (n=85), two (samples collected <52 (n=34) vs. 52-104 (n=51) weeks prior to diagnosis), or four cohorts (samples collected <26 (n=13) vs. 26-52 (n=21) vs. 52-78 (n=21) vs. 78-104 (n=39) weeks prior to diagnosis). In one set of analysis, the BC patients were first filtered for tumor size 20 mm), before the remaining patients were divided into three cohorts (samples collected <35 (n=16) vs. 35-69 (n=16) vs. 70-104 (n=25) weeks prior to diagnosis). All statistical analysis was based on two-group comparisons. In an attempt to classify BC, and subsets thereof, vs. N (n=170), we used support vector machine (SVM), a supervised learning method in R (40-42) that creates a hyperplane between two pre-defined groups of data. The SVM was trained using leave-one-out (LOO) cross-validation, where one sample is left out while creating the hyperplane, after which the classifier tried to correctly classify the left-out sample. After each iteration, a decision value was calculated based on the distance between the sample and the hyperplane. Based on the decision values, a receiver operating characteristics (ROC) curve was constructed and area under the curve (AUC) value was calculated. No filtration of the data was performed before training the data, i.e. data from all antibodies on the arrays were included in the analysis. ROC AUC values were also calculated for each single antibody using the array signal intensities. Given the expression values for a given antibody, then each sample was classified as either positive or negative by introducing a cut such that e.g. the sample was positive if the signal was larger than this cut. Thus, a specific cut resulted in a sensitivity-specificity pair by comparing with the true sample labels. A ROC curve was then computed by considering all possible cuts for this antibody. Significantly up- or down-regulated analytes (p<0.05) were defined based on relative protein levels and identified using Wilcoxon's signed-rank test. The Benjamini-Hochberg procedure was used for false discovery rate control (q-values) (43). Clinical parameter was mapped onto the BC samples, and visualized/stratified using PCA analysis (QIucore). In order to identify panels of antibodies with the most discriminatory power between two groups, a cross-validated backward elimination strategy was applied, as described previously (21). Briefly, the strategy involved identifying members (antibodies) recognizing orthogonal patterns in the dataset, and removing members which did not contribute to the discriminatory power, in an iterative manner, resulting in a list with a minimal number of members (antibodies) which discriminate the two groups most efficiently.
[0233] Results
[0234] In order to characterize the serological profile of early breast cancer, we performed immunoprofiling of breast cancer patients (n=85) vs. controls (n=170) for which the serum samples were collected prior 104 weeks) to clinical breast cancer diagnosis. To this end, we performed protein expression profiling of crude, biotinylated serum samples using recombinant antibody microarrays targeting predominantly immunoregulatory and cancer-associated analytes.
[0235] Classification of Early BC Vs. N
[0236] In an attempt to classify early breast cancer (BC) from healthy controls (N), SVM LOO cross-validation on unfiltered data was performed. The results showed that the classification was moderate, as illustrated by a ROC AUC of 0.65 (
[0237] In order to investigate the relevance of the apparent early cancer-associated signature, we compared it to two known serum protein signatures found to be associated with BC at the time of diagnosis (
[0238] Refined Classification of Early BC Vs. N
[0239] To refine the classification of early BC vs. N, the BC samples were divided into two or four cohorts based on the time of sample collection prior to diagnosis, and SVM LOO cross-validation on unfiltered data was performed. Dividing the samples in two cohorts resulted in ROC AUC values of 0.59 (weeks 0-52) and 0.69 (weeks 52-104), respectively (Table 3). Adopting four BC cohorts, the results indicated that the classification was poor to moderate (ROC AUC of 0.5 to 0.72), but improved the earlier the samples had been collected prior to diagnosis with 78-104 weeks >52-78 weeks <26-52 weeks >0-26 weeks (Table 3).
