NZ771630B2 - Methods of detecting prostate cancer - Google Patents
Methods of detecting prostate cancer Download PDFInfo
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- NZ771630B2 NZ771630B2 NZ771630A NZ77163014A NZ771630B2 NZ 771630 B2 NZ771630 B2 NZ 771630B2 NZ 771630 A NZ771630 A NZ 771630A NZ 77163014 A NZ77163014 A NZ 77163014A NZ 771630 B2 NZ771630 B2 NZ 771630B2
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Abstract
Disclosed is a method for diagnosing or aiding in the diagnosis of prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of one or more markers selected from the group consisting of PC-MACRO/PC-NEUTRO 1-200 in a population of the subject's macrophage cells; b) measuring the levels of the one or more selected PC-MACRO/PC-NEUTRO markers in a population of the subject's non-phagocytic cells; and c) identifying a difference between the measured levels of the one or more selected PC-MACRO/PC-NEUTRO markers in steps a) and b), wherein the identified difference indicates that the subject has the prostate cancer.
Description
S OF DETECTING PROSTATE CANCER Related Applications This application is a divisional application of New Zealand Patent Application No. 737187, filed on 10 November 2017 which is a divisional of New Zealand Patent Application No. 631091, filed on 7 March 2014, and is related to International Patent Application No. , filed on 7 March 2014, and claims priority and benefit from U.S. Provisional Patent Application , 559, filed 9 March 2013, the ts and disclosures of which are incorporated by reference in their entirety.
Field of the Invention This invention relates generally to using biological markers for the diagnosis, prognosis, and monitoring of prostate cancer.
Background of the Invention Early diagnosis of prostate cancer often increases the likelihood of sful treatment or cure of such disease. Current diagnostic methods, however, depend largely on population-derived average values obtained from healthy individuals. Personalized diagnostic methods are needed that enable the diagnosis, especially the early diagnosis, of the presence of te cancer in individuals who are not known to have the cancer or who have recurrent prostate cancer.
Leukocytes begin as pluripotent hematopoietic stem cells in the bone marrow and develop along either the myeloid lineage (monocytes, macrophages, neutrophils, eosinophils, and basophils) or the lymphoid e (T and B lymphocytes and natural killer . The major function of the myeloid lineage cells (e.g., neutrophils and macrophages) is the phagocytosis of ious organisms, live unwanted damaged cells, senescent and dead cells otic and necrotic), as well as the clearing of cellular debris. Phagocytes from healthy animals do not ate and are diploid, i.e., have a DNA t of 2n. On average, each cell contains <10 ng DNA, <20 ng RNA, and <300 ng of protein.
Non-phagocytic cells are also diploid and are not involved in the internalization of dead cells or infectious organisms and have a DNA index of one.
The lifetime of various white blood cell subpopulations varies from a few days (e. g., neutrophils) to several months (e. g., macrophages). Like other cell types, leukocytes age and eventually die. During their aging process, human blood- and tissue-derived phagocytes (e. g., neutrophils) t all the classic markers of programmed cell death (i.e., apoptosis), including caspase tion, pyknotic nuclei, and chromatin fragmentation. These cells also display a number of "eat-me" ?ags (e. g., phosphatidylserine, sugars) on the extracellular es of their plasma membranes. Consequently, dying and dead cells and lular fragments thereof are cleared from tissues and blood by other phagocytic cells.
The prostate speci?c antigen is currently one of the most widely used diagnostic measures used to detect prostate cancer. However, false ves and false negatives are common, resulting in mistreatment of patients with no prostate cancer or overtreatment of patients with non-lethal prostate cancer. Thus, improved methods for detecting te cancer are needed.
Summary of the Invention In one aspect, the present invention provides methods for detecting or diagnosing prostate cancer by using at least one or more markers selected from the RO 1-100 (Table 2) and/or PC-MACRO 101-200 (Table 3) and/or PC- NEUTRO 1-100 (Table 4) and/or PC-NEUTRO 101-200 (Table 5). Levels (e.g., gene expression levels, protein expression levels, or activity levels) of the selected s may be measured from macrophages or neutrophils, respectively, and from non-phagocytes, from a t. Such levels then can be compared, e. g., the levels of the selected markers in the phagocytic cells and in the non-phagocytic cells to identify one or more differences between the measured levels, indicating whether the subject has prostate cancer. The identi?ed difference(s) can also be used for assessing the risk of developing prostate cancer, prognosing prostate cancer, monitoring prostate cancer progression or regression, assessing the y of a treatment for te , or identifying a compound capable of ameliorating or treating prostate cancer.
In yet another aspect, the levels of the selected markers in the phagocytic cells may be compared to the levels of the selected markers in a control (e.g., a normal or healthy control subject, or a normal or healthy cell from the subject) to identify one or more differences between the measured levels, indicating whether the subject has prostate cancer, the prognosis of the cancer and the monitoring of the cancer. The identi?ed difference(s) can also be used for ing the risk of developing te cancer, prognosing prostate cancer, monitoring prostate cancer progression or regression, assessing the efficacy of a treatment for prostate cancer, or identifying a compound capable of ameliorating or treating prostate cancer. id="p-9" id="p-9" id="p-9" id="p-9" id="p-9"
id="p-9"
[0009] Some embodiments of this invention are as follows: 1. A method for diagnosing or aiding in the sis of prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of one or more markers selected from the group consisting of PC-MACRO 1-200 in a tion of the subject’s hage cells; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells; and c) identifying a difference between the measured levels of the one or more selected PC-MACRO markers in steps a) and b), n the identified difference tes that the subject has said te cancer. 2. A method for assessing the risk of developing prostate cancer in a t, the method comprising the steps of: a) measuring the levels of one or more s ed from the group consisting of PC-MACRO 1-200 in a population of the subject’s macrophage cells; b) ing the levels of the one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells; and c) identifying a difference between the measured levels of the one or more selected PC-MACRO markers in steps a) and b), wherein the identified difference indicates that the subject has a risk of developing said prostate cancer. 3. A method for prognosing or aiding in the prognosis of prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject’s macrophage cells; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells; and c) identifying a difference between the measured levels of the one or more selected PC-MACRO markers in steps a) and b), wherein the identified ence is tive of the prognosis of said prostate cancer in the subject. 4. A method for ing the cy of a treatment for prostate cancer in a subject comprising: a) measuring the levels of one or more s selected from the group consisting of PC-MACRO 1-200 in a population of the subject’s macrophage cells before the treatment; b) measuring the levels of the one or more selected RO markers in a population of the subject’s non-phagocytic cells before the treatment; c) identifying a first difference between the measured levels of the one or more selected PC-MACRO markers in steps a) and b); d) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s macrophage cells after the treatment; e) measuring the levels of the one or more selected PC-MACRO s in a population of the subject’s non-phagocytic cells after the treatment; f) identifying a second difference n the measured levels of the one or more selected PC-MACRO markers in steps d) and e); and g) identifying a difference between the first difference and the second ence, wherein the difference identified in g) is indicative of the efficacy of the treatment for said prostate cancer in the subject. 5. A method for monitoring the ssion or regression of prostate cancer in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject’s macrophage cells at a ?rst time point; b) measuring the levels of the one or more selected RO markers in a tion of the subject’s non-phagocytic cells at the ?rst time point; c) identifying a ?rst difference n the measured levels of the one or more selected PC-MACRO markers in steps a) and b); d) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s macrophage cells at a second time point; e) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells at the second time point; f) identifying a second difference between the measured levels of the one or more selected PC-MACRO markers in steps d) and e); and g) identifying a difference between the ?rst ence and the second ence, wherein the difference identi?ed in g) is indicative of the progression or regression of said prostate cancer in the subject. 6. A method for identifying a compound capable of ameliorating or treating prostate cancer in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject’s macrophage cells before stering the compound to the subject; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells before administering the compound to the subject; c) fying a ?rst ence between the measured levels of the one or more ed PC-MACRO markers in steps a) and b); d) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s macrophage cells after the administration of the compound; e) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells after the administration of the f) fying a second difference between the ed levels of the one or more selected PC-MACRO markers in steps d) and e); and g) identifying a difference between the ?rst difference and the second difference, wherein the ence identi?ed in g) indicates that the compound is capable of ameliorating or treating said prostate cancer in the subject. 7. A method for diagnosing or aiding in the sis of prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of one or more markers selected from the group ting of PC-NEUTRO 1-200 in a population of the subject’s neutrophil cells; b) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells; and c) identifying a difference between the measured levels of the one or more selected PC-NEUTRO markers in steps a) and b), wherein the identi?ed difference indicates that the subject has said prostate cancer. 8. A method for assessing the risk of developing te cancer in a subject, the method comprising the steps of: a) ing the levels of one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject’s neutrophil cells; b) measuring the levels of the one or more selected TRO markers in a population of the subject’s non-phagocytic cells; and c) identifying a difference between the measured levels of the one or more selected PC-NEUTRO markers in steps a) and b), wherein the ?ed difference indicates that the subject has a risk of developing said prostate cancer. 9. A method for prognosing or aiding in the prognosis of prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of one or more markers selected from the group ting of PC-NEUTRO 1-200 in a population of the subject’s neutrophil cells; b) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells; and c) identifying a difference between the measured levels of the one or more ed PC-NEUTRO s in steps a) and b), wherein the identi?ed difference is indicative of the prognosis of said prostate cancer in the subject.
. A method for assessing the ef?cacy of a treatment for prostate cancer in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject’s neutrophil cells before the treatment; b) ing the levels of the one or more selected PC-NEUTRO s in a tion of the subject’s non-phagocytic cells before the treatment; c) identifying a ?rst difference between the measured levels of the one or more selected PC-NEUTRO s in steps a) and b); d) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject’s neutrophil cells after the treatment; e) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells after the treatment; f) identifying a second difference n the measured levels of the one or more selected PC-NEUTRO markers in steps d) and e); and g) identifying a difference between the ?rst difference and the second difference, wherein the difference identi?ed in g) is indicative of the ef?cacy of the ent for said prostate cancer in the subject. ll. A method for ring the progression or regression of prostate cancer in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject’s neutrophil cells at a ?rst time point; b) ing the levels of the one or more ed PC-NEUTRO markers in a population of the t’s non-phagocytic cells at the ?rst time point; c) identifying a ?rst difference between the measured levels of the one or more selected PC-NEUTRO markers in steps a) and b); d) ing the levels of the one or more selected PC-NEUTRO markers in a population of the subject’s neutrophil cells at a second time point; e) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells at the second time point; f) identifying a second ence between the measured levels of the one or more selected PC-NEUTRO markers in steps d) and e); and g) identifying a difference between the ?rst difference and the second difference, wherein the difference identi?ed in g) is indicative of the progression or regression of said prostate cancer in the subject. 12. A method for identifying a compound capable of ameliorating or treating prostate cancer in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject’s neutrophil cells before administering the compound to the subject; b) measuring the levels of the one or more ed TRO markers in a population of the subject’s non-phagocytic cells before administering the compound to the subject; c) identifying a ?rst difference between the measured levels of the one or more selected PC-NEUTRO markers in steps a) and b); d) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject’s neutrophil cells after the administration of the compound; e) measuring the levels of the one or more selected PC-NEUTRO s in a population of the subject’s non-phagocytic cells after the administration of the compound; f) fying a second difference n the measured levels of the one or more selected PC-NEUTRO markers in steps d) and e); and g) identifying a difference between the first difference and the second difference, wherein the difference identified in g) indicates that the compound is capable of ameliorating or treating said te cancer in the subject. 13. A method for sing or aiding in the diagnosis of prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 in a tion of the t’s macrophage cells, and measuring the levels of at least one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject’s neutrophil cells; b) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells; and measuring the levels of the at least one or more selected TRO markers in a population of the subject’s non-phagocytic cells; c) identifying a difference between the measured levels of the at least one or more ed PC-MACRO markers in steps a) and b); and d) identifying a difference between the measured levels or activities the at least one or more selected PC-NEUTRO markers in steps a) and b); wherein the differences identified in c) and d) indicate that the subject has said prostate cancer. 14. A method for assessing the risk of developing prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject’s hage cells, and measuring the levels of at least one or more s selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject’s neutrophil cells; b) measuring the levels of the at least one or more ed PC-MACRO markers in a population of the subject’s non-phagocytic cells; and measuring the levels of the at least one or more ed PC-NEUTRO markers in a population of the subject’s non-phagocytic cells; c) fying a difference between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and b); and d) identifying a difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps a) and b); wherein the differences identi?ed in c) and d) indicate that the subject has a risk of developing said prostate cancer.
. A method for prognosing or aiding in the prognosis of prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject’s macrophage cells, and measuring the levels of at least one or more markers selected from the group ting of PC-NEUTRO 1-200 in a population of the subject’s neutrophil cells; b) measuring the levels of the at least one or more ed PC-MACRO markers in a population of the t’s non-phagocytic cells; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s agocytic cells; c) identifying a difference between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and b); and d) identifying a difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps a) and b); wherein the differences identi?ed in c) and d) are indicative of the prognosis of said te cancer in the subject. 16. A method for assessing the ef?cacy of a treatment for prostate cancer in a subject comprising: a) measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject’s macrophage cells before the treatment, and measuring the levels of at least one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject’s neutrophil cells before the treatment; b) measuring the levels of the at least one or more selected PC-MACRO markers in a tion of the t’s non-phagocytic cells before the treatment; measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s agocytic cells before the treatment; c) identifying a first difference between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and b); and identifying a second difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps a) and b); d) measuring the levels of the at least one or more selected PC-MACRO marker in a population of the subject’s hage cells after the treatment, and measuring the levels of the at least one or more ed PC-NEUTRO marker in a population of the subject’s neutrophil cells after the treatment; e) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells after the treatment; measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the t’s non-phagocytic cells after the treatment; f) identifying a third difference between the measured levels of the at least one or more selected RO markers in steps d) and e); and g) identifying a fourth difference between the ed levels of the at least one or more selected PC-NEUTRO markers in steps d) and e); h) identifying a difference between the first and second differences; and i) identifying a difference between the third and fourth differences, wherein the ences fied in h) and i) are indicative of the efficacy of the treatment for said prostate cancer in the subject. 17. A method for monitoring the progression or sion of prostate cancer in a subject comprising: a) ing the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject’s macrophage cells at a ?rst time point, and measuring the levels of at least one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject’s neutrophil cells at the ?rst time point; b) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells at the ?rst time point; measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the t’s non-phagocytic cells at the ?rst time point; c) identifying a ?rst difference between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and b); and identifying a second difference between the measured levels of the at least one or more ed PC-NEUTRO markers in steps a) and b); d) ing the levels of the at least one or more selected RO markers in a population of the subject’s macrophage cells at a second time point, and measuring the levels of the at least one or more selected PC-NEUTRO s in a population of the subject’s neutrophil cells at the second time point; e) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells at the second time point; and ing the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells at the second time point; f) identifying a third difference between the measured levels of the at least one or more selected PC-MACRO markers in steps d) and e); and g) identifying a fourth difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps d) and e); h) identifying a difference between the first and second differences; and i) identifying a difference between the third and fourth differences, wherein the differences identi?ed in h) and i) are indicative of the progression or regression of said prostate cancer in the subject. 18. A method for identifying a compound capable of ameliorating or treating prostate cancer in a subject comprising: a) measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the t’s macrophage cells before administering the compound to the subject, and measuring the levels of at least one or more s selected from the group consisting of PC-NEUTRO 1-200 in a population of the t’s neutrophil cells before administering the compound to the t; b) measuring the levels of the at least one or more selected PC-MACRO markers in a tion of the subject’s non-phagocytic cells before administering the compound to the subject; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells before administering the compound to the subject; c) identifying a first difference between the measured levels of the at least one or more selected RO markers in steps a) and b); and identifying a second difference between the measured levels of the at least one or more selected TRO markers in steps a) and b); d) ing the levels of the at least one or more selected PC-MACRO markers in a population of the subject’s macrophage cells after administering the compound to the subject, and measuring the levels of the at least one or more selected TRO markers in a population of the subject’s neutrophil cells after administering the compound to the subject; e) measuring the levels of the at least one or more ed PC-MACRO markers in a population of the subject’s non-phagocytic cells after administering the compound to the subject; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells after stering the compound to the subject; f) identifying a third difference between the measured levels of the at least one or more selected PC-MACRO markers in steps d) and e); and g) identifying a fourth difference between the measured levels of the at least one or more ed PC-NEUTRO markers in steps d) and e); h) fying a difference between the ?rst and second differences; and i) identifying a difference between the third and fourth ences, wherein the differences identi?ed in h) and i) te that the compound is e of ameliorating or treating said prostate cancer in the subject. 19. The method of any one of embodiments 1-18, further comprising measuring at least one standard parameter ated with said prostate cancer.