[0240] In order to define the smallest panel of antibodies required to achieve the best classification (minimized error) of the early BC cohorts vs. N, a backward elimination strategy was implemented. The results indicated that a panel of 45 antibodies (0-52 weeks vs. N) and 58 antibodies (52-104 weeks vs. N) achieved the best classification (minimized error) of early BC vs. N when BC was divided into two cohorts, respectively (Supplementary
[0241] Since the early BC sample cohorts were defined based on time of sample collection prior to diagnosis, the cohorts were, as could be expected, found to be heterogeneous with respect to tumor size (2-120 mm) (Supplementary
[0242] Next, we mapped key clinical parameters, such as oestrogen receptor (ER) status, progesterone receptor (PgR) status, histological grade, pre-/post-menopausal status, and BMI, on the BC samples, and examined whether they could be stratified. Albeit limited by the sample number and that all clinical parameters were not recorded for all patients, the results indicated that neither ER status, PgR status, histological grade, pre-/post-menopausal status, nor BMI could be pin-pointed as confounding factors (Supplementary
[0243] Immunoprofiling of Early BC Vs. N
[0244] In order to decipher biological differences between early BC vs. N, their serological immunoprofiles were compared and evaluated in terms of the identity, nature and number of differentially (p<0.05) expressed analytes. When the early BC samples were divided into two cohorts, 34 (p<0.05, q<0.3) (0-52 weeks) (
[0245] This scenario could be even further refined by dividing the early BC sample into four cohorts and re-running the analysis (cfs. Table 3 and
[0246] In an attempt to examine the influence of the tumor size, which is part of determining the stage of the cancer, on the observed biological differences, we again only included patients with 2-20 mm sized tumors, divided into three cohorts, and re-run the immunoprofiling of early BC vs. N. The results showed that the number of differentially expressed analytes decreased in the order of 33 (70-104 week) >1 (0-35) >0 (36-59 weeks) (Table 3 and Supplementary
[0247] Immunoprofiling of Early BC
[0248] In order to further unravel the molecular pattern of early BC, we compared the serological immunoprofiles of the four BC sub-cohorts, divided based on the time of sample collection prior to diagnosis. Running SVM LOO cross-validation on unfiltered data showed on poor to moderate classification, as illustrated by ROC AUC values of 0.50 to 0.71 (
[0249] When comparing the biological differences in terms of number of differentially expressed analytes, an intricate pattern of numerous up- and down-regulated analytes was observed (
[0250] The top 15 most differentially expressed analytes are displayed for each comparison in
DISCUSSION
[0251] Major proteomic efforts have been made to decipher BC-associated biomarkers, but a majority of these have used biological samples collected at or after diagnosis (44, 45). To the best of our knowledge, only a few studies have so far been designed to target serum samples collected prior to diagnosis (1, 2, 46), which could open up novel avenues for decoding serological biomarker panels reflecting early BC. Using a mass spectrometry-based discovery approach, Opstal-van Winden and co-workers have indicated a handful of serum biomarkers to be de-regulated in early BC up to three years before diagnosis, such C3a des-arginine anaphylatoxin and apolipoprotein C-I (1, 2). In comparison, our data pin-pointed C3 to be de-regulated in early BC vs. N up to two years before diagnosis. C3 plays a central role in the complement system and contributes to innate immunity, and is proteolytically cleaved to C3a and C3b upon activation of the complement cascade. Further, the authors concluded the need for additional efforts, in particular studies using other analytical techniques (better suited for profiling crude serum samples) to generate more data on early BC (1, 2). In a follow up study, Opstal-van Winden and co-workers then used a bead-based multiplexed immunoassay to target ten pre-selected markers (e.g. CA 19-9, CEA, CA-125, haptoglobin, and leptin), known to be associated with diagnosed BC, to analyse early BC serum samples (46). While the assay worked satisfactorily, the data showed that early BC vs. controls could not be differentiated based on these analytes, indicating the need of defining early BC markers.
[0252] In this study, we have for the first time used recombinant antibody microarrays to perform serum protein expression profiling of early human BC, by targeting crude, i.e. non-fractionated, serum samples collected up to two years before diagnosis. In our focused discovery approach, we harvested the immune system as an early sensor for disease by targeting a large set of predominantly immunoregulatory analytes and cancer-associated markers. Our results showed that several de-regulated analytes could be defined in serum samples collected up to two years prior to diagnosis, clearly indicating the applicability of our approach for deciphering early BC associated serum biomarkers.
[0253] The analysis showed that early BC vs. N could be classified with moderate performance, illustrated by ROC AUC values of 0.67 (all BC samples), 0.72 (samples collected 70-104 weeks before diagnosis), and 0.71 (tumors 20 mm, and collected 70-104 weeks before diagnosis). Hence, the data indicated novel opportunities for early detection and diagnosis, up to 70-104 weeks before clinical diagnosis. Reviewing the list of de-regulated analytes, many of the proteins have previously been shown to be associated with diagnosed BC (e.g. C3, IL-7, IL-8, and IL-18) (20, 21, 44, 45, 47) and/or early BC (e.g. IL-10 and IL-12) (4, 5, 7, 10, 11), clearly demonstrating the relevance of our findings. It should, however, be noted that these markers were pin-pointed mainly as single or low-plex markers in the previous studies, and not as part of large multiplexed serum biomarker panels as in our study, in particular for early human BC.
[0254] Notably, examining the immunoprofiles of early human BC, and cohorts thereof, in more detail revealed several serum biomarkers that have been described as markers for disease progression by the cancer immunoediting concept in mice and/or humans (4, 5, 7, 9-11). In more detail, a pattern of deregulated key cytokines with both anti-tumor properties (e.g. IL-1, IL-1, IL-12, and IFN-) and tumor promoting, i.e. immunosuppressive, properties (e.g. IL-10, TGF-, and VEGF) were observed. To the best of our knowledge, this is the first time such detailed multiplexed immunoprofiles of crude serum samples have been described for early human BC, targeting samples collected up to two years before clinical diagnosis.
[0255] Compared to the healthy controls, a pattern of mainly up- (in 3 cohorts) or down-regulated (1 cohort) analytes was observed over time when dividing the early BC samples into four time-dependent cohorts. The largest differences, with respect to the number of differentially expressed proteins, was observed for samples collected 26-52 weeks before diagnosis. Hence, the data implied significant immunoregulatory and/or cancer-associated processes taking place in early breast cancer over time, potentially peaking 26-52 weeks before diagnosis. Considering the nature of key de-regulated analytes (e.g. IL-10, IL-12, and IFN- etc), this might be interpreted in terms of cancer immunoediting processes (4, 5, 7, 9-11). However, tumors of different sizes, 2-120 mm, were analysed, which might impact the results, since the size is part of determining the stage of the cancer, in other words, in which phase of cancer immunoediting each individual tumor might be. In accordance, the data also indicated that significant immunological processes involving similar analytes occurred in tumors of different sizes (2-20 mm vs. 2-120 mm), but at different timelines (70-104 weeks vs. 26-52 weeks).
[0256] When comparing the four cohorts of early BC samples with each other with respect to the nature and number of differentially expressed analytes, the data indicated, as might be expected (4, 5, 7, 9-11), that different and significant immunoregulatory and/or cancer-associated process took place during the progression of the disease towards clinically diagnosed BC, in particular during weeks 26-52. Again, many analytes known to be involved in the cancer immunoediting process (4, 5, 7, 9-11), including cytokines with both anti-tumor properties (e.g. IL-1, IL-1, IL-12, IFN-, and TNF-) and immunosuppressive properties (e.g. IL-10, TGF-, and VEGF) were found to be de-regulated. Many of these key counteracting analytes, such as IL10, IL-12, and IFN- were found to display similar expression patterns over time. Although the profiles revealed large changes occurring over time, the ratio (balance) of IL-10 vs. IL-12 or IFN- did not change significantly. The balance of these analytes is essential for estimating in which phase of the cancer immunoediting process a specific tumor is (4, 10).
[0257] In the first elimination phase, the balance is displaced towards IL-12 and IFN-, promoting tumor immunity (4, 10). In the second equilibrium phase, there is a balance between the tumor promoting and immunosuppressive cytokines, while the balance is displaced towards IL-10 (immunosuppression) in the final escape phase (4, 10). Still, the data indicated the potential of studying the detailed serological profile of early BC samples using affinity proteomics, and highlighted the need for analyzing numerous additional well-characterized early BC samples in order to pre-validate the results and to take the data analysis to the next level of resolution.
[0258] Taken together, this study demonstrated that a minimally invasive blood sample harbored disease-specific information, i.e., biomarkers reflecting early human BC and key associated biological processes thereof up to two years before diagnosis. Hence, the observed serological profiles sheds further light on biological processes involved in early BC, such as cancer immunoediting, and provides novel opportunities for early BC diagnosis and classification.