. The method of embodiment 19, wherein the rd parameter is selected from the group consisting of tumor stage, tumor grade, tumor size, tumor visual characteristics, tumor growth, tumor thickness, tumor progression, tumor metastasis tumor distribution within the body, odor, molecular pathology, genomics, or tumor angiograms. 21. The method of any one of embodiments 13-18, n the selected PC- MACRO markers and the selected PC-NEUTRO markers are measured from the same population of non-phagocytic cells in steps b) or e). 22. The method of any one of embodiments 13-18, wherein the selected PC- MACRO markers and the selected PC-NEUTRO are from different populations of non-phagocytic cells in steps b) or e). 23. The method of any one of embodiments 1-6 and 13-18, wherein at least two, three, four, or ?ve markers are selected from PC-MACRO 1-200. 24. The method of any one of embodiments 1-6 and 13-18, wherein the ed PC-MACRO markers comprise one or more markers selected from the group consisting of P2RYlO, TNFAIP3, CXCRl, , and CHI3Ll. 25. The method of any one of embodiments 1-6 and 13-18, wherein the selected PC-MACRO markers are up-regulated or activated in the macrophage cells compared to the non-phagocytic cells. 26. The method of embodiment 1-6 and 13-18, wherein the ed PC- MACRO markers are up-regulated or activated in the macrophage cells compared to the non-phagocytic cells. 27. The method of any one of embodiments 1-6 and 13-18, wherein the selected PC-MACRO markers are down-regulated or inhibited in the macrophage cells compared to the non-phagocytic cells. 28. The method of any one of ments 1-6 and 13-18, wherein the selected PC-MACRO s are down-regulated or inhibited in the macrophage cells ed to the non-phagocytic cells. 29. The method of any one of embodiments 7-18, wherein at least two, three, four, ?ve, six, seven, eight, nine, ten, or eleven markers are selected from PC- NEUTRO 1-200.
. The method of any one of embodiments 7-18, wherein the selected PC- NEUTRO markers comprise one or more PC-NEUTRO markers selected from the group consisting of EIF3S5, EEEFlAl, RPL23A, RPLl4, RPL23A, RPL3, RPS28, and PTMA. 3 l. The method of any one of embodiments 7-18, n the selected PC- NEUTRO markers comprise one or more markers selected from the group consisting of PC-NEUTRO 1-200 and wherein the selected PC-NEUTRO markers are down-regulated or inhibited in the neutrophil cells compared to the non- ytic cells. 32. The method of any one of embodiments 7-18, wherein the selected PC- NEUTRO markers are down-regulated or inhibited in the neutrophil cells compared to the agocytic cells. 33. The method of any one of embodiments 1-6 and 13-18, further comprising lysing the macrophage cells and the non-phagocytic cells before a). 34. The method of any one of embodiments 1-6 and 13-18, r comprising extracting the cellular contents from the macrophage cells and the non-phagocytic cells before a). 35. The method of any one of embodiments 7-18, further comprising lysing the neutrophil cells and the non-phagocytic cells before a). 36. The method of any one of ments 7-18, r comprising extracting the cellular contents from the neutrophil cells and the non-phagocytic cells before a). 37. The method of embodiment 34, n the cellular contents of the macrophage cells comprise Viable diseased cells, dead ed cells, apoptotic diseased cells, circulating tumor cells, infectious agents, fetal cells, trophoblasts, or fragments thereof. 38. The method of ment 36, wherein the cellular contents of the neutrophil cells comprise Viable diseased cells, dead diseased cells, apoptotic diseased cells, ating tumor cells, infectious agents, fetal cells, blasts, or fragments thereof. 39. The method of embodiment 34, wherein the selected one or more markers are present in the cellular contents of the macrophage cells. 40. The method of embodiment 34, wherein the selected one or more markers are not present in the cellular contents of the non-phagocytic cells. 41. The method of any one of embodiments 1-6 and 13-18, wherein the macrophage cells express the one or more selected PC-MACRO markers. 42. The method of embodiment 36, wherein the ed one or more markers are present in the cellular contents of the neutrophil cells. 43. The method of embodiment 36, wherein the selected one or more markers are not present in the cellular contents of the non-phagocytic cells. 44. The method of any one of embodiments 7-l8, wherein the neutrophil cells express the one or more selected PC-NEUTRO s. 45. The method of any one of embodiments l-l8, wherein the non-phagocytic cells are T cells, B cells, null cells, ils, or mixtures thereof. 46. The method of any one of embodiments 1-6 and 13-18, wherein the macrophage cells are isolated from a bodily ?uid sample, tissues, or cells of the subject. 47. The method of any one of embodiments 7-l8, wherein the neutrophil cells are isolated from a bodily ?uid sample, tissues, or cells of the subject. 48. The method of any one of embodiments l-l8, wherein the non-phagocytic cells are isolated from a bodily ?uid sample, tissues, or cells of the subject. 49. The method of any one of embodiments 46-48, wherein the bodily ?uid sample is blood, urine, stool, saliva, lymph ?uid, cerebrospinal ?uid, synovial ?uid, cystic ?uid, ascites, pleural effusion, ?uid obtained from a pregnant woman in the ?rst ter, ?uid obtained from a pregnant woman in the second trimester, ?uid obtained from a nt woman in the third trimester, maternal blood, ic ?uid, chorionic villus sample, ?uid from a lantation embryo, maternal urine, maternal saliva, placental sample, fetal blood, lavage and cervical vaginal ?uid, interstitial ?uid, or ocular ?uid. 50. The method of any one of embodiments 1-6 and 13-18, n the macrophage cells are isolated using antibodies, using a ligand that binds to a molecular receptor expressed on the plasma membranes of white blood cells, or by ?ow cytometry, cence activated cell sorting, ?ltration, gradient-based centrifugation, n, micro?uidics, magnetic separation technique, ?uorescent- magnetic separation technique, nanostructure, quantum dots, high throughput microscope-based platforms, or a combination f. 51. The method of any one of embodiments 7-18, wherein the neutrophil cells are isolated using antibodies, using a ligand that binds to a molecular receptor expressed on the plasma membranes of white blood cells, or by ?ow cytometry, ?uorescence activated cell sorting, ?ltration, gradient-based centrifugation, n, uidics, magnetic separation technique, ?uorescent-magnetic separation technique, nanostructure, quantum dots, high throughput microscope-based platforms, or a combination thereof. 52. The method of any one of embodiments 1-18, wherein the non-phagocytic cells are isolated using antibodies, using a ligand that binds to a molecular receptor sed on the plasma membranes of white blood cells, or by ?ow cytometry, ?uorescence activated cell sorting, ?ltration, gradient-based centrifugation, elution, micro?uidics, magnetic separation technique, ?uorescent-magnetic separation technique, nanostructure, quantum dots, high throughput microscope-based platforms, or a combination thereof. 53. The method of any one of embodiments 1-6 and 13-18, wherein the hage cells are isolated using a product secreted by the macrophage cells. 54. The method of any one of embodiments 7-l8, wherein the neutrophil cells are isolated by using a product secreted by the neutrophil cells. 55. The method of any one the embodiments 1-6 and 13-18, wherein the macrophage cells are isolated by using a cell surface target on the surface of macrophage cells. 56. The method of any one of embodiments 7-18, wherein the neutrophil cells are isolated by using a cell surface target on the surface of neutrophil cells. 57. The method of embodiment 55, wherein the target is expressed by the macrophage cells. 58. The method of embodiment 55, wherein the target is not expressed by the macrophage cells. 59. The method of embodiment 56, wherein the target is expressed by the neutrophil cells. 60. The method of embodiment 56, wherein the target is not expressed by the neutrophil cells. 61. The method of any one of ments 55-60, wherein the target is a marker of said prostate cancer. 62. The method of any one of embodiments 1-18, wherein the measured levels are gene expression levels. 63. The method of any one of ments 1-18, n the measured levels are n expression levels. 64. The method of any one of the embodiment l-l8, wherein the levels or activities are measured by a ative assay, a quantitative assay, or a combination thereof. 65. The method of embodiment 64, wherein the quantitative assay uses sequencing, direct sequencing, RNA sequencing, whole transcriptome shotgun sequencing, random shotgun sequencing, Sanger dideoxy ation sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, ary electrophoresis, gel electrophoresis, duplex sequencing, cycle cing, single- base ion sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, on PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, read sequencing, -molecule sequencing, sequencing-by-synthesis, real-time cing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD® sequencing, MS-PET sequencing, mass spectrometry, matrix assisted laser desorption/ionization-time of ?ight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser deorption/ionization-time of ?ight (SELDI-TOF) mass spectrometry, quadrupole-time of ?ight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass ometry, secondary ion mass spectrometry (SIMS), polymerase chain reaction (PCR) analysis, tative PCR, real-time PCR, ?uorescence assay, metric assay, uminescent assay, or a combination thereof. 66. The method of embodiment 62, wherein the gene expression levels are measured by polymerase chain reaction (PCR) analysis, sequencing analysis, electrophoretic analysis, restriction fragment length polymorphism (RFLP) analysis, Northern blot analysis, quantitative PCR, reverse-transcriptase-PCR analysis (RT-PCR), allele-speci?c oligonucleotide hybridization analysis, ative genomic hybridization, heteroduplex mobility assay (HMA), single strand conformational polymorphism (SSCP), denaturing gradient gel electrophisis (DGGE), RNAase mismatch analysis, mass spectrometry, tandem mass spectrometry, matrix assisted laser desorption/ionization-time of ?ight (MALDI- TOF) mass spectrometry, electrospray tion (ESI) mass spectrometry, e-enhanced laser deorption/ionization-time of ?ight -TOF) mass spectrometry, quadrupole-time of ?ight (Q-TOF) mass spectrometry, atmospheric re photoionization mass ometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), e plasmon resonance, Southern blot analysis, in situ hybridization, ?uorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), microarray, comparative c hybridization, karyotyping, multiplex ligation- dependent probe ampli?cation (MLPA), Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF), microscopy, methylation c PCR (MSP) assay, HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay, radioactive e labeling assays, colorimetric DNA acetylation assay, chromatin immunoprecipitation combined with microarray (ChIP-on-chip) assay, restriction landmark genomic scanning, Methylated DNA immunoprecipitation (MeDIP), molecular break light assay for DNA adenine methyltransferase activity, chromatographic separation, methylation-sensitive restriction enzyme is, bisulfite-driven conversion of non-methylated ne to uracil, methyl-binding PCR analysis, or a combination thereof. 67. The method of embodiment 62, wherein the gene expression levels are measured by a sequencing technique selected from the group consisting of direct sequencing, RNA sequencing, whole transcriptome shotgun sequencing, random shotgun sequencing, Sanger dideoxy ation cing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel ophoresis, duplex sequencing, cycle sequencing, single- base extension cing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, sequencing by reversible dye ator, paired-end sequencing, near-term cing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD® sequencing, MS-PET sequencing, mass spectrometry, and a combination thereof. 68. The method of embodiment 63, n the protein expression levels are measured by an immunohistochemistry assay, an enzyme-linked immunosorbent assay (ELISA), in situ hybridization, chromatography, liquid chromatography, size exclusion chromatography, high performance liquid chromatography (HPLC), gas chromatography, mass spectrometry, tandem mass spectrometry, matrix assisted laser tion/ionization-time of ?ight -TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser deorption/ionization-time of ?ight (SELDI-TOF) mass spectrometry, quadrupole- time of ?ight (Q-TOF) mass spectrometry, heric re photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser tion/ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), radioimmunoassays, microscopy, micro?uidic chip-based assays, surface plasmon resonance, sequencing, Western blotting assay, or a combination thereof. 69. The method of any one the embodiments 1-68, wherein the subject is a mammal. 70. The method of embodiment 69, wherein the t is a human. 71. The method of any one the embodiments 1-18, wherein the difference is greater than a 1-fold difference. 72. The method of embodiment 71, wherein the difference is at least 1.05- fold, l.l-fold, l.2-fold, l.3-fold, ld, ld, 2-fold, 2.5-fold, , , 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold difference. 73. A kit for measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200, comprising reagents for specifically measuring the levels of the selected PC-MACRO . 74. A kit for measuring the levels of at least one or more markers selected from the group consisting of TRO 1-200, sing reagents for speci?cally measuring the levels of the selected PC-NEUTRO marker. 75. A kit for measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 and at least one or more markers ed from the group consisting of PC-NEUTRO 1-200, comprising reagents for specifically measuring the levels of the selected PC-MACRO marker and reagents for speci?cally measuring the levels of the selected PC-NEUTRO . 76. The kit of embodiment 73 or 75, wherein the selected PC-MACRO markers comprise one or more markers selected from the group consisting of P2RY10, 3, CXCRl, DNAJBl, and CHI3Ll. 77. The kit of embodiment 74 or 75, wherein the selected PC-NEUTRO markers comprise one or more markers selected from the group consisting of EIF3S5, EEEFlAl, RPL23A, RPLl4, RPL23A, RPL3, RPSZ8, and PTMA. 78. The kit of any one of embodiments 73-77, wherein the reagents comprise one or more antibodies or fragments thereof, oligonucleotides, or aptamers. 79. A method of treating or preventing prostate cancer in a subject comprising administering to said subject an agent that modulates the activity or expression of at least one or more markers ed from the group consisting of PC-MACRO l- 80. A method of treating or preventing prostate cancer in a t comprising administering to said subject an agent that modulates the activity or expression of at least one or more markers selected from the group consisting of PC-NEUTRO l- 200. 81. The method of embodiment 79 or 80, wherein the agent is a small molecule modulator, siRNA, or an dy or fragment thereof Brief Description of the Drawings Figure 1 depicts a diagram of a 3-fold cross validation method. id="p-11" id="p-11" id="p-11" id="p-11" id="p-11"
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[0011] Figure 2 depicts exemplary te cancer genes identified from macrophages. M, macrophage; TC, T cell; DE, differential expression.
Figure 3 depicts the ion of a prostate cancer marker from among potential markers analyzed in macrophages vs. T cells.
Figure 4 depicts genetic signature for prostate cancer markers form macrophages in head and neck cancer patients.
Figure 5 depicts a summary of te cancer markers fied from macrophage vs. T cell and neutrophil vs. T cell comparisons in prostate cancer patients.
Figure 6 depicts genetic signature for prostate cancer markers from phils in head and neck cancer ts.
Figure 7 depicts a comparison of cancer detection using s identified from macrophages and neutrophils vs. T cells, as compared to detection when the phagocyte gene expression is not compared to T cell gene expression.
Figure 8 depicts a puri?cation method for validating methods of detecting prostate cancer.
Figure 9 depicts a comparison of purification methods in validating a method of detecting prostate cancer.
Detailed Description of the Invention The present ion provides biological markers and methods of using them to detect a cancer. More speci?cally, the present invention provides kers that are speci?c for prostate cancer.
As used here in, a "biomarker" or "marker" refers to an analyte (e. g., a nucleic acid, DNA, RNA, e, protein, or metabolite) that can be objectively measured and evaluated as an tor for a biological process. In some embodiments, a marker is differentially detectable in phagocytes and is indicative of the presence or absence of prostate cancer. An analyte is differentially detectable if it can be distinguished quantitatively or qualitatively in phagocytes compared to a control, e.g., a normal or healthy control or non-phagocytic cells.
The present invention is based on the discovery that one or more markers selected from Tables 2 and 3 (PC-MACRO markers) or Tables 4 and 5 (PC- NEUTRO markers) are useful in diagnosing prostate cancer. By measuring the levels of the biomarkers (e.g., gene expression , protein expression levels, or protein activity levels) in a population of phagocytes (e.g., macrophage or neutrophils) from a human subject, one can provide a reliable diagnosis for prostate cancer.