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TABLES
[0306]
TABLE-US-00003 TABLE A # Biomarker Table A(i) - Core biomarkers 1. CIMS - SGSG-EDFR 2. CIMS - SGSG-TEEQLK 3. CIMS - SGSG-LSADHR Table A(ii) - Preferred biomarkers 4. AKT3 5. Angiomotin 6. Apo-A1 7. C1s 8. C1q 9. CDK2 10. CIMS - SGSG-DFAEDK 11. CIMS - SGSG-EPFR 12. CIMS - SGSG-FLLMQYGGMDEHAR 13. CIMS - SGSG-GIVKYLYEDEG 14. CIMS - SGSG-LNVWGK 15. CIMS - SGSG-LTEFAK 16. CIMS - SGSG-LWETVQKWREYRRQ 17. CIMS - SGSG-LYEIAR 18. CIMS - SGSG-QEASFK 19. CIMS - SGSG-SEAHLR 20. CIMS - SGSG-SSAYSR 21. CIMS - SGSG-SYVSLK 22. CIMS - SGSG-TLYVGK 23. CIMS - SGSG-WDSR 24. CIMS - SGSG-WTRNSNMNYWLIIRL 25. CSF2 26. CT17 27. Cystatine C 28. Digoxin 29. EGFR 30. FASN 31. GAK 32. GM-CSF 33. HADH2 34. Her2/ErbB-2 35. HLA-DR 36. ICAM-1 37. IgM 38. IL-11 39. IL-2 40. Integrin alpha-11 41. JAK3 42. Keratin19 43. KSYK 44. Leptin 45. Lewis y 46. LUM 47. MATK 48. MK01 49. MK08 50. Mucin-1 51. ORP-3 52. Osteopontin 53. P85A 54. Procathepsin W 55. Properdine 56. PSA 57. PTK6 58. PTPN1 59. RPS6KA2 60. STAP2 61. Surface antigen X 62. TENS4 63. TNF-a 64. TNFRSF14 65. TNFRSF3 66. UBC9 67. UBE2C 68. UCHL5 Table A(iii) - Optional biomarkers 69. Apo-A4 70. ATP5B 71. BTK 72. C1 esterase inhibitor 73. C3 74. 75.
76. CD40 77. CD40L 78. CHX10 79. Eotaxin 80. Factor B 81. GLP-1 82. IFN- 83. IL-10 84. IL-12 85. IL-13 86. IL-16 87. IL-18 88. IL-1a 89. IL-1b 90. IL-1-ra 91.
92. IL-4 93.
94. IL-6 95.
96. IL-8 97. IL-9 98. Integrin alpha-10 99. LDL 100. Lewis x 101. MCP-1 102. MCP-3 103. MCP-4 104. MYOM2 105. OSBPL3 106. RANTES 107. Sialle Lewis x 108. TBC1D9 109. TGF-1 110. TM peptide 111. TNF-b 112. UPF3B 113. VEGF 114. -galactosidase
TABLE-US-00004 TABLE B Sub-table (Related (figure(s)) i ii iii iv v vi vii viii (1B, (2A, (2B, (3A, (3B, (3C, (3D, (S3A, ix x xi xii # Biomarker S5A) S5B) S5C) S5B) S5D) S5E) S5F) S5G) (S3B) (S4A) (S4B) (S4C) 1. AKT3 d/u 2. Angiomotin d/u u u d/u d u d 3. Apo-A1 d d/u u 4. Apo-A4 d/u d/u u u d/u 5. ATP5B d/u d/u d d/u d u d 6. BTK d/u d/u u u d 7. C1 esterase inhibitor u u u u u u d/u u d 8. C1q d/u 9. C1s d/u d/u d/u d/u d 10. C3 d d/u d d d/u d d d u 11. C4 d/u d/u d/u d d d 12. C5 d/u d/u d/u d 13. CD40 d/u d/u u d 14. CD40L u u d/u 15. CDK2 d/u d/u d/u u d/u d d/u u 16. CHX10 d u d 17. CIMS - d/u d SGSG-FLLMQYGGMDEHAR 18. CIMS - d/u u u u u u d SGSG-GIVKYLYEDEG 19. CIMS - d/u u u d u SGSG- LWETVQKWREYRRQ 20. CIMS - d/u u d/u u u d u SGSG-WTRNSNMNYWLIIRL 21. CIMS - SGSG-DFAEDK d/u u d d/u.sup. u/d u d d/u d/u u d 22. CIMS - SGSG-EDFR d/u u d u 23. CIMS - SGSG-EPFR d u d u 24. CIMS - SGSG-LNVWGK u u d u 25. CIMS - SGSG-LSADHR d/u u u d u d 26. CIMS - SGSG-LTEFAK d/u u d u d 27. CIMS - SGSG-LYEIAR u u 28. CIMS - SGSG-QEASFK u u u d u 29. CIMS - SGSG-SEAHLR d/u u u u d u d 30. CIMS - SGSG-SSAYSR u d u 31. CIMS - SGSG-SYVSLK u d d u 32. CIMS - SGSG-TEEQLK d/u u u d 33. CIMS - SGSG-TLYVGK u d u 34. CIMS - SGSG-WDSR d/u d d 35. CSF2 u u u 36. CT17 d/u d/u d/u 37. Cystatine C d/u d/u d 38. Digoxin d/u d/u u d 39. EGFR d/u d/u d/u u 40. Eotaxin u u d/u u d/u u u d u d 41. Factor B d/u d d u 42. FASN d/u d/u d/u u 43. GAK d/u d/u u u 44. GLP-1 d/u d d/u d/u d 45. GM-CSF u u u u d 46. HADH2 d/u d/u d/u u d 47. Her2/ErbB-2 d/u u d/u u d/u u u 48. HLA-DR u d u 49. ICAM-1 d/u d d/u d u d 50. IFN- u u u u 51. IgM u u d/u d/u u d d/u u d u d 52. IL-10 u u d/u u d u d/u d u d 53. IL-11 u u u u u u 54. IL-12 u u d/u d/u u d d/u d u d 55. IL-13 d/u d d/u d d/u u 56. IL-16 u u u d u d u d 57. IL-18 u u d/u u u d u d 58. IL-1a d/u d d u d 59. IL-1b d/u u d/u u d u 60. IL-1-ra u u d u 61. IL-2 d/u u 62. IL-3 d/u d/u u d 63. IL-4 d/u u d/u u d u d u d 64. IL-5 d/u d/u d/u u d/u d 65. IL-6 d/u d/u d/u d/u d/u d/u d u d 66. IL-7 u u u u d u 67. IL-8 u u d/u u d u u d u d 68. IL-9 u u d/u d/u u u u d u 69. Integrin alpha-10 u u u 70. Integrin alpha-11 u d u 71. JAK3 u u d u 72. Keratin 19 d/u u d/u 73. KSYK d/u d/u u u d 74. LDL d/u d/u d 75. Leptin d/u u d u 76. Lewis x u u u u u 77. Lewis y d/u u d/u u 78. LUM d/u d 79. MATK d/u d 80. MCP-1 u u d/u d/u u d u u d u d 81. MCP-3 d/u d/u d/u u d/u u u 82. MCP-4 d/u u u d 83. MK01 d/u d/u d/u d d/u d u d d/u d 84. MK08 d/u u d/u u d d/u u d d 85. Mucin-1 d/u d/u d/u u d 86. MYOM2 d/u u d d 87. ORP-3 u u d u d 88. OSBPL3 d/u 89. OSTP d/u u d/u u d/u u 90. P85A d/u d d u d 91. Procathepsin W d/u d d/u d d 92. Properdine u d 93. PSA d/u u d/u d u 94. PTK6 d/u d/u u 95. PTPN1 d/u 96. RANTES d/u d/u u d/u d/u d u 97. RPS6KA2 d 98. Sialle Lewis x u u u 99. STAP2 d/u u 100. Surface antigen X u d d u d 101. TBC1D9 u u u d u 102. TENS4 d/u 103. TGF-1 d/u d/u d/u u d d/u d u 104. TM peptide u u u u u 105. TNF-a d/u d/u d/u d/u u u d 106. TNF-b u u d/u d/u u d u u d u d 107. TNFRSF14 d/u u d 108. TNFRSF3 d/u d/u d u u d 109. UBC9 d/u u d/u u d/u u 110. UBE2C u d 111. UCHL5 d/u d 112. UPF3B d/u d 113. VEGF d/u d/u u d u u d u d 114. -galactosidase u u u u Total 114 68 60 43 21 78 40 37 1 38 63 82 47
TABLE-US-00005 TABLE 1 Demographic data of the patients included in the study. Parameter Breast Cancer Controls No. of samples 85 170 Age, mean (range) 56.8 (45-71.3) 56.4 (45.8-71.8) Collection time before diagnosis 59.8 (1-103) (weeks), mean (range) Tumor size (mm), mean (range) 19.9 (2-120) Oestrogen receptor (+//n.d.) 61/6/18 Progesterone receptor (+//n.d.) 45/22/18 Grade (1/2/3/n.d.) 13/35/19/18 Pre/Post menopausal 26/59 48/122 BMI, mean (range) 25.4 (18.0-40.2) 25.4 (16.7-47.1) n.d. = not determined