As used herein, a " of a marker of this invention can be qualitative (e. g., ce or absence) or quantitative (e.g., amounts, copy numbers, or dosages). In some embodiments, a level of a marker at a zero value can indicate the absence of this marker. The levels of any marker of this invention can be measured in various forms. For example, the level can be a gene expression level, a RNA transcript level, a n expression level, a protein activity level, an enzymatic activity level.
The markers of this invention can be used in methods for diagnosing or aiding in the diagnosis of prostate cancer by ing levels (e. g., gene expression levels, or n expression levels, or protein activities) of one or more prostate cancer markers (e. g., nucleic acids or proteins) between phagocytes (e. g., hages or neutrophils) and non-phagocytic cells taken from the same individual. This ion also provides s for assessing the risk of developing prostate , prognosing said cancer, monitoring said cancer progression or regression, assessing the efficacy of a treatment, or identifying a compound capable of ameliorating or treating said cancer.
The methods of this invention can be applied to prostate cancer. As used herein, "prostate cancer" means any cancer of the prostate including, but not limited to, arcinoma and small cell carcinoma.
In a first , the methods (e.g., sis of prostate , prognosis of prostate cancer, or assessing the risk of developing prostate cancer) provided in the invention comprise: a) measuring the levels of one or more markers selected from Tables 2 and 3 (PC-MACRO markers) in a population of a subject’s macrophage cells; b) measuring the levels of one or more of the selected markers in a population of a subject’s non-phagocytic cells (e. g., T-cells, B-cells, null cells, basophils or the mixtures of two more non-phagocytic cells); comparing the measured levels in step a) to the measured levels in step b) and further identify a ence between the measured levels of a) and b). The identified difference is tive of the diagnosis (e. g., presence or absence), prognosis (e.g., lethal outcome, or tumor stage), or the risk of ping prostate cancer.
In a second aspect, the methods (e.g., diagnosis of prostate cancer, prognosis of prostate cancer, or assessing the risk of developing prostate cancer) provided in the invention comprise: a) measuring the levels of one or more markers selected from Tables 2 and 3 (PC-MACRO markers) in a population of a t’s macrophage cells; identifying a ence between the measured levels of the selected markers in step a) and the levels of the selected markers in a control (e. g., a healthy control cell, or a control cell from a healthy subject). The identified difference is indicative of the diagnosis (e.g., ce or absence), prognosis (e.g., lethal e, or tumor stage), or the risk of developing prostate cancer.
In the first and second aspects, the selected markers comprise one or more (e.g., two, three, four, or five) of PC-MACRO Markers 1-4 and 105, i.e., P2RY10, 3, CXCRl, DNAJBI, and CHI3L1. In some embodiments, the selected markers are up-regulated (see Tables 2 and/or 3 for up-regulated s) in prostate cancer patients. In some embodiments, the selected markers are down- ted (see Tables 2 and/or 3 for down-regulated markers) in prostate cancer patients. In some embodiments, the selected markers comprise at least one PC- MACRO Marker that is up-regulated and at least one PC-MACRO Marker that is down-regulated. In some embodiments, the selected markers consist of P2RY10, TNFAIP3, CXCR1, and DNAJBl or of PZRYlO, TNFAIP3, CXCRl, CHI3Ll and DNA]B l.
In a third aspect, the methods (e.g., diagnosis of te cancer, prognosis of prostate cancer, or assessing the risk of developing prostate cancer) provided in the invention comprise: a) measuring the levels of one or more markers selected from Tables 4 and 5 (PC-NEUTRO Markers) in a population of a subject’s neutrophil cells; b) measuring the levels of one or more of the selected markers in a population of a subject’s non-phagocytic cells (e. g., T-cells, B-cells, null cells, basophils or the mixtures of two more agocytic cells); comparing the measured levels in step a) to the measured levels in step b) and further identify a difference between the measured levels of a) and b). The identi?ed difference is indicative of the diagnosis (e. g., presence or absence), prognosis (e.g., lethal outcome, or tumor stage), or the risk of developing te .
In a fourth aspect, the methods (e. g., diagnosis of prostate cancer, prognosis of prostate cancer, or assessing the risk of developing prostate cancer) provided in the invention comprise: a) measuring the levels of one or more markers selected from Tables 4 and 5 (PC-NEUTRO Markers) in a population of a subject’s neutrophil cells; identifying a ence between the measured levels of the selected markers in step a) and the levels of the selected markers in a l (e.g., a healthy control cell, or a control cell from a healthy subject). The identified difference is indicative of the diagnosis (e.g., presence or absence), prognosis (e.g., lethal outcome, or tumor , or the risk of ping prostate cancer. id="p-30" id="p-30" id="p-30" id="p-30" id="p-30"
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[0030] In the third and fourth aspects, the selected markers comprise one or more (e.g., two, three, four, ?ve, six, seven, eight, nine, ten, or eleven) PC-NEUTRO Markers 1-11, e.g., , EEEFlAl, RPL23A, RPL14, RPL23A, RPL3, RPSZ8, and PTMA). In some ments, the selected markers are up-regulated (see Tables 4 and 5 for up-regulated markers) in prostate cancer patients. In some embodiments, the ed markers are down-regulated (see Tables 4 and 5 for egulated markers) in prostate cancer patients. In some embodiments, the selected s comprise at least one PC-NEUTRO Marker that is up-regulated and at least one TRO Marker that is down-regulated. In some embodiments, the selected markers consist of EIF3 SS, EEEFlAl, RPL23A, RPLl4, RPL23A, RPL3, RPSZS, and PTMA.
In a ?fth aspect, the methods (e. g., diagnosis of prostate cancer, prognosis of prostate cancer, or assessing the risk of ping prostate cancer) provided in the invention comprise: a) measuring the levels of one or more markers selected from Tables 4 and 5 (PC-NEUTRO markers) in a population of a subject’s neutrophil cells and the levels of one or more markers selected from Tables 2 and 3 (PC-MACRO markers) in a tion of a subject’s hage cells; b) measuring the levels of one or more of the selected PC-MACRO markers and the levels one or more of the selected PC-NEUTRO s in a population of a subject’s non-phagocytic cells (e.g., T-cells, s, null cells, basophils or the mixtures of two more non-phagocytic cells); identifying a difference between the measured levels of the ed PC-NEUTRO markers of steps a) and b) and identifying a difference between the ed levels of the selected PC-MACRO s of steps a) and b). The identified differences are indicative of the diagnosis (e. g., presence or e), prognosis (e. g., lethal outcome, or tumor stage), or the risk of developing prostate cancer.
In a sixth , the methods (e. g., diagnosis of prostate cancer, prognosis of prostate cancer, or assessing the risk of developing prostate cancer) provided in the invention comprise: a) measuring the levels of one or more markers selected from Tables 4 and 5 (PC-NEUTRO markers) in a population of a subject’s neutrophil cells and the levels of one or more markers selected from Tables 2 and 3 (PC-MACRO s) in a population of a subject’s macrophage cells; identifying a difference between the measured levels of the selected PC-NEUTRO markers of steps a) and the levels of the selected PC-NEUTRO markers in a control (e. g., a healthy control cell, or a control cell from a healthy t) and identifying a difference between the measured levels of the selected PC-MACRO markers of step a) and the levels of the selected PC-MACRO markers in a control (e.g., a healthy control cell, or a control cell from a healthy subject). The ?ed differences are indicative of the diagnosis (e.g., presence or absence), prognosis (e. g., lethal outcome, or tumor stage), or the risk of developing prostate cancer.
In the ?fth and sixth aspects, the selected PC-NEUTRO s se one or more (e.g., two, three, four, ?ve, six, seven, eight, nine, ten, or eleven) PC- NEUTRO Markers 1-1 1, e.g., EIF3 SS, EEEFlAl, RPL23A, RPL14, RPL23A, RPL3, RPSZS, and PTMA and the selected PC-MACRO markers comprise one or more (e.g., two, three, four, or ?ve) PC-MACRO Markers 1-4 and 105, i.e., , TNFAIP3, CXCRl, DNAJBl, and CHI3Ll. In some embodiments, the selected markers are up-regulated (see Tables 2-5 for up-regulated markers) in te cancer patients. In some embodiments, the selected markers are down- ted (see Tables 2-5 for down-regulated s) in te cancer patients.
In some embodiments, the selected markers comprise at least one marker (PC- MACRO or PC-NEUTRO marker) that is ulated and at least one marker (PC-MACRO or TRO marker) that is down-regulated. In some embodiments, the selected s consist of EIF3 SS, EEEFlAl, RPL23A, RPL14, RPL23A, RPL3, RPSZS, PTMA, P2RYlO, TNFAIP3, CXCRl, , and CHI3Ll.
In a seventh aspect, the methods provided in this invention for assessing the ef?cacy of a treatment for prostate cancer, monitoring the progression or regression of prostate cancer, or identifying a compound capable of ameliorating or treating prostate cancer, respectively, in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of PC-MACRO 1-200 (Tables 2 and 3) in a population of the subject’s macrophage cells before the treatment, or at a ?rst time point, or before administration of the compound, respectively; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells before the treatment, or at the ?rst time point, or before administration of the compound, respectively; c) identifying a ?rst ence between the measured levels of the one or more selected PC-MACRO markers in steps a) and b); d) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s macrophage cells after the treatment, or at a second time point, or after administration of the compound, respectively; e) measuring the levels of the one or more selected PC- MACRO markers in a population of the subject’s non-phagocytic cells after the treatment, or at the second time point, or after administration of the compound, tively; f) fying a second difference between the measured levels of the one or more ed PC-MACRO markers in steps d) and e); and g) identifying a difference between the ?rst difference and the second difference, wherein the difference identi?ed in g) is indicative of the ef?cacy of the treatment for the prostate cancer, or the progression or sion of the prostate cancer, or whether the compound is capable of ameliorating or treating the prostate cancer, respectively, in the subject.
In a eighth aspect, the methods ed in this invention for assessing the ef?cacy of a treatment for prostate cancer, monitoring the progression or regression of prostate cancer, or identifying a compound capable of rating or treating prostate cancer, respectively, in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of PC-MACRO 1-200 (Tables 2 and 3) in a population of the subject’s macrophage cells before the treatment, or at a ?rst time point, or before administration of the compound, respectively; b) identifying a ?rst difference between the ed levels of the one or more ed RO markers in step (a) and the levels of the one or more selected PC-MACRO markers in a control (e.g., a healthy control cell, or a control cell from a healthy subject) before the treatment, or at the ?rst time point, or before administration of the compound, respectively; c) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s macrophage cells after the treatment, or at a second time point, or after stration of the compound, respectively; d) identifying a second difference between the measured levels of the one or more selected PC-MACRO markers in step c) and the levels of the one or more selected PC-MACRO markers in a control after the treatment, or at the second time point, or after administration of the compound, respectively; and e) identifying a difference between the ?rst difference and the second difference, wherein the difference identi?ed in e) is tive of the ef?cacy of the ent for the prostate cancer, or the progression or regression of the prostate cancer, or whether the compound is capable of ameliorating or treating the prostate cancer, tively, in the subject.
In the seventh and eighth aspects, the selected markers se one or more (e.g., two, three, four, or ?ve) PC-MACRO Markers 1-4 and 105, i.e., P2RYlO, TNFAIP3, CXCRl, DNAJBl, and CHI3Ll. In some embodiments, the selected s are up-regulated (see Tables 2 and 3 for up-regulated markers) in prostate cancer ts. In some embodiments, the selected markers are down- regulated (see Tables 2 and 3 for down-regulated markers) in prostate cancer patients. In some embodiments, the selected markers comprise at least one PC- MACRO Marker that is up-regulated and at least one PC-MACRO Marker that is down-regulated. In some embodiments, the selected markers consist of , TNFAIP3, CXCRl, DNAJB l, and CHI3Ll In a ninth aspect, the methods provided in this ion for assessing the ef?cacy of a treatment for prostate cancer, monitoring the progression or regression of prostate cancer, or identifying a compound capable of ameliorating or treating prostate cancer, tively, in a subject comprising: a) measuring the levels of one or more markers selected from the group ting of PC-NEUTRO 1-200 (Table 4 and 5) in a population of the t’s neutrophil cells before the treatment, or at a ?rst time point, or before administration of the compound, respectively; b) measuring the levels of the one or more selected PC-NEUTRO markers in a tion of the subject’s non-phagocytic cells before the treatment, or at the ?rst time point, or before administration of the compound, respectively; c) identifying a ?rst difference between the measured levels of the one or more selected PC-NEUTRO markers in steps a) and b); d) measuring the levels of the one or more selected PC-NEUTRO s in a tion of the subject’s neutrophil cells after the treatment, or at a second time point, or after administration of the compound, respectively; e) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the t’s non- phagocytic cells after the treatment, or at the second time point, or after administration of the compound, respectively; f) fying a second difference between the measured levels of the one or more selected PC-NEUTRO markers in steps d) and e); and g) identifying a difference between the ?rst difference and the second difference, wherein the difference identi?ed in g) is indicative of the ef?cacy of the treatment for the prostate cancer, or the progression or regression of the prostate cancer, or r the compound is capable of ameliorating or treating the prostate cancer, respectively, in the subject.
In a tenth aspect, the methods provided in this invention for assessing the efficacy of a treatment for te cancer, monitoring the progression or regression of prostate cancer, or identifying a compound capable of ameliorating or treating prostate cancer, respectively, in a subject comprising: a) measuring the levels of one or more markers selected from the group ting of PC-NEUTRO 1-200 s 4 and 5) in a tion of the subject’s neutrophil cells before the treatment, or at a first time point, or before administration of the compound, respectively; b) identifying a first difference between the measured levels of the one or more selected PC-NEUTRO markers in step (a) and the levels of the one or more selected PC-NEUTRO markers in a l (e.g., a control cell from a healthy subject, or a normal or healthy cell from the subject) before the treatment, or at the first time point, or before administration of the compound, respectively; c) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject’s neutrophil cells after the treatment, or at a second time point, or after administration of the compound, tively; d) fying a second ence between the measured levels of the one or more selected PC- NEUTRO markers in step c) and the levels of the one or more selected PC- NEUTRO markers in a control after the treatment, or at the second time point, or after administration of the compound, respectively; and e) identifying a difference between the ?rst difference and the second difference, wherein the ence identified in e) is tive of the efficacy of the treatment for the te cancer, or the progression or regression of the prostate cancer, or whether the compound is capable of ameliorating or treating the prostate cancer, respectively, in the subject.
In the ninth and tenth aspects, the selected markers comprise one or more (e.g., two, three, four, ?ve, six, seven, eight, nine, ten, or 11) PC-NEUTRO Markers 1-1 1, e.g., EIF3SS, EEEFlAl, RPL23A, RPLl4, RPL23A, RPL3, RPSZS, and PTMA. In some embodiments, the selected markers are up-regulated (see Tables 4 and 5 for up-regulated markers) in prostate cancer patients. In some embodiments, the selected markers are down-regulated (see Tables 4 and 5 for egulated markers) in prostate cancer patients. In some ments, the selected s comprise at least one PC-NEUTRO Marker that is up-regulated and at least one PC-NEUTRO Marker that is down-regulated. In some embodiments, the ed markers consist of EIF3 SS, EEEFlAl, , RPL14, RPL23A, RPL3, RPSZS, and PTMA.
In an eleventh , the methods provided in this invention for assessing the cy of a treatment for prostate cancer, monitoring the progression or regression of prostate cancer, or identifying a compound capable of ameliorating or treating prostate , respectively, in a subject comprising: a) measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject’s macrophage cells before the treatment, at a first time point, or before administration of the nd, respectively, and measuring the levels of at least one or more markers ed from the group consisting of PC-NEUTRO 1-200 in a population of the subject’s neutrophil cells before the treatment, at the first time point, or before administration of the compound, respectively; b) measuring the levels of the at least one or more selected PC-MACRO markers in a tion of the subject’s non-phagocytic cells before the treatment, at the first time point, or before stration of the compound, respectively; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells before the treatment, at the first time point, or before administration of the compound, respectively; c) identifying a first difference between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and b); and identifying a second difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps a) and b); d) measuring the levels of the at least one or more selected PC-MACRO marker in a population of the subject’s macrophage cells after the treatment, at a second time point, or after administration of the compound, respectively, and measuring the levels of the at least one or more selected PC-NEUTRO marker in a population of the subject’s neutrophil cells after the treatment, at the second time point, or after stration of the compound, respectively; e) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells after the treatment, at the second time point, or after administration of the compound, respectively; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells after the treatment, at the second time point, or after administration of the compound, respectively; f) identifying a third difference between the measured levels of the at least one or more selected PC-MACRO markers in steps d) and e); and g) identifying a fourth difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps d) and e); h) identifying a difference n the first and second differences; and i) identifying a difference n the third and fourth differences, wherein the differences identified in h) and i) are indicative of the efficacy of the treatment for the prostate , or the progression or sion of the prostate cancer, or whether the compound is capable of ameliorating or ng the te cancer, respectively, in the subject.
In an twelfth aspect, the methods provided in this invention for assessing the efficacy of a treatment for prostate cancer, monitoring the ssion or regression of prostate cancer, or identifying a compound capable of ameliorating or treating prostate cancer, tively, in a subject comprising: a) measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject’s macrophage cells before the treatment, at a first time point, or before stration of the compound, respectively, and measuring the levels of at least one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a tion of the subject’s neutrophil cells before the ent, at the first time point, or before administration of the compound, respectively; b) identifying a first difference between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and the levels of the at least one or more selected PC-MACRO markers in a control before the treatment, at the first time point, or before administration of the compound, respectively; and identifying a second difference between the measured levels of the at least one or more selected PC-NEUTRO s in steps a) and the levels of the at least one or more selected PC-NEUTRO markers in a control before the treatment, at the first time point, or before administration of the compound, respectively; c) measuring the levels of the at least one or more selected PC-MACRO marker in a population of the subject’s macrophage cells after the treatment, at a second time point, or after administration of the compound, respectively, and measuring the levels of the at least one or more selected PC-NEUTRO marker in a population of the t’s neutrophil cells after the treatment, at the second time point, or after administration of the compound, respectively; d) identifying a third difference between the measured levels of the at least one or more selected PC-MACRO markers in steps c) and the levels of the at least one or more selected PC-MACRO markers in a control after the ent, at the second time point, or after administration of the compound, respectively; and e) identifying a fourth difference between the measured levels of the at least one or more selected PC-NEUTRO s in steps c) and the levels of the at least one or more selected PC-NEUTRO markers in a l after the treatment, at the second time point, or after administration of the compound, respectively; f) identifying a difference between the first and second differences; and g) identifying a difference between the third and fourth differences, n the differences identified in f) and g) are indicative of the efficacy of the treatment for the prostate cancer, or the progression or sion of the prostate cancer, or whether the compound is capable of ameliorating or treating the prostate , respectively, in the subject. id="p-42" id="p-42" id="p-42" id="p-42" id="p-42"
id="p-42"
[0042] In the fifth and sixth aspects, the selected PC-NEUTRO markers comprise one or more (e.g., two, three, four, ?ve, six, seven, eight, nine, ten, or eleven) PC- NEUTRO Markers 1-1 1, e.g., EIF3 SS, EEEFlAl, RPL23A, RPLl4, RPL23A, RPL3, RPSZS, and PTMA and the selected PC-MACRO s comprise one or more (e.g., two, three, four, or ?ve) RO Markers 1-4 and 105, i.e., P2RYlO, TNFAIP3, CXCRl, DNAJBl, and CHI3Ll. In some ments, the selected markers are up-regulated (see Tables 2-5 for ulated markers) in prostate cancer patients. In some embodiments, the selected markers are down- regulated (see Tables 2-5 for down-regulated markers) in prostate cancer patients.
In some embodiments, the selected markers comprise at least one marker (PC- MACRO or TRO marker) that is ulated and at least one marker (PC-MACRO or PC-NEUTRO marker) that is down-regulated. In some embodiments, the selected markers consist of EIF3 SS, EEEFlAl, RPL23A, RPLl4, RPL23A, RPL3, RPSZ8, PTMA, P2RYlO, TNFAIP3, CXCRl, DNAJBl, and CHI3Ll.
In some embodiments, two sub-populations of phagocytic cells are used in the methods of this invention, i.e., phagocytic cells that have a DNA content greater than 2n (the >2n phagocytic cells) and ytic cells that have a DNA content of 2n (the =2n phagocytic cells). In those embodiments, the levels of the selected markers in the >2n phagocytic cells are compared to the =2n phagocytic cells to identify one or more difference. The identi?ed ences indicate whether the subject has prostate cancer, or has a risk of developing prostate cancer, or has a progressing or ssive prostate cancer.
In some ments, the levels of two, three, four, ?ve, six, seven, eight, nine, ten, ll, l2, l3, l4, l5, l6, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more markers selected from Tables 2-5 are measured. In some embodiments, one or more marker selected from Tables 2 and 3 and one or more marker selected from Tables 4 and 5 are measured.
In various ments of the present invention, at least one or more of the selected markers (PC-MACRO markers or PC-NEUTRO markers) may be substituted with a biological marker different from any of the selected markers. In some embodiments, such biological markers may be known markers for prostate cancer. In some embodiments, such biological markers and the substituted selected markers may belong to the same signaling or biological pathway (e.g., a protein synthesis y, Thl cytokine production pathway, transcription pathway, programmed cell death pathway), or may have similar biological function or activity (e. g., protein synthesis, Thl cytokine production, nucleotide binding, protein binding, transcription, a receptor for purines coupled to eins, inhibition of programmed cell death, phil tion, an IL-8 receptor, an interacting n, stimulating ATPase activity), or may be regulated by a common n, or may belong to the same protein x (e.g., an HSP70 protein complex).
In various embodiments of the present ion, a population of the subject’s hage cells is used as the selected phagocytic cells for measuring the levels of the selected markers (e. g., PC-MACRO s) and a population of the subject’s T-cells is used as the ed non-phagocytic cells for measuring the levels of the ed markers (e.g., PC-MACRO markers).
In various embodiments of the present invention, a population of the subject’s neutrophil cells is used as the selected phagocytic cells for ing the levels of the selected markers (e.g., PC-NEUTRO markers) and a population of the subject’s T-cells is used as the selected non-phagocytic cells for measuring the levels of the selected markers (e. g., PC-NEUTRO markers).
The gene names/descriptions provided in Tables 2-5 are merely illustrative. The markers of this invention encompass all forms and variants of any speci?cally described markers, including, but not limited to, polymorphic or c variants, isoforms, mutants, derivatives, precursors including nucleic acids and pro-proteins, cleavage products, and structures comprised of any of the markers as constituent subunits of the ?Jlly assembled structure.
A "patient", "subject", or "individual" are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., s, felines) and rodents (e. g., mice and rats).
As used herein, the terms "normal control", "healthy control", and "not- diseased cells" se mean a sample (e.g., cells, serum, tissue) taken from a source (e.g., subject, control subject, cell line) that does not have the condition or disease being assayed and therefore may be used to determine the baseline for the condition or disorder being measured. A control subject refers to any individual that has not been diagnosed as having the disease or condition being assayed. It is also understood that the control subject, normal control, and healthy control, e data obtained and used as a standard, i.e. it can be used over and over again for multiple different subjects. In other words, for e, when comparing a subject sample to a control sample, the data from the control sample could have been obtained in a different set of experiments, for example, it could be an average ed from a number of healthy subjects and not ly ed at the time the data for the subject was obtained.
The term "diagnosis" as used herein refers to methods by which the skilled artisan can estimate and/or determine r or not a patient is suffering from a given disease or condition. In some ments, the term osis" also refers to staging (e.g., Stage I, II, III, or IV) of . The skilled n often makes a diagnosis on the basis of one or more diagnostic indicators, e.g., a marker, the presence, absence, amount, or change in amount of which is indicative of the presence, severity, or absence of the condition.
The term "prognosis" as used herein refers to is used herein to refer to the likelihood of prostate cancer progression, including recurrence of prostate cancer.
The disclosure of the International Applications PCT/USl 1/44969, PCT/USl 1/45018, and PCT/USO9/3 1395 and US. Provisional Applications 6l/660,518 and 61/660,427 are incorporated herein by reference for all purposes.
Each embodiment described herein may be combined with any other embodiment described herein.
Methods using the prostate cancer markers described herein provide high speci?city, sensitivity, and accuracy in detecting and diagnosing prostate cancer.
The methods also eliminate the "inequality of baseline" that is known to occur among individuals due to intrinsic (e.g., age, gender, ethnic background, health status and the like) and temporal variations in marker expression. Additionally, by using a comparison of phagocytes and non-phagocytes from the same individual, the methods also allow detection, sis, and treatment to be personalized to the individual. Accordingly, in some ments, the invention provides non- invasive assays for the early detection of te , i.e., before the prostate cancer can be diagnosed by conventional diagnostic techniques, e. g., imaging techniques, and, therefore, provide a foundation for improved decision-making relative to the needs and strategies for intervention, prevention, and treatment of individuals with such disease or condition.
The methods described herein are supported by whole genome microarray data of total RNA s isolated from macrophages and neutrophils and from non-phagocytic T cells. The samples were obtained from human subjects with and without te cancer. The data from these microarray experiments demonstrate that macrophage-T cell and neutrophil-T cell comparisons easily and accurately differentiate between prostate cancer patients and human subjects without prostate cancer.
The methods of this invention can be used together with any known diagnostic methods, such as physical inspection, visual inspection, biopsy, ng, histology, radiology, imaging, ultrasound, use of a commercial kit, genetic testing, immunological testing, analysis of bodily ?uids, or monitoring neural activity.
Phagocytic cells that can be used in the methods of this invention include all types of cells that are capable of ingesting s types of substances (e. g., apoptotic cells, infectious agents, dead cells, viable cells, cell-free DNAs, cell-free RNAs, cell-free proteins). In some embodiments, the phagocytic cells are neutrophils, macrophages, monocytes, dendritic cells, foam cells, mast cells, eosinophils, or keratinocytes. In some embodiments, the phagocytic cells can be a mixture of different types of phagocytic cells. In some embodiments, the ytic cells can be activated ytic cells, e. g., activated hages or neutrophils. In some embodiments, a phagocyte is a histiocyte, e. g., a Langerhans cell.
As used herein, ing" prostate cancer refers to taking steps to obtain beneficial or d results, including clinical s. Beneficial or desired clinical results e, but are not limited to, alleviation or amelioration of one or more symptoms associated with diseases or conditions. id="p-60" id="p-60" id="p-60" id="p-60" id="p-60"
id="p-60"
[0060] As used , "administering" or "administration of’ a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be stered, intravenously, arterially, intradermally, intramuscularly, intraperitonealy, intravenously, subcutaneously, ly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorbtion, e.g., through a skin duct). A nd or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow, or controlled e of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In some aspects, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a t to dminister a drug, or to have the drug stered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient. In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion, or intravenously, e.g., to a subject by injection. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.
In certain embodiments, markers used in the methods of ion are up- regulated or activated in phagocytes (e.g., macrophages or neutrophils) compared to non-phagocytes. In certain ments, markers used in the methods of invention are down-regulated or inhibited in ytes (e.g., macrophages or neutrophils) compared to non-phagocytes. As used herein, "up-regulation or up- regulated" can refer to an increase in expression levels (e.g., gene expression or protein sion), gene copy numbers, gene dosages, and other qualitative or quantitative detectable state of the markers. Similarly, regulation or down- regulated" can refer to a decrease in expression levels, gene copy numbers, gene dosages, and other ative or quantitative detectable state of the markers. As used herein, "activation or activated" can refer to an active state of the marker, e.g., a phosphorylation state, a DNA methylation state, or a DNA acetylation state.
Similarly, "inhibition or inhibited" can refer to a sed state or an inactivated state of the marker, e. g., a de-phosphorylation state, a ubiquitination state, or a DNA de-methylation state.
In certain embodiments, methods of this invention also comprise at least one of the following steps before determination of various : i) lysing the phagocytic or non-phagocytic cells; and ii) extracting cellular contents from the lysed cells. Any known cell lysis and extraction methods can be used herein. In certain embodiments, at least one or more prostate cancer markers are t in the phagocytes. In certain embodiments, there is no marker present in the cellular contents of the non-phagocytic cells.
In certain embodiments, the phagocytic cells and/or non-phagocytic cells are isolated from a bodily ?uid sample, tissues, or population of cells. Exemplary bodily ?uid samples can be whole blood, urine, stool, saliva, lymph ?uid, cerebrospinal ?uid, synovial ?uid, cystic ?uid, ascites, pleural effusion, ?uid obtained from a pregnant woman in the first trimester, ?uid obtained from a pregnant woman in the second trimester, ?uid obtained from a pregnant woman in the third trimester, maternal blood, amniotic ?uid, chorionic villus sample, ?uid from a preimplantation embryo, maternal urine, maternal saliva, placental sample, fetal blood, lavage and cervical vaginal ?uid, interstitial ?uid, buccal swab sample, sputum, bronchial lavage, Pap smear sample, or ocular ?uid. In some embodiments, the phagocytic cells or agocytic cells are isolated from white blood cells.
In the s of this invention, cell separation/isolation/purification methods are used to isolate populations of cells from bodily ?uid , cells, or s of a subject. A skilled worker can use any known cell separation/isolation/purification techniques to isolate phagocytic cells and non- phagocytic cells from a bodily ?uid. Exemplary techniques include, but are not limited to, using antibodies, ?ow cytometry, ?uorescence activated cell sorting, filtration, gradient-based centri?lgation, elution, micro?uidics, ic separation technique, ?uorescent-magnetic separation technique, nanostructure, quantum dots, high throughput microscope-based platform, or a combination thereof. id="p-65" id="p-65" id="p-65" id="p-65" id="p-65"
id="p-65"
[0065] In certain embodiments, the phagocytic cells and/or non-phagocytic cells are isolated by using a t secreted by the cells. In certain embodiments, the phagocytic cells and/or non-phagocytic cells are isolated by using a cell surface target (e. g., or protein) on the e of the cells. In some embodiments, the cell surface target is a protein that has been ed by phagocytic cells. In some embodiments, the cell surface target is expressed by cells on their plasma membranes. In some embodiments, the cell surface target is an exogenous protein that is ocated on the plasma nes, but not expressed by the cells (e.g., the phagocytic cells). In some embodiments, the cell surface target is a marker of te cancer.
In certain aspects of the methods described herein, analytes include nucleic acids, proteins, or any combinations thereof. In certain aspects of the methods described herein, markers include nucleic acids, proteins, or any combinations thereof. As used herein, the term "nucleic acid" is intended to e DNA molecules (e. g., cDNA or genomic DNA), RNA molecules (e. g., mRNA), DNA-RNA hybrids, and s of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be a nucleotide, oligonucleotide, double-stranded DNA, single-stranded DNA, stranded DNA, complementary DNA, genomic DNA, non-coding DNA, messenger RNA (mRNAs), microRNA s), small nucleolar RNA As), mal RNA (rRNA), transfer RNA (tRNA), small ering RNA (siRNA), geneous nuclear R,\IAs (hnRNA), or small hairpin RNA (shRNA). In some embodiments, the c acid is a transrenal nucleic acid. A transrenal nucleic acid is an extracellular nucleic acid that is excreted in the urine. See, e. g., US.
Patent Publication No. 2010006871 1 and US. Patent Publication No. 20120021404.
As used herein, the term "amino acid" includes organic compounds containing both a basic amino group and an acidic carboxyl group. Included within this term are natural amino acids (e.g., L-amino acids), modi?ed and unusual amino acids (e.g., D-amino acids and B-amino acids), as well as amino acids which are known to occur biologically in free or combined form but usually do not occur in proteins. Natural protein occurring amino acids e alanine, arginine, gine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, ine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, ine, tyrosine, tryptophan, proline, and valine. Natural non-protein amino acids include arginosuccinic acid, citrulline, cysteine sul?lric acid, 3,4-dihydroxyphenylalanine, homocysteine, homoserine, omithine, 3-monoiodotyrosine, 3,5-diiodotryosine, 3, 5, 5-triiodothyronine, and 3,3 ’,5,5 ’- tetraiodothyronine. Modi?ed or unusual amino acids include D-amino acids, hydroxylysine, 4-hydroxyproline, N-Cbz- protected amino acids, 2,4-diaminobutyric acid, homoarginine, norleucine, N- methylaminobutyric acid, naphthylalanine, phenylglycine, (x-phenylproline, tert- leucine, 4-aminocyclohexylalanine, N—methyl-norleucine, 3 ydroproline, N,N—dimethylaminoglycine, N-methylaminoglycine, 4-aminopiperidine-4- carboxylic acid, 6-aminocaproic acid, trans-4-(aminomethyl)- cyclohexanecarboxylic acid, 2-, 3-, and 4-(aminomethyl)- benzoic acid, 1- aminocyclopentanecarboxylic acid, 1-aminocyclopropanecarboxylic acid, and 2- benzyl-5-aminopentanoic acid.