TABLE-US-00006 TABLE 2 Antigens targeted on the antibody microarray No of antibody Protein Full name clones Uniprot Entry Angiomotin Angiomotin 2 Q4VCS5 Apo-A1 Apolipoprotein A1 3 P02647 Apo-A4 Apolipoprotein A4 3 P06727 ATP-5B ATP synthase subunit beta, 3 P06576 mitochondrial b- Beta-galactosidase 1 P16278 galactosidase BTK Tyrosine-protein kinase BTK 4 Q06187 C1 inhibitor Plasma protease C1 inhibitor 4 P05155 C1q* Complement C1q 1 P02745/6/7 C1s Complement C1s 1 P09871 C3* Complement C3 6 P01024 C4* Complement C4 4 P0COL4/5 C5* Complement C5 3 P01031 CD40 CD40 protein 4 Q6P2H9 CD40L CD40 ligand 1 P29965 CDK-2 Cyclin-dependent kinase 2 2 P24941 CHX10 Visual system homeobox 2 3 P58304 CIMS** Context indepndent peptide 31 Peptide motifs - not applicable motifs (4 tp 6 amino acid residues long) CT Cholera toxin subunit B 1 P01556 (control) Cystatin C Cystatin C 4 P01034 Digoxin Digoxin (control) 1 no protein, i.e. not applicable DUSP9 Dual specificity protein 1 Q99956 phosphatase 9 EGFR Epidermal growth factor 1 P00533 receptor Eotaxin Eotaxin 3 P51671 Factor B* Complement factor B 4 P00751 FASN Fatty acid synthase 4 Q6PJJ3 GAK Cyclin G-associated kinase 3 Q5U4P5 GLP-1 Glucagon-like peptide-1 1 P01275 GLP-1R Glucagon-like peptide 1 1 P43220 receptor GM-CSF Granulocyte-macrophage 6 P04141 colony-stimulating factor HADH2 3-hydroxyacyl-CoA 4 Q6IBS9 dehydrogenase type-2 Her2/ErbB-2 Receptor tyrosine-protein 4 P04626 kinase erbB-2 HLA-DR/DP HLA-DR/DP 1 P01903/P01911/P13762/Q30154/P20036/P0440 ICAM-1 Intercellular adhesion 1 P05362 molecule 1 IFN-g Interferon gamma 3 P01579 IgM Immunoglobulin M 5 e.g. P01871 (not complete protein); isotype- specific for IgM on Ramos B cells.sup.1) IL-10* Interleukin-10 3 P22301 IL-11 Interleukin-11 3 P20809 IL-12* Interleukin-12 4 P29459/60 IL-13* Interleukin-13 3 P35225 IL-16 Interleukin-16 3 Q14005 IL-18 Interleukin-18 3 Q14116 IL-1a* Interleukin-1 alpha 3 P01583 IL-1b Interleukin-1 beta 3 P01584 IL-1ra Interleukin-1 receptor 3 P18510 antagonist protein IL-2 Interleukin-2 3 P60568 IL-3 Interleukin-3 3 P08700 IL-4* Interleukin-4 4 P05112 IL-5* Interleukin-5 3 P05113 IL-6* Interleukin-6 8 P05231 IL-7 Interleukin-7 2 P13232 IL-8* Interleukin-8 3 P10145 IL-9 Interleukin-9 3 P15248 Integrin a- Integrin alpha-10 1 O75578 10 Integrin a- Integrin alpha-11 1 Q9UKX5 11 JAK3 Tyrosine-protein kinase JAK3 1 P52333 Keratin19 Keratin, type I cytoskeletal 19 3 P08727 KSYK Tyrosine-protein kinase SYK 2 P43405 LDL Apolipoprotein B-100 2 P04114 Leptin Leptin 1 P41159 Lewis x Lewis x 2 carbohydrate, i.e. not applicable Lewis y Lewis y 1 carbohydrate, i.e. not appliable Lumican Lumican 1 P51884 MAPK1 Mitogen-activated protein 4 P28482 kinase 1 MAPK8 Mitogen-activated protein 3 P45983 kinase 8 MATK Megakaryocyte-associated 3 P42679 tyrosine-protein kinase MCP-1* C-C motif chemokine 2 9 P13500 MCP-3 C-C motif chemokine 7 3 P80098 MCP-4 C-C motif chemokine 13 3 Q99616 MUC-1 Mucin-1 