As used herein, the term "peptide" es nds that consist of two or more amino acids that are linked by means of a peptide bond. Peptides may have a molecular weight of less than 10,000 Daltons, less than 5,000 Daltons, or less than 2,500 Daltons. The term de" also includes compounds containing both peptide and non-peptide components, such as pseudopeptide or peptidomimetic residues or other non-amino acid components. Such compounds containing both peptide and non-peptide components may also be referred to as a "peptide analog." As used herein, the term "protein" includes compounds that t of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Proteins used in methods of the invention include, but are not limited to, amino acids, peptides, dies, antibody fragments, cytokines, oteins, or glycoproteins.
As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies (including ?lll length antibodies which have an immunoglobulin Fc region), dy compositions with polyepitopic speci?city, multispecif1c antibodies (e. g., bispecif1c antibodies, diabodies, and single-chain molecules, and antibody nts (e.g., Fab or F(ab’)2, and FV). For the structure and properties of the different classes of antibodies, see e. g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and r 6.
As used herein, the term "cytokine" refers to a secreted protein or active fragment or mutant thereof that tes the actiVity of cells of the immune system. es of cytokines include, without limitation, interleukins, interferons, chemokines, tumor necrosis factors, colony-stimulating s for immune cell precursors, and the like.
As used herein, the term "lipoprotein" includes vely d compositions that comprise a core of hydrophobic cholesteryl esters and triglyceride surrounded by a e layer of amphipathic phospholipids with which free cholesterol and apolipoproteins are associated. Lipoproteins may be characterized by their density (e.g. very-low-density lipoprotein (VLDL), low- density lipoprotein (LDL) and high density lipoprotein (HDL)), which is determined by their size, the relative amounts of lipid and n. oteins may also be characterized by the presence or absence of ular modifications (e.g. oxidization, acetylation, or glycation).
As used herein, the term protein" includes glycosides which have one or more oligo- or polysaccharides covalently attached to a peptide or protein.
Exemplary glycoproteins can include, without limitation, immunoglobulins, members of the major histocompatibility complex, collagens, mucins, glycoprotein IIb/IIIa, glycoprotein-41 (gp41) and glycoprotein-120 (gpl2), follicle-stimulating hormone, alpha-fetoprotein, erythropoietin, transferrins, alkaline phosphatase, and lectins.
In some embodiments of the invention, a sample may comprise one or more izers for a cell or an analyte such as DNA, RNA, and/or protein. For example, a sample may comprise a DNA stabilizer, an RNA stabilizer, and/or a protein stabilizer. Stabilizers are well known in the art and include, for e, DNAse inhibitors, RNAse inhibitors, and protease inhibitors or equivalents thereof. id="p-75" id="p-75" id="p-75" id="p-75" id="p-75"
id="p-75"
[0075] In some embodiments of the invention, levels of at least one or more prostate cancer markers are compared. This comparison can be quantitative or qualitative. Quantitative measurements can be taken using any of the assays described herein. For example, sequencing, direct sequencing, random shotgun sequencing, Sanger y termination sequencing, targeted sequencing, whole- genome sequencing, sequencing by hybridization, quencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle cing, single- base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co-ampli?cation at lower denaturation temperature-PCR (COLD-PCR), sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, -molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer cing, SOLiD® sequencing, MS-PET sequencing, mass spectrometry, matrix assisted laser desorption/ionization-time of ?ight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser deorption/ionization-time of ?ight (SELDI-TOF) mass ometry, quadrupole- time of ?ight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), polymerase chain on (PCR) is, quantitative PCR, ime PCR, ?uorescence assay, colorimetric assay, chemiluminescent assay, or a combination thereof.
Quantitative comparisons can include tical analyses such as t-test, ANOVA, Krustal-Wallis, on, Mann-Whitney, and odds ratio. Quantitative differences can include differences in the levels of markers n levels or differences in the numbers of markers present between , and combinations thereof. Examples of levels of the markers can be, without limitation, gene sion levels, nucleic acid , and protein levels. Qualitative differences can include, but are not limited to, activation and inactivation, protein degradation, nucleic acid degradation, and covalent modifications.
In certain embodiments of the invention, the level is a nucleic acid level or a protein level, or a combination thereof The level can be qualitatively or quantitatively determined.
A nucleic acid level can be, t limitation, a genotypic level, a single nucleotide polymorphism level, a gene mutation level, a gene copy number level, a DNA methylation level, a DNA acetylation level, a chromosome dosage level, a gene expression level, or a combination thereof The nucleic acid level can be determined by any methods known in the art to detect pes, single nucleotide polymorphisms, gene mutations, gene copy numbers, DNA ation states, DNA acetylation , chromosome s.
Exemplary methods include, but are not limited to, polymerase chain reaction (PCR) analysis, sequencing analysis, electrophoretic analysis, restriction fragment length polymorphism (RFLP) analysis, Northern blot analysis, quantitative PCR, reverse-transcriptase-PCR is (RT-PCR), -specific oligonucleotide ization analysis, comparative genomic hybridization, heteroduplex mobility assay (HMA), single strand conformational polymorphism (SSCP), denaturing gradient gel electrophisis (DGGE), RNAase mismatch analysis, mass spectrometry, tandem mass spectrometry, matrix assisted laser desorption/ionization-time of ?ight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser deorption/ionization-time of ?ight (SELDI-TOF) mass spectrometry, quadrupole- time of ?ight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier orm mass ometry , matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), e plasmon resonance, Southern blot analysis, in situ hybridization, ?uorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), microarray, comparative genomic hybridization, karyotyping, multiplex ligation-dependent probe ampli?cation (MLPA), Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF), microscopy, methylation specific PCR (MSP) assay, HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay, ctive acetate labeling assays, colorimetric DNA acetylation assay, chromatin immunoprecipitation combined with microarray (ChIP-on-chip) assay, restriction landmark genomic scanning, Methylated DNA immunoprecipitation ), lar break light assay for DNA adenine methyltransferase activity, chromatographic separation, methylation-sensitive restriction enzyme analysis, bisulf1te-driven conversion of non-methylated cytosine to uracil, co-amplif1cation at lower denaturation ature-PCR (COLD-PCR), multiplex PCR, methyl-binding PCR analysis, or a combination f.
As used herein, the term ncing" is used in a broad sense and refers to any technique known in the art that allows the order of at least some utive tides in at least part of a c acid to be identi?ed, including without limitation at least part of an extension product or a vector . Exemplary cing techniques include targeted sequencing, single molecule real-time sequencing, whole transcriptome shotgun sequencing ("RNA-seq"), electron microscopy-based sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, exon sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high- throughput cing, massively parallel signature sequencing, on PCR, co-amplif1cation at lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, sequencing by ible dye terminator, paired-end cing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD® sequencing, MS-PET sequencing, mass spectrometry, and a ation thereof. In some embodiments, sequencing comprises an detecting the sequencing product using an instrument, for example but not limited to an ABI PRISM® 377 DNA Sequencer, an ABI PRISM® 310, 3100, 3100-Avant, 3730, or 3730xI Genetic Analyzer, an ABI PRISM® 3700 DNA Analyzer, or an Applied Biosystems SOLiDTM System (all from Applied Biosystems), a Genome Sequencer 20 System (Roche Applied Science), or a mass spectrometer. In certain embodiments, sequencing comprises emulsion PCR. In certain embodiments, sequencing comprises a high throughput sequencing technique, for example but not limited to, massively parallel signature cing (MPSS).
In ?arther embodiments of the invention, a protein level can be a protein expression level, a protein activation level, or a combination thereof In some ments, a protein activation level can comprise determining a phosphorylation state, an ubiquitination state, a myristoylation state, or a conformational state of the protein.
A protein level can be detected by any methods known in the art for detecting protein expression levels, protein phosphorylation state, protein ubiquitination state, protein myristoylation state, or protein conformational state.
In some embodiments, a protein level can be determined by an immunohistochemistry assay, an enzyme-linked immunosorbent assay ), in situ hybridization, chromatography, liquid chromatography, size exclusion chromatography, high performance liquid tography (HPLC), gas chromatography, mass spectrometry, tandem mass spectrometry, matrix assisted laser desorption/ionization-time of ?ight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser deorption/ionization-time of ?ight (SELDI-TOF) mass spectrometry, quadrupole- time of ?ight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), r transform mass ometry (FTMS), matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass ometry, secondary ion mass spectrometry (SIMS), radioimmunoassays, microscopy, micro?uidic chip-based assays, surface n resonance, cing, Western blotting assay, or a ation thereof As used herein, the "difference" between different levels detected by the methods of this invention can refer to different gene copy numbers, different DNA, RNA, or protein expression levels, different DNA methylation states, different DNA acetylation states, and different protein modification states. The difference can be a difference greater than 1 fold. In some embodiments, the ence is a l.05-fold, l.l-fold, l.2-fold, ld, l.4-fold, l.5-fold, 2-fold, 2.5-fold, 3-fold, 4- fold, 5-fold, , , 8-fold, 9-fold, or 10-fold difference. In some embodiments, the difference is any fold difference between 1- l 0, 2-10, 5-10, 10- , or lO-lOO fold. id="p-84" id="p-84" id="p-84" id="p-84" id="p-84"
id="p-84"
[0084] In some embodiments, the difference is differential gene expression (DGE), e. g. DGE of phagocytes vs. non-phagocytes. DGE can be measured as X = log2(Yp) — log2(YNp). The DGE may be any number, provided that it is signi?cantly ent between the phagocytes and the non-phagocytes. For example, a 2-fold increased in gene expression could be represented as X = log2(Yp) — log2(YNp) = log2(Yp/YNP) =log2(2) = 1, while a 2-fold decrease in gene expression could be represented as X = log2(Yp) — log2(YNp) = p/YNP) =log2(l/2) = -l. Down-regulated genes have X < 0, while up-regulated genes have X > 0. See, e.g., Efron, J Am Stat Assoc 104: 1015-1028 (2009).
A general principle of assays to detect markers involves preparing a sample or reaction mixture that may contain the marker (e.g., one or more of DNA, RNA, or protein) and a probe under appropriate conditions and for a time sufficient to allow the marker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways.
For example, one method to conduct such an assay would involve anchoring the marker or probe onto a solid phase support, also referred to as a substrate, and detecting target marker/probe complexes ed on the solid phase at the end of the reaction. In one embodiment of such a method, a sample from a subject, which is to be assayed for presence and/or concentration of marker, can be anchored onto a carrier or solid phase t. In r embodiment, the reverse situation is le, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored ent of the assay.
There are many established methods for anchoring assay components to a solid phase. These include, without limitation, marker or probe molecules which are immobilized h ation of biotin and streptavidin. Such biotinylated assay components can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In certain embodiments, the surfaces with immobilized assay components can be prepared in advance and stored. id="p-88" id="p-88" id="p-88" id="p-88" id="p-88"
id="p-88"
[0088] Other le carriers or solid phase supports for such assays include any material e of binding the class of le to which the marker or probe belongs. Well known supports or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, l and modi?ed celluloses, polyacrylamides, gabbros, and magnetite.
In order to conduct assays with the above ned approaches, the non- immobilized component is added to the solid phase upon which the second component is anchored. After the reaction is complete, uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase. The detection of marker/probe complexes anchored to the solid phase can be accomplished in a number ods outlined herein. id="p-90" id="p-90" id="p-90" id="p-90" id="p-90"
id="p-90"
[0090] In certain exemplary embodiments, the probe, when it is the unanchored assay component, can be d for the purpose of ion and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are nown to one skilled in the art.
It is also possible to directly detect /probe complex formation without r manipulation or labeling of either component (marker or probe), for example by utilizing the technique of ?uorescence energy er (see, for example, US. Patent Nos. 169 and 4,868,103). A ?uorophore label on the first, ‘donor’ molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted ?uorescent energy will be absorbed by a ?uorescent label on a second ‘acceptor’ molecule, which in turn is able to ?uoresce due to the absorbed energy. Altemately, the ‘donor’ protein molecule may simply utilize the natural ?uorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the ?uorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET g event can be conveniently measured through standard ?uorometric ion means well known in the art (e.g., using a ?uorimeter).
In r embodiment, determination of the ability of a probe to recognize a marker can be accomplished t labeling either assay component (probe or marker) by utilizing a technology such as real-time Biomolecular Interaction is (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C, 1991, Anal.
Chem. 8 2345 and Szabo et al, 1995, Curr. Opin. Struct. Biol. 5:699 705). As used herein, "BIA" or "surface plasmon resonance" is a logy for studying biospecific ctions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the e (the optical phenomenon of surface n resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules. atively, in r embodiment, analogous diagnostic and prognostic assays can be conducted with marker and probe as solutes in a liquid phase. In such an assay, the complexed marker and probe are separated from uncomplexed components by any of a number of standard ques, including but not limited to: differential centri?lgation, chromatography, ophoresis and immunoprecipitation. In differential centrifugation, marker/probe complexes may be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas and Minton (1993) Trends Biochem. Sci. 18:284). Standard chromatographic techniques may also be utilized to separate complexed les from uncomplexed ones. For example, gel ?ltration chromatography separates molecules based on size, and through the utilization of an appropriate gel ?ltration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components. Similarly, the vely different charge properties of the marker/probe complex as compared to the uncomplexed components may be exploited to differentiate the complex from uncomplexed components, for example through the utilization of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard (1998) J. Mol. Recognit. 11 :141; Hage and Tweed (1997) J.
Chromatogr. B. Biomed. Sci. Appl. 12:499). Gel ophoresis may also be ed to separate complexed assay components from unbound components (see, e.g., Ausubel et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 1999). In this technique, protein or nucleic acid complexes are separated based on size or , for e. In order to maintain the g interaction during the electrophoretic process, non-denaturing gel matrix materials and conditions in the absence of reducing agent are typically preferred. Appropriate conditions to the particular assay and components thereof will be well known to one d in the art.
In certain ary embodiments, the level ofmRNA corresponding to the marker can be determined either by in situ and/or by in Vitro formats in a biological sample using methods known in the art. Many expression detection methods use ed RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation ofmRNA can be utilized for the puri?cation ofRNA from blood cells (see, e. g., Ausubel et al, ed., Current Protocols in Molecular y, John Wiley & Sons, New York 1987 1999).
Additionally, large numbers of cells and/or samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single- step RNA isolation process of Chomczynski (1989, US. Patent No. 4,843,155).
Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, rn or Northern analyses, rase chain reaction analyses and probe . In certain exemplary embodiments, a diagnostic method for the detection ofmRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a ?Jll-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to speci?cally hybridize under stringent ions to an mRNA or genomic DNA encoding a marker of the present invention. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe tes that the marker in question is being expressed. id="p-96" id="p-96" id="p-96" id="p-96" id="p-96"
id="p-96"
[0096] In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the s), for example, in a gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present invention.
An alternative method for ining the level ofmRNA ponding to a marker of the present invention in a sample involves the process of nucleic acid ampli?cation, e. g., by RT-PCR (the experimental embodiment set forth in US. Patent Nos. 4,683,195 and 4,683,202), COLD-PCR (Li et al. (2008) Nat.
Med. 14:579), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl.
Acad. Sci. USA 87:1874), transcriptional ampli?cation system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1 173), Q- Beta Replicase (Lizardi et al. (1988) Bio/Technology 6: 1 197), rolling circle replication (US. Patent No. 033) or any other nucleic acid ampli?cation method, followed by the detection of the ampli?ed molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the ion of nucleic acid molecules if such molecules are present in very low s. As used herein, ampli?cation primers are de?ned as being a pair of nucleic acid molecules that can anneal to 5’ or 3’ regions of a gene (plus and minus strands, respectively, or Vice-versa) and contain a short region in between. In general, ampli?cation primers are from about 10 to 30 nucleotides in length and ?ank a region from about 50 to 200 nucleotides in length. Under riate conditions and with appropriate reagents, such primers permit the ampli?cation of a c acid molecule sing the nucleotide sequence ?anked by the primers. id="p-98" id="p-98" id="p-98" id="p-98" id="p-98"
id="p-98"
[0098] For in situ methods, mRNA does not need to be ed from the sample (e.g., a bodily ?uid (e.g., blood ) prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, lly a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the marker. id="p-99" id="p-99" id="p-99" id="p-99" id="p-99"
id="p-99"
[0099] As an alternative to making determinations based on the absolute expression level of the marker, determinations may be based on the normalized expression level of the marker. Expression levels are normalized by correcting the absolute expression level of a marker by comparing its sion to the sion of a gene that is not a marker, 6.g. , a housekeeping gene that is constitutively expressed. le genes for ization include housekeeping genes such as the actin gene, or epithelial cell- speci?c genes. This normalization allows the comparison of the expression level in a patient sample from one source to a patient sample from another source, e.g., to compare a population of phagocytic from an individual to a population of non-phagocytic cells from the individual.
In one embodiment of this invention, a protein or polypeptide corresponding to a marker is detected. In n embodiments, an agent for detecting a protein or ptide can be an antibody capable of binding to the polypeptide, such as an antibody with a detectable label. As used herein, the term "labeled," with regard to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another t that is ly labeled. Examples of indirect labeling include detection of a primary antibody using a ?uorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with ?uorescently labeled streptavidin. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab’)2) can be used. In one format, antibodies, or antibody fragments, can be used in methods such as Western blots or immuno?uorescence techniques to detect the sed ns. In such uses, it is generally preferable to immobilize either the antibody or proteins on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well known ts or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, magnetite and the like.
A variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats e, but are not d to, competitive and non-competitive immunoassay, enzyme immunoassay (EIA), radioimmunoassay (RIA), antigen capture assays, two-antibody sandwich assays, Western blot analysis, enzyme linked immunoabsorbant assay (ELISA), a planar array, a colorimetric assay, a chemiluminescent assay, a ?uorescent assay, and the like. Immunoassays, including radioimmmunoassays and enzyme- linked immunoassays, are useful in the methods of the present invention. A skilled artisan can readily adapt known n/antibody detection methods for use in determining whether cells (e.g., bodily ?uid cells such as blood cells) express a marker of the t invention.
One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such t for use with the present invention. For e, protein isolated from cells (e. g., bodily ?uid cells such as blood cells) can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the ably labeled antibody.
The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount ofbound label on the solid support can then be detected by conventional means.
In certain exemplary embodiments, assays are ed for diagnosis, prognosis, assessing the risk of ping prostate cancer, assessing the efficacy of a treatment, monitoring the ssion or regression of prostate cancer, and identifying a compound capable of ameliorating or treating prostate cancer. An exemplary method for these methods involves obtaining a bodily ?uid sample from a test subject, isolating phagocytes and non-phagocytes, and contacting the phagocytes and non-phagocytes with a compound or an agent capable of detecting one or more of the markers of the disease or condition, e.g., marker nucleic acid (e.g., mRNA, c DNA), marker peptide (e.g., polypeptide or protein), marker lipid (e.g., cholesterol), or marker metabolite (e.g. such that the , creatinine) presence of the marker is detected. In one embodiment, an agent for detecting marker mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to marker mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length marker nucleic acid or a n thereof. Other le probes for use in the diagnostic assays of the ion are described herein.
As used herein, a compound capable of ameliorating or treating prostate cancer can include, without limitations, any substance that can e symptoms or prognosis, prevent progression of the prostate cancer, promote regression of the prostate cancer, or eliminate the te cancer.
The methods of the invention can also be used to detect genetic alterations in a marker gene, thereby ining if a t with the altered gene is at risk for developing prostate cancer characterized by misregulation in a marker protein activity or nucleic acid expression. In certain embodiments, the methods include detecting, in phagocytes, the presence or absence of a genetic alteration characterized by an alteration affecting the integrity of a gene encoding a marker peptide and/or a marker gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from one or more marker genes; 2) an addition of one or more nucleotides to one or more marker genes; 3) a substitution of one or more nucleotides of one or more marker genes, 4) a chromosomal rearrangement of one or more marker genes; 5) an alteration in the level of a messenger RNA transcript of one or more marker genes; 6) aberrant modi?cation of one or more marker genes, such as of the methylation pattern of the genomic DNA; 7) the ce of a non-wild type ng n of a messenger RNA transcript of one or more marker genes; 8) a ld type level of a one or more marker proteins; 9) allelic loss of one or more marker genes; and 10) inappropriate post-translational modi?cation of one or more marker proteins. As described , there are a large number of assays known in the art which can be used for detecting tions in one or more marker genes. id="p-106" id="p-106" id="p-106" id="p-106" id="p-106"
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[0106] In certain embodiments, detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., US. Patent Nos. 4,683,195, 4,683,202 and 5,854,033), such as real-time PCR, COLD-PCR (Li et al. (2008) Nat. Med. ), anchor PCR, recursive PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e. g., Landegran et al. (1988) Science 241 : 1077; Prodromou and Pearl (1992) Protein Eng. 5:827; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91 :360), the latter of which can be particularly useful for detecting point mutations in a marker gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675). This method can include the steps of collecting a sample of cell free bodily ?uid from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which speci?cally hybridize to a marker gene under conditions such that hybridization and cation of the marker gene (if present) occurs, and detecting the presence or absence of an ampli?cation product, or ing the size of the ampli?cation product and ing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary ampli?cation step in conjunction with any of the techniques used for detecting ons described herein.
Alternative ampli?cation methods include: self sustained sequence replication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874), transcriptional ampli?cation system (Kwoh et al., (1989) Proc. Natl. Acad. Sci.
USA 86:1173), Q Beta Replicase di et al. (1988) chnology 6: 1 197), or any other nucleic acid ampli?cation method, followed by the detection of the ampli?ed molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low s.
In an alternative embodiment, mutations in one or more marker genes from a sample can be identi?ed by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is ed, optionally ampli?ed, digested with one or more restriction endonucleases, and fragment length sizes are ined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
Moreover, the use of sequence speci?c mes (see, for example, US. Pat. No. ,498,531) can be used to score for the presence of speci?c ons by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in one or more of the markers described herein can be identi?ed by hybridizing a sample and control c acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al. (1996) Human Mutation 7: 244; Kozal et al. (1996) Nature Medicine 2:753). For example, genetic mutations in a marker nucleic acid can be ?ed in two dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Brie?y, a ?rst hybridization array of probes can be used to scan through long stretches ofDNA in a sample and control to identify base changes n the ces by making linear arrays of sequential overlapping probes. This step allows the identi?cation of point mutations. This step is followed by a second ization array that allows the characterization of c mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other mentary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence a marker gene and detect mutations by comparing the sequence of the sample marker gene with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and t ((1977) Proc.
Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA ). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when ming the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36: 127-162; and Grif?n et al. (1993) Appl. Biochem. Biotechnol. 38:147).
Other s for detecting mutations in a marker gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242).
In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild- type marker sequence with ially mutant RNA or DNA obtained from a tissue sample. The double-stranded es are treated with an agent which cleaves single-stranded s of the duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be d with hydroxylamine or osmium tetroxide and with piperidine in order to digest ched regions. After ion of the mismatched regions, the resulting al is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Sci.
USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217286. In one embodiment, the control DNA or RNA can be d for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in de?ned systems for detecting and mapping point mutations in marker cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657). According to an ary embodiment, a probe based on a marker sequence, e. g., a wild-type marker sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from ophoresis protocols or the like. See, for example, US. Patent No. id="p-113" id="p-113" id="p-113" id="p-113" id="p-113"
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[0113] In other embodiments, tions in electrophoretic mobility will be used to identify mutations in marker genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc.
Natl. Acad. Sci. USA 862766, see also Cotton (1993) Mutat. Res. 285:125; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73). Single-stranded DNA fragments of sample and control marker nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting tion in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be ed by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double ed heteroduplex les on the basis of changes in ophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
In yet another embodiment the movement of mutant or wild-type fragments in rylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be d to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of elting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265:12753).
Examples of other techniques for ing point mutations include, but are not limited to, selective oligonucleotide hybridization, selective ampli?cation or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163; Saiki et al. (1989) Proc. Natl. Acad. Sci.
USA 86:6230). Such allele speci?c oligonucleotides are hybridized to PCR ampli?ed target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele speci?c ampli?cation logy which depends on selective PCR ampli?cation may be used in conjunction with the t ion.
Oligonucleotides used as primers for speci?c ampli?cation may carry the mutation of interest in the center of the molecule (so that ampli?cation depends on differential hybridization) (Gibbs et al. (1989) Nucl. Acids Res. 17:2437) or at the extreme 3 ’ end of one primer where, under appropriate conditions, ch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 1 1:23 8). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments cation may also be performed using Taq ligase for ampli?cation (Barany (1991) Proc. Natl. Acad.
Sci. USA 88: l 89). In such cases, ligation will occur only if there is a perfect match at the 3’ end of the 5’ sequence making it possible to detect the presence of a known mutation at a speci?c site by g for the presence or e of ampli?cation.
An exemplary method for detecting the presence or absence of an analyte (e.g., DNA, RNA, protein, polypeptide, or the like) corresponding to a marker of the invention in a biological sample involves obtaining a bodily ?uid sample (6.g. blood) from a test t and ting the bodily ?uid sample with a compound or an agent capable of detecting one or more markers. Detection methods described herein can be used to detect one or more markers in a biological sample in vitro as well as in viva. For example, in vitro techniques for detection ofmRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a polypeptide ponding to a marker of the invention e enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immuno?uorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. Furthermore, in viva techniques for detection of a polypeptide corresponding to a marker of the invention include introducing into a subject a d antibody directed against the polypeptide. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Because each marker is also an analyte, any method described herein to detect the presence or absence of a marker can also be used to detect the presence or e of an analyte.
The markers useful in the methods of the invention can include any mutation in any one of the markers. Mutation sites and sequences can be identi?ed, for example, by databases or repositories of such information, e.g., The Human Gene on Database (www.hgmd.cf.ac.uk), the Single Nucleotide Polymorphism Database , www.ncbi.nlm.nih.gov/projects/SNP), and the Online Mendelian Inheritance in Man (OMIM) e (www.ncbi.nlm.nih.gov/omim).
The t invention also provides kits that comprise marker detection agents that detect at least one or more of the prostate cancer markers described herein.
The present invention also provides methods of treating or preventing prostate cancer in a subject comprising administering to said subject an agent that modulates the activity or expression or disrupts the function of at least one or more of the markers of this invention.
The one or more markers identi?ed by this invention (e.g., markers in Tables 2-5) may be used in the treatment of prostate cancer. For example, a marker (e.g., a protein or gene) identi?ed by the present invention may be used as a molecular target for a therapeutic agent. A marker identi?ed by the invention also may be used in any of the other s of the invention, e.g., for monitoring the progression or regression of a disease or condition. In certain embodiments, the one or more markers identi?ed by the methods of this invention may have eutic potential. For example, if a marker is identi?ed as being up-regulated (or down-regulated), see, for example, the up-regulated (or down-regulated) markers in Tables 2-5, or ted (or inhibited) in phagocytic cells from a subject having prostate cancer, a compound or an agent that is e of down-regulating (or ulating) or inhibiting (or activating) said marker may be useful in ng prostate cancer. Similarly, a gene protein expression level, a protein expression level, or a combination thereofmay be useful in this aspect of the invention.
Unless otherwise de?ned herein, scienti?c and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, lar y, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and c acid chemistry, described herein, are those well known and commonly used in the art. id="p-123" id="p-123" id="p-123" id="p-123" id="p-123"
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[0123] All of the above, and any other publications, patents and published patent applications referred to in this application are cally incorporated by reference herein. In case of t, the present cation, including its c de?nitions, will control.
Throughout this speci?cation, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or ents).
The singular forms "a," "an," and "the" include the plurals unless the context clearly dictates otherwise. id="p-126" id="p-126" id="p-126" id="p-126" id="p-126"
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[0126] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.
It is to be understood that the ments of the present invention which have been described are merely illustrative of some of the applications of the principles of the present invention. Numerous modi?cations may be made by those skilled in the art based upon the teachings presented herein without departing from the true spirit and scope of the invention.
The following examples are set forth as being representative of the present invention. These examples are not to be construed as limiting the scope of the invention as these and other lent embodiments will be apparent in view of the present disclosure and accompanying claims.
Examples Example 1: Microarray analysis of prostate cancer patients Study Population Blood samples were collected from 62 prostate cancer patients. To meet the criteria of ing blood only from na'ive patients who had not undergone any treatment prior to blood draw, four patients who had received either chemo and/or radiation therapy prior to blood draw were excluded. Embedded tumor and adjacent "normal prostate" s were available from 42 of the 58 patients.
Gleason scores, staging information, and serum PSA levels (determined prior to y) were available from all (Table 1). Approximately 10 ml of blood was collected from each patient into purple top blood collection EDTA tubes (BD ences, CA) one to two weeks before l prostatectomy. Within 3 hours, macrophages, phils and T cells were isolated from each blood sample and total RNA was extracted and puri?ed on the same day. Healthy l blood samples were obtained from apheresis collars of anonymous platelet donors.
Gender determination of the blood donors was performed by PCR using two sets of primers, SRY primers (Forward: 5'—CAG TGT GAA ACG GGA GAA AAC AG— 3'; Reverse: 5'—ACT TCG CTG CAG AGT ACC GAA G—3') amplifying a 336 bp fragment on Y some and AR6 primers (Forward: 5'—CAA TCA GAG ACA TTC CCT CTG G—3'; Reverse: 5'—AGT GGT CCT CTC TGA ATC TC—3') amplifying a 267 bp fragment on X chromosome (males have both fragments lO ampli?ed; females have only one). The PCR (36 cycles) was done under the ions of 95 0C for 45 seconds, 56 0C for 45 seconds, and 72 0C for 45 seconds.
Table 1: Characteristics of 58 prostate cancer patients Parameter No. of patients Median (range) 60 (48-73) Race N/A White 87.9 Non-white 5.2 - PSA (ng/ml) 4.77 (ODS-23.80) Pathologic staging Isolation ofMacrophages (M), Neutrophl'ls (N), and T Cells (TC) from Whole Blood 7 mL of 1X PBS containing 2% FBS and 2 mM EDTA were added to approximately 5 mL of whole blood, the sample centrifuged (2,000 RPM, 10 minutes at 20°C). The buffy coat was removed and centrifuged (2,000 RPM, 10 minutes at 20°C). The cell pellet was then suspended in ~1 mL of PBS and transferred to a 1.5 mL microfuge tube. Next, macrophages, neutrophils, and T cells were isolated using ic beads coated with dies speci?c to each of the three cell types (positive cell depletion). Cells were separated from the buffy coat always in the following sequence: 1) macrophages; 2) phils; and 3) T cells (changing the order did not alter the RNA yield and quality). The y isolated white blood cell samples (in ~1 mL PBS) were incubated (25 min, 4°C, constant shaking) first with anti-monocyte coated Dynabeads® (CD14 - Cat. No. 11149D, Life Technologies), then with anti-neutrophil coated Dynabeads® (CD15 - Cat. No. 11137D, Life Technologies), and ?nally with anti- T cell coated Dynabeads® (CD2 Pan T - Cat. No. 11159D, Life Technologies). Following each incubation, the bead-bound cells were separated using a . The purity of these white blood cell subpopulations, which per manufacturer's speci?cations (Life Technologies) is >95%, was t from the unique gene expression pattern obtained (cluster analysis). As soon as each white blood cell subpopulation was isolated, the magnetic bead bound cells were washed with 1x PBS and lysed in Trizol®. The fractionation and subsequent lysis of all the three types of cells were completed in less than 2 hours after the ion of the buffy coat.
Total RNA Isolation Total RNA was extracted from cells and tissues with Trizol® and the Pure-Link RNA isolation kit (Cat. # 12183018A, Life Technologies). The quantity and purity of the RNA s were determined on a Bioanalyzer 2100 (Agilent Technologies) and the Degradometer software on 1.41). In general, the RIN and 28s/18s ratios were always found to be in the satisfactory range, 29 and 21.9, respectively.
Whole Genome Microarray Data is Total RNA from macrophages, neutrophils, T cells, tumor tissue (TT), and "normal" prostate tissue (NT) of prostate cancer patients, and from macrophages, neutrophils, and T cells extracted from healthy male blood donors were used in gene expression pro?ling. Biotinylated cDNA probes were prepared from 100 ng of each RNA , fragmented, and hybridized with Human Gene 1.0 ST chip (Affymetrix). Array signals of ?uorescence were scanned, captured and recorded as CEL ?les. All the processing and analysis of the data were done using R 49 and Bioconductor software packages. To obtain the log2 transformed expression levels, the raw data ?les obtained in CEL ?le format were pre- processed using the oligo package and the RMA (robust hip average algorithm) routine to background correct, le normalize and summarize at the core level.