6 P15941 Myomesin-2 Myomesin-2 2 P54296 ORP-3 Oxysterol-binding protein- 2 Q9H4L5 related protein 3 Osteopontin Osteopontin 3 P10451 P85A Phosphatidylinositol 3-kinase 3 P27986 regulatory subunit alpha PKB RAC-gamma serine/threonine- 2 Q9Y243 gamma protein kinase Procathepsin W Procathepsin W 1 P56202 Properdin* Properdin 1 P27918 PSA Prostate-specific antigen 1 P07288 PTK-6 Protein-tyrosine kinase 6 1 Q13882 PTP-1B Tyrosine-protein phosphatase 3 P18031 non-receptor type 1 RANTES C-C motif chemokine 5 3 P13501 RPS6KA2 Ribosomal protein S6 kinase 3 Q15349 alpha-2 Sialyl Lewis x Sialyl Lewis x 1 carbohydrate, i.e. not applicable Sox11A Transcription factor SOX-11 1 P35716 STAP2 Signal-transducing adaptor 4 Q9UGK3 protein 2 STAT1 Signal transducer and activator 2 P42224 of transcription 1-alpha/beta Surface Ag X Surface Ag X 1 not applicable TBC1D9 TBC1 domain family member 9 3 Q6ZT07 TENS4 Tensin-4 1 Q8IZW8 TGF-b1 Transforming growth factor 3 P01137 beta-1 TM peptide Transmembrane peptide 1 peptide antigen, notapplicable TNF-a Tumor necrosis factor 3 P01375 TNF-b* Lymphotoxin-alpha 4 P01374 TNFRSF14 Tumor necrosis factor receptor 2 Q92956 superfamily member 14 TNFRSF3 Tumor necrosis factor receptor 3 P36941 superfamily member 3 UBC9 SUMO-conjugating enzyme 3 P63279 UBC9 UBE2C Ubiquitin-conjugating enzyme 2 O00762 E2 C UCHL5 Ubiquitin carboxyl-terminal 1 Q9Y5K5 hydrolase isozyme L5 UPF3B Regulator of nonsense 2 Q9BZI7 transcripts 3B VEGF* Vascular endothelial growth 4 P15692 factor *Antibody specificity determined by protein arrays, MSD, ELISA, blocking/spiking experiments, and/or mass spectrometry. **31 CIMS clones selected against 18 motifs. Specification of the clones (clone name/linker sequence/selection motif/no. of clones against the motif); CIMS 1-SGSG-FLLMQYGGMDEHAR (1); CIMS 2-SGSG-LWETVQKWREYRRQ (1); CIMS 3-SGSG-GIVKYLYEDEG (2); CIMS 4-SGSG-WTRNSNMNYWLIIRL (2); CIMS 5-SGSG-EDFR (2); CIMS 6-SGSG-LYEIAR (1); CIMS 7-SGSG-DFAEDK (1); CIMS 8-SGSG-LTEFAK (1); CIMS 9-SGSG-TEEQLK (3); CIMS 10-SGSG-SSAYSR (2); CIMS 11-SGSG-SYVSLK (1); CIMS 12-SGSG-TLYVGK (1); CIMS 13-SGSG-EPFR (2); CIMS 14-SGSG-LNVWGK (1); CIMS 15-SGSG-QEASFK (2); CIMS 16-SGSG-LSADHR (2); CIMS 17-SGSG-SEAHLR (4); CIMS 18-SGSG-WDSR (2).
TABLE-US-00007 TABLE 3 Classification of early BC patients vs. N after dividing all BC patients into two or four cohorts based on time of sample collection prior to clinical diagnosis. The BC patients were also filtered for tumor size (20 mm) and divided into three cohorts, and re-compared to the controls. BC cohort, weeks No. of differentially prior to diagnosis ROC AUC expressed analytes All BC samples 0-52 0.59 33 52-104 0.67 5 0-26 0.50 5 26-52 0.51 52 52-78 0.57 21 78-104 0.72 18 BC samples with tumor 20 mm 0-35 0.44 1 35-69 0.38 0 70-104 0.66 33