Example 2: Statistical analysis of microarray data Working with rray data can be challenging because large numbers of genes can increase the likelihood of false positives, while a small number of samples can lead to over?tting. These issues can be overcome by using tical methods to reduce the false rate of positives and using independent training and test data sets (e. g., cross-validation) to avoid over?tting. In particular, instead of using a "typical" 5% signi?cance level, the false discovery rate (FDR) can be controlled to ensure that only 5% of the genes that are discovered are false positives, and Empirical Bayesian estimates can be used to e test statistics.
Because an over?t model will perform poorly on an independent test set, a good test of the ?t of a model is how well is performs on an independent test set.
For small sample sizes, splitting data into test and training sets may leave too small of a data set for good training. This issue can be solved by using cross-validation, which splits the data into K-folds, trains the method on K-l of the folds, and tests the method on the last fold. Figure 1 depicts a m of a three-fold cross validation, wherein the diagnostic accuracy is averaged from the three splits. The ideal split for cross-validation is 10-fold for te and precise estimates of diagnostic cy. In a 10-fold cross validation, however, there are more than 10 splits because there are many choices for which data points go into the folds. For example, with the microarray data collected as bed above, there are 50,979,600 ways to form 90% training/10% testing data sets.
The Empirical Bayesian method was used as follows: 1. The ential gene expression (DE) of phagocytes (macrophages or neutrophils) vs. T cells was calculated for each gene. DE is expressed as the log of the ratio of phagocyte to T cell expression: DE = log(GEp/GETC), where GEP is phagocyte gene expression and GETC is T cell gene expression. 2. The mean DE was compared in cancer and control patients with a two- sample t-test. Empirical Bayes estimates of the test statistics "shrink" these toward zero. An ordered list ofthese test statistics was created, as shown by the exemplary prostate cancer macrophage genes in Figure 2. 3. Calculate a diagnostic ure with K genes: .5 = Z"? (DE — pg.) If S>0, then the t was diagnosed with cancer. 4. The number of genes K to include in the signature was determined by ing misclassi?cation rates in independent test sets with cross- validation.
Errors were calculated using an average of l-sensitiVity and ificity, and the cross-validated error was used to select markers (Figure 3). id="p-136" id="p-136" id="p-136" id="p-136" id="p-136"
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[0136] Using the above methods, the markers associated with prostate cancer in macrophages vs. T cells (Tables 2 and 3) and the markers associated with prostate cancer in neutrophils vs. T cells (Tables 4 and 5) were ?ed. Of these, specific signatures of four s (for macrophages) and 11 markers (for neutrophils) also were identi?ed that give especially high sensitiVity and specificity. For example, a four-marker signature (PC-MACRO 1-4) from macrophages has a sensitivity of 100% and a city of 99.4%. The four genes identi?ed were: 1. P2RYlO: purinergic receptor P2Y (associated with leukemia, lymphoma, pancreatic and soft tissue/muscle tissue tumors); 2. TNFAIP3: tumor necrosis factor, alpha-induced protein 3 iated with adrenal, breast, cervical, ctal, gastrointestinal, germ cell, prostate, kidney, liver, lung, ovarian, atic, primitive neuroectodermal, skin, soft tissue/muscle, e tumors, leukemia, chondrosarcoma, lymphoma, glioma, non-neoplasia) inhibitor of programmed cell death; 3. CSCRl: chemokine (C-X-C motif) receptor 1 (associated with ia) interleukin-8 receptor (in?ammatory response); and 4. DNAJBl: DnaJ homolog, ily B, member 1 (associated with breast, al, colorectal, esophageal, gastrointestinal, germ cell, prostate, , liver, lung, ovarian, pancreatic, primitive neuroectodermal, skin, soft /muscle, and uterine tumors, leukemia, prostate cancer, chondrosarcoma, lymphoma, glioma, non-neoplasia, bladder carcinoma, retinoblastoma) heat shock protein binding, chaperone mediated protein folding requiring cofactor.
Figure 5 shows a summary of the prostate cancer markers identi?ed from macrophages and from neutrophils, as compared to T cells from the same individuals, for the PC-MACRO l- l 00, PC-MACRO 101-200, PC-NEUTRO l- 100, and TRO 101-200 markers. Speci?cally, average error, sensitivity, and speci?city values are given for a four marker panel from PC-MACRO l-lOO (PC-MACRO l-4), a ?ve marker panel from PC-MACRO 101-200 (PC-MACRO 101-105), an 11 marker panel from PC-NEUTRO l-lOO (PC-NEUTRO l-l l), and a ?ve marker panel from PC-NEUTRO 101-200 (PC-NEUTRO 101-105). Figure 7 demonstrates the power of a paired within-subj ect (phagocyte to agocyte) comparison to detect prostate cancer as compared to phagocytes not paired with T cell data for comparison. The paired approach (comparing macrophage or phils to T cell expression) is better than the phagocyte gene expression alone.
Table 2: Macrophage prostate cancer markers (PC-MACRO) l-lOO PC- Transcript Control Cancer Pattern 1 8168524 0.02960015 0.17371456 cancer 8122265 0.2072961 0.64660566 cancer 8058905 9747 9.01125215 cancer 8034837 0.30827525 0.59910162 cancer 7961371 0079 0.35670899 cancer 7903592 0.13849989 1.06724942 cancer 6 0.0203181 0.04210984 cancer 7930413 0.55491407 1.29068269 cancer 7952036 0.55537645 1.25160155 cancer 8174361 0.61722782 0.99292328 cancer 8037205 0.58997357 2.11572726 cancer 7920575 0.39329959 0.62620162 cancer 8119016 0.36673125 0.69477584 cancer 8052654 2.17872272 5.21317113 cancer 7923547 1.25355687 8.47033089 cancer PC- Transcript Control Cancer Pattern 16 8083569 0.47715218 1.04210643 cancer 7923917 002901112 0.0622508 cancer 8072328 0.40442136 0.82657902 cancer 8003601 7628 1.16646463 cancer 8000702 0.77897628 1.16646463 cancer 8146550 2.75450762 4.74699662 cancer 8001317 0.80092363 1.41111983 cancer 7926916 0.11459634 0.31950013 cancer 8097461 3.60707506 1.37545092 cancer 8095728 105690588 161196238 cancer 8070720 3.2097594 1.47073612 cancer 8063382 2.40434209 6762 cancer 7972805 5.28713747 2.35650501 cancer 9 2725 2.28292516 cancer 7900426 1.28882569 1.93805477 cancer PC- Transcript Control Cancer Pattern 31 7922474 0.76633493 1.34565158 cancer 7899253 0.69522433 1.75493876 cancer 8048227 1.44379397 9.95849988 cancer 7929032 0.58060723 1.01380895 cancer 7904361 0.08449196 2318 cancer 8179263 087218945 33 cancer 8177983 087218945 2.232133 cancer 8118142 087218945 2.232133 cancer 7961142 474853476 1.1168988 cancer 7958262 0.79461476 1.39716641 cancer 7898693 1.15920613 6.21826436 cancer 8130768 1.37675482 2.14622801 cancer 8036710 1.01224589 1.46693108 cancer 8116983 4.10900898 1.91359618 cancer 2 776 1.3563131 cancer PC- ript Control Cancer Pattern 46 7978595 1.08988349 1.72579173 cancer 8078014 2.64661452 4.48150446 cancer 1 075839436 1.1521868 cancer 7987192 1.41678085 2.19019682 cancer 8103226 3.21824227 5.86388463 cancer 8114010 0.99048925 1.76524384 cancer 8025672 0.55755809 1.01345746 cancer 8016540 118977345 4.2507857 cancer 7945169 0.32493882 0.99411563 cancer 7999642 0.84943451 1.19058275 cancer 8075316 3.9521795 1.67634544 cancer 8114572 128027337 397022993 cancer 0.69601899 1.27835261 cancer 8019877 0.64706614 1.14703044 cancer 7974920 0.03658486 0.10583053 cancer PC- ript Control Cancer Pattern 61 8092691 4.31398434 8.88039871 cancer 8124280 0.56453876 0.85399528 cancer 8062927 1.10649927 5.23596961 cancer 0 1.17446597 1.72020153 cancer 7974870 1.83021593 1.13565448 cancer 7929616 1.46879694 2.74881528 cancer 8119898 7.26271784 3.23095057 cancer 7945944 1.63739015 2.27047063 cancer 8024582 0.43651409 2531 cancer 7940287 3959 0.81965175 cancer 7946559 2.03180864 3.35098913 cancer 7924603 1.01883127 1.48695002 cancer 8066905 0.89267917 1.25423704 cancer 7923233 0.31387565 0.49612974 cancer 8072346 0.60513857 0.85285694 cancer PC- Transcript Control Cancer Pattern 76 8127145 1.30383618 0.89535391 cancer 8026564 0.82494247 2655 cancer 7922846 202777211 0.8219638 cancer 7956819 2.14980211 1.22057225 cancer 8105778 042568173 0.2740702 cancer 8104492 2.19340942 0.74828404 cancer 8072744 2444 4.38539283 cancer 7973352 1.62434306 0.87111779 cancer 8064438 2.1394305 1.41278384 cancer 8094743 0.06926233 0.03501436 cancer 8093993 0389 0.80768602 cancer 8177951 1.30231682 0.78155345 cancer 8061373 1.41841367 9805 cancer 8086125 0.71037012 0.39842202 cancer 8026047 3.06943153 1.50812928 cancer PC- Transcript Control Cancer Pattern ----91 8090577 677 1 04964813 cancer ----92 8117330 080516115 0 04 cancer ----93 7954711 085606841 0 49112958 cancer ----94 7909610 387 2 22353869 cancer 95 7897482 120067741. 4585 cancer upregulated h---8156848 104872188 722 cancer ----97 8099797 218785448 141539173 cancer h---8078214 1.4099199 109850026 cancer h---7901054 207831718 10928931 cancer ----100 8008870 3.86152022 2.72090652 cancer Table 3: Macrophage prostate cancer markers (PC-MACRO) 101-200 Transcript Gene Name Pattern Cluster 101 8168524 P2RY10 0.152005 0029718. cancer upregulated 102 8122265 TNFAIP3 0 611381 0.207375 cancer upregulated 103 8058905 CXCRl 9.262282 0792206 Transcript Gene Name Pattern Cluster 8034837 DNAJBl 0.577777 0.306905 cancer upregulated 7923547 CHI3L1 9.146662 33 cancer 7903592 KIAA1324 1.149342 0.13814 cancer 8037205 CEACAMl 2.222375 0.575345 cancer 8083569 TIPARP 1.072432 0.472752 cancer 7898693 ALPL 6.626161 1.146884 cancer 7962516 SLC38A1 0.04104 0.020194 cancer 7920575 PBXIP1 0.617361 0.3943 cancer 7961371 DUSP16 0.330336 0.152073 cancer 8179263 TNF 2.369374 0.866687 cancer 8177983 TNF 2.369374 0.866687 cancer 8118142 TNF 2.369374 87 cancer 7899253 8 1.744595 0.688354 cancer 7930413 DUSP5 55 0.55359 cancer Transcript Gene Name Pattern Cluster 8062927 P13 5.154207 1.0788 cancer 8016540 PHOSPHOl 43 1.194592 cancer 7952036 MPZL3 1.249639 0.548515 cancer 8048227 CXCR2 10.73569 1.46568 cancer 7923917 FAIM3 0.057531 0.029321 cancer 8119016 MAPK13 0.664511 0.35986 cancer 8104492 ROPN1L 2.327292 0.73435 cancer 8174361 TSC22D3 0.981877 34 cancer 7996100 GPR97 4.78039 1.14386 cancer 7904361 FAM46C 53 0.081631 cancer 8114572 --downregulatedHBEGF 3.818586 13.18658 cancer 8095728 --downregulatedEREG 1.674886 11.07804 cancer 7974870 --downregulatedSNAP 1.085902 96 cancer 7972805 RAB20 2.212362 5.314486 cancer downregulated 7972557 GPR183 0.336149 1.149753 cancer Transcript Gene Name Pattern Cluster 7955589 NR4A1 32 6.651218 cancer downregulated 1 --downregulatedCCRN4L 1.379503 36 cancer 8070720 --downregulatedICOSLG 1.531107 3.198553 cancer 8001317 N4BP1 1.327524 0.790791 cancer 8146550 SDCBP 4.50276 2.731552 cancer 7 SMCHD1 1.134906 0.650561 cancer 8061373 --downregulatedGZF1 1.419186 2.453877 cancer 8063382 --downregulatedSNAII 1.188284 2.400909 cancer 8083494 MME 7.315031 1.208894 cancer 8063115 MMP9 4.024209 1.304142 cancer 7922474 KIAA0040 1.259402 0.759183 cancer 8026564 KLF2 68 0.528515 cancer 7926916 ZEB1 0.284048 0.11094 cancer 8156848 --downregulatedNR4A3 1.050414 2.67113 cancer Transcript Gene Name Pattern Cluster 8073148 ATF4 1.496953 1.069621 cancer 8000482 XPO6 76 0.731566 cancer 8078014 SLC6 4.293247 2.663884 cancer 7973352 LRP10 1.565493 0.874717 cancer 8052654 PELI 4.641692 2.197016 cancer 7961142 regulatedOLR1 1.156079 4.848597 cancer 8026456 CYP4F 5.935402 1.367724 cancer 7922846 FAM129A 2.077295 0.821 cancer 7954711 C12orf35 0.86259 0.488389 cancer 7987192 SLC12A6 2.074418 1.423051 cancer 8019885 SMCHD1 96 0.691931 cancer 8092691 BCL6 9.149487 4.324519 cancer 7998931 ZNF200 1.133831 0.752141 cancer 7945169 B 0.885462 0.323788 cancer upregulated 8072328 SEC14L2 0.764754 0.403596 cancer Transcript Gene Name Pattern 7937335 IFITM1 0.362774 0.115083 cancer upregulated 8044049 IL18RAP -upregulated0.30373 0.049551 cancer 7946559 GNG10 3.195841 2.039353 cancer 8145244 TNFRSF10C 5.007134 1.784866 cancer 0 IRFl 1.714693 0.991787 cancer 8 EGLN1 1.783661 1.202735 cancer 7924603 LBR 1.446919 17 cancer 8177951 HCG27 1.293374 0.794496 cancer 7958600 3A 1.640972 1.172031 cancer 8075316 --downregulatedOSM 1.762227 3.980639 cancer 8117330 HIST1H3A 0.808308 0.379302 cancer 8090577 MBD4 1.42938 1.048742 cancer 8064438 NSFLlC 2.080207 1.414354 cancer 8127145 ELOVL5 1.251642 0.886912 cancer Transcript Gene Name n Cluster 8025672 SLC44A2 0.969342 0.555724 cancer 8105778 PIK3R1 0.421185 0.272848 cancer 7974920 SYNE2 ulated0.094911 0.03597 cancer 8086125 TRANK1 -upregulated0.716401 0.399926 cancer 8026047 JUNB -upregulated2.974865 1.490665 cancer 7945944 RHOG 2.197395 1.627514 cancer 8068761 ABCG1 0.985942 0.582396 cancer 8119898 --downregulatedVEGFA 3.47377 7.314255 cancer 6 FRAT1 -upregulated2.715937 1.48299 cancer 8128111 UBE2J1 2.280333 28 cancer 8032127 C190rf2 1.389324 0.85144 cancer 0 --downregulatedPDE4D 0.545705 0.908491 cancer 7940287 MS4A1 0.865272 0.300979 cancer 7992811 MMP25 3.585842 1.509232 cancer upregulated 7929032 0.97455 0.580443 cancer Transcript Gene Name Cluster 7993035 UBN1 1.892712 1.228282 cancer 8103226---TMEM154 5.549365 22 cancer 8079140---SNRK 0.804865 0.595551 cancer 8116983---CD83 2.156174 4.156219 cancer 8117071 ---FAM8A1 1.317348 0.866955 cancer 7904465---HIST2H2BA 0.972877 0.687243 cancer 7961365---MANSCl 5.080479 1.822875 cancer 8124280---FAM65B 0.84345 0.566881 cancer 7978595---BAZlA 44 1.089146 cancer 8066905---ZNFX 1.210468 0.893689 cancer Table 4: 1\eutrophil prostate cancer markers (PC-NEUTRO) 1-100 PC- Transcript Control Cancer Pattern NEUTRO Cluster ID mean mean 1 8180410 3484 0.19397697 cancer gulated 2 8158952 0.6875578 0.24194927 cancer downregulated PC- Transcript Control Cancer Pattern 3 8138531 0.7122055 0.26055464 cancer 8091806 0742 0.20402938 cancer 8005943 0.55224767 3405 cancer 7956743 0.47917225 0.14307559 cancer 8026440 0.58374878 0.20494112 cancer 8076209 0.39282293 0.08752956 cancer 7942824 0.59451215 0.16395325 cancer 8107470 0.80083176 0.34060884 cancer 8005471 0.58174315 0.16062621 cancer 0.58587709 0.16124473 cancer 8180297 0.47149462 3531 cancer 7998655 0.53911452 0.22118994 cancer 8013348 0.2186433 0.53383007 cancer -7983843 0.18561995 0.54618191 cancer downregulated 8180355 0.19440318 0.59101735 cancer PC- Transcript l Cancer Pattern 18 7920317 015604404 056532741 cancer 8026868 016320999 058908022 cancer 8154394 0.20189545 0.74519071 cancer 7986323 0.10306287 0.41147346 cancer 7946812 013349395 057522988 cancer 8116929 018102731 055370767 cancer 8030351 020773723 06 cancer 7961022 0.35396993 0.75191636 cancer 8061136 0.2926624 0.79565411 cancer 8085026 0.16197889 0.48683008 cancer 8153903 154 0.6269282 cancer 8115158 020196131 668 cancer 8101429 0.07289442 0.48628779 cancer 8177003 0.18190856 0.73530108 cancer 8171111 0.18190856 0.73530108 cancer PC- Transcript l Cancer Pattern 33 7900585 0.36257439 0.84416026 cancer 8173513 0.10395144 0.41660967 cancer 7954006 035364421 0.7468305 cancer 8024299 022346081 0.6228029 cancer 8115234 0.08063857 0.33222302 cancer 8164100 0.0914208 0.38215761 cancer 7948679 2613 0.57794944 cancer 8076511 0.1676643 0.48723858 cancer 8043100 6421 0.74881525 cancer 8174710 022337229 195 cancer 7990965 0.10872074 0.39812056 cancer 7990916 0.10872074 0.39812056 cancer 8051066 0.2069229 0.79589283 cancer 8127526 0.2185427 0.49610551 cancer 7986765 0.16454363 0.52908894 cancer PC- Transcript Control Cancer Pattern 48 8034416 0.1365427 0.47170321 cancer 8092457 027929283 081493697 cancer 7899160 0.13609987 0.61167474 cancer 7954997 0.23835321 0.87499649 cancer 7966534 0.21611199 0.52322977 cancer 8109750 0.06776826 0.36450083 cancer 7968872 0.15717923 0.57732615 cancer 8038086 0.13287674 0.56174531 cancer 8099887 0.18843453 0.44463292 cancer 4 0.1922139 0.45416067 cancer 7990898 0.18632151 0.44425134 cancer 1 0.20793347 0.69036634 cancer 7973056 0.13506675 0.48508632 cancer 7990949 0.18326745 0.43844761 cancer 8022170 1473 0.52059361 cancer PC- ript Control Cancer Pattern 63 8116520 0.13806378 0.61288083 cancer 7912956 0.4490642 0.97752282 cancer 2 021362909 951 cancer 8009561 0.26753012 0.58406567 cancer 7899957 0.32670929 0.71219915 cancer 7944152 0.15977809 0.61855823 cancer 8050215 0.2762267 0.56213724 cancer 8027778 0.29827452 0.62982603 cancer 7948667 0.11071476 0.53165314 cancer 7966996 0.14171537 0.44740386 cancer 8007441 0.32635716 0.62715565 cancer 8036602 0.17588633 0.64662967 cancer 0.28983952 0.64953767 cancer 7999520 0.10457296 0.45991055 cancer 8028916 0.2090505 0.60393557 cancer PC- Transcript Control Cancer Pattern 78 8172154 0.30916584 0.59966248 cancer 8109222 0.23220579 0.54727725 cancer 8178220 0.19535983 1.32225018 cancer 7917906 0.23715116 0.55094415 cancer 7937476 0.09528914 0.45042024 cancer 8154727 0.20024719 0.65593494 cancer 7 0.22640003 0.84722905 cancer 7903010 0.21683704 0.48614067 cancer 8036777 0.10972586 0.34434732 cancer 8125750 0.180311 0.455186% cancer 8180402 0.36726964 0.68339044 cancer 8117377 0.22475425 4616 cancer 7901038 0.19506976 0.50446547 cancer 7954063 6204 0.40193484 cancer 7933760 0.18249417 0.69607118 cancer PC- Transcript Control Cancer n IIII93 8051204 0.17089002 7142 cancer IIII94 8047635 019882641 048315169 cancer IIII95 8151376 023801921 377 cancer IIII8084488 0.13506115 0.39473505 cancer IIII97 8008132 0.18825496 0.56719612 cancer IIII7905099 0.22074548 0.63249579 cancer IIII8118594 0.19038692 1.37544631 cancer 100 III7906564 0.37291778 1.44546045 cancer Table 5: Neutrophil prostate cancer markers (PC-NEUTRO) 101-200 PC- Transcript Gene Name Control Cancer Pattern 101 IIIII8158952 EEFlAl 0.272609 0.69519 cancer 102 IIIII8138531 EEF1A1 0.292444 0.718089 cancer 103 091806 RPL23A 0.217459 0.602422 cancer 104 IIIII8026440 RPL23A 0.217383 0.591544 cancer 105 IIIII8005943 RPL23A 0.202168 0.559676 cancer PC- Transcript Gene Name Control Cancer Pattern cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated RP" cancer downregulated "5 cancer downregulated cancer downregulated cancer downregulated RP" cancer gulated cancer downregulated cancer downregulated cancer downregulated PC- Transcript Gene Name Control Cancer Pattern cancer downregulated cancer downregulated cancer downregulated m cancer gulated cancer downregulated "5 cancer downregulated m cancer downregulated cancer downregulated W cancer downregulated RP" cancer downregulated cancer downregulated RP" cancer downregulated cancer downregulated cancer downregulated cancer downregulated PC- Transcript Gene Name Control Cancer Pattern cancer downregulated m cancer downregulated cancer downregulated cancer downregulated cancer downregulated W cancer downregulated cancer downregulated RP" cancer downregulated cancer gulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated PC- Transcript Gene Name Control Cancer Pattern RP" cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated "5 cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer gulated cancer downregulated RP" cancer downregulated PC- Transcript Gene Name Control Cancer Pattern cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer gulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated PC- Transcript Gene Name Control Cancer Pattern cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer gulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated cancer downregulated PC- Transcript Gene Name Control Cancer Pattern 196 IIIII7996947 CYB5B 0.258857 0.497964 cancer 197 IIIII7971134 C13orf2 0.267679 53 cancer 198 IIIII7944152 IL10RA 0.185371 0.619532 cancer 199 IIIII7999520 RSL1D1 0.109754 91 cancer 200 IIIII7993349 NPIP 0.286955 0.524779 cancer Example 4: Detecting head and neck cancer After identifying markers useful for detecting prostate cancer, PC- MACRO 1-4 were tested for their ability to also diagnose head and neck cancer.
Specifically, the genetic signature S was calculated for each patient as a weighted average of the genes in the signature, wherein S > 0 implies cancer and S < 0 implies no cancer (healthy). The prostate cancer markers accurately segregated ts into "healthy" and "cancer" groups (Figure 4). The PC-NEUTRO 1-11 markers also were tested for their ability to diagnose head and neck cancer by calculating S for each t. In this experiment, only a single "healthy" patient was gnosed (Figure 6).
Example 5: Additional validation Additional validation is performed by validating the gene signature on a new data set of ~50 prostate cancer cases and 50 controls. Final validation is performed by estimating the sensitivity and icity of the final gene signature on a large sample. For example, 195 cases and 195 controls can be used to estimate a sensitivity/specificity of at least 97.5% with a 95% margin of error no more than 5%. A challenge in ing a final tion study is that although cancer patients are pure, controls may have up to 20% false negatives. A statistical issue is that, while estimating sensitivity is not a problem, specificity has an upper bound of 80%. The solution is to purify the l set of patients. A puri?cation method uses secondary screening of all controls, wherein three methylated gene marker tests are used to purify the control test set: 1. GST-Pi (sensitivity = 95%, speci?city =85%) 2. RAR-Zb (sensitivity = 95%, speci?city =48%) 3. APC (sensitivity = 95%, speci?city =50%) in men with two serial negative biopsies. A second puri?cation method is ed in Figure 8, and a comparison of puri?cation methods is shown in Figure 9.
Other embodiments of the ion as described herein are defined in the following paragraphs: 1. A method for diagnosing or aiding in the diagnosis of prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject's macrophage cells; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the t's non-phagocytic cells; and c) identifying a difference n the ed levels of the one or more selected PCMACRO markers in steps a) and b), wherein the identified difference tes that the subject has said prostate cancer. 2. A method for assessing the risk of developing prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject's macrophage cells; b) measuring the levels of the one or more selected PC-MACRO markers in a tion of the subject's non-phagocytic cells; and c) identifying a difference between the measured levels of the one or more selected PCMACRO markers in steps a) and b), n the identified difference indicates that the subject has a risk of developing said prostate cancer. 3. A method for prognosing or aiding in the prognosis of prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the t's macrophage cells; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject's agocytic cells; and c) identifying a difference between the measured levels of the one or more selected PCMACRO markers in steps a) and b), wherein the identified difference is indicative of the prognosis of said prostate cancer in the subject. 4. A method for assessing the efficacy of a treatment for te cancer in a subject comprising: a) ing the levels of one or more markers selected from the group consisting of PC-MACRO 1-200 in a tion of the t's macrophage cells before the treatment; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject's non-phagocytic cells before the treatment; c) identifying a first difference between the measured levels of the one or more selected PC-MACRO markers in steps a) and b); d) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject's macrophage cells after the treatment; e) measuring the levels of the one or more ed PC-MACRO markers in a population of the subject's non-phagocytic cells after the treatment; f) identifying a second difference between the measured levels of the one or more selected PC-MACRO markers in steps d) and e); and g) fying a difference between the first ence and the second difference, wherein the difference identified in g) is indicative of the efficacy of the treatment for said prostate cancer in the subject.
. A method for monitoring the progression or sion of prostate cancer in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject's hage cells at a first time point; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject's non-phagocytic cells at the first time point; c) fying a first difference between the measured levels of the one or more selected RO markers in steps a) and b); d) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject's macrophage cells at a second time point; e) measuring the levels of the one or more selected PC-MACRO s in a tion of the subject's non-phagocytic cells at the second time point; f) identifying a second difference between the measured levels of the one or more selected PC-MACRO markers in steps d) and e); and g) identifying a difference n the first difference and the second difference, wherein the difference identified in g) is indicative of the progression or regression of said prostate cancer in the subject. 6. A method for identifying a compound capable of ameliorating or treating prostate cancer in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject's macrophage cells before administering the compound to the subject; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject's non-phagocytic cells before stering the compound to the subject; c) identifying a first difference between the ed levels of the one or more selected PC-MACRO markers in steps a) and b); d) measuring the levels of the one or more ed PC-MACRO markers in a population of the t's macrophage cells after the administration of the compound; e) measuring the levels of the one or more selected PC-MACRO markers in a tion of the subject's non-phagocytic cells after the administration of the compound; f) identifying a second difference between the measured levels of the one or more selected PC-MACRO markers in steps d) and e); and g) identifying a ence n the first difference and the second difference, wherein the difference identified in g) indicates that the compound is capable of ameliorating or treating said prostate cancer in the subject. 7. A method for diagnosing or aiding in the diagnosis of prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of one or more s selected from the group consisting of PC-NEUTRO 1-200 in a population of the t's neutrophil cells; b) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject's non-phagocytic cells; and c) identifying a difference between the ed levels of the one or more selected PCNEUTRO markers in steps a) and b), n the identified difference indicates that the subject has said prostate cancer. 8. A method for assessing the risk of developing prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of one or more s selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject's neutrophil cells; b) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject's non-phagocytic cells; and c) identifying a difference between the measured levels of the one or more selected PCNEUTRO markers in steps a) and b), wherein the identified difference indicates that the subject has a risk of developing said prostate cancer. 9. A method for prognosing or aiding in the prognosis of prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject's neutrophil cells; b) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject's non-phagocytic cells; and c) identifying a difference between the measured levels of the one or more selected PCNEUTRO s in steps a) and b), wherein the identified difference is tive of the prognosis of said prostate cancer in the subject.
. A method for assessing the cy of a treatment for prostate cancer in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject's neutrophil cells before the treatment; b) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject's non-phagocytic cells before the treatment; c) identifying a first difference between the measured levels of the one or more selected PC-NEUTRO markers in steps a) and b); d) measuring the levels of the one or more selected PC-NEUTRO markers in a tion of the subject's phil cells after the treatment; e) measuring the levels of the one or more selected PC-NEUTRO s in a population of the subject's non-phagocytic cells after the treatment; f) identifying a second difference between the measured levels of the one or more selected TRO markers in steps d) and e); and g) identifying a difference between the first ence and the second difference, n the difference identified in g) is indicative of the efficacy of the treatment for said prostate cancer in the subject. 11. A method for monitoring the progression or regression of prostate cancer in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject's neutrophil cells at a first time point; b) measuring the levels of the one or more selected PC-NEUTRO s in a population of the subject's agocytic cells at the first time point;\ c) identifying a first ence between the measured levels of the one or more selected PC-NEUTRO markers in steps a) and b); d) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject's neutrophil cells at a second time point; e) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject's agocytic cells at the second time point; f) identifying a second difference between the measured levels of the one or more selected PC-NEUTRO markers in steps d) and e); and g) identifying a difference between the first difference and the second difference, wherein the difference identified in g) is tive of the progression or regression of said prostate cancer in the subject. 12. A method for fying a compound capable of ameliorating or ng prostate cancer in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject's neutrophil cells before administering the compound to the subject; b) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject's non-phagocytic cells before administering the compound to the subject; c) identifying a first ence between the measured levels of the one or more selected PC-NEUTRO markers in steps a) and b); d) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject's neutrophil cells after the administration of the compound; e) measuring the levels of the one or more selected PC-NEUTRO markers in a population of the subject's non-phagocytic cells after the administration of the compound; f) identifying a second ence between the measured levels of the one or more ed PC-NEUTRO markers in steps d) and e); and g) identifying a difference between the first difference and the second difference, wherein the difference identified in g) indicates that the compound is capable of rating or treating said prostate cancer in the subject. 13. A method for diagnosing or aiding in the diagnosis of prostate cancer in a subject, the method comprising the steps of: a) ing the levels of at least one or more markers selected from the group consisting of RO 1-200 in a population of the subject's macrophage cells, and measuring the levels of at least one or more markers ed from the group consisting of PCNEUTRO 1-200 in a population of the subject's neutrophil cells; b) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject's non-phagocytic cells; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject's agocytic cells; c) identifying a ence between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and b); and d) identifying a difference between the measured levels or activities the at least one or more selected PC-NEUTRO markers in steps a) and b); wherein the differences fied in c) and d) indicate that the subject has said prostate cancer. 14. A method for assessing the risk of developing prostate cancer in a subject, the method comprising the steps of: a) measuring the levels of at least one or more markers selected from the group ting of PC-MACRO 1-200 in a population of the subject's macrophage cells, and measuring the levels of at least one or more markers selected from the group consisting of PCNEUTRO 1-200 in a tion of the subject's neutrophil cells; b) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject's non-phagocytic cells; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject's non-phagocytic cells; c) identifying a difference between the measured levels of the at least one or more ed PC-MACRO markers in steps a) and b); and d) identifying a difference n the measured levels of the at least one or more ed PC-NEUTRO s in steps a) and b); wherein the differences identified in c) and d) indicate that the subject has a risk of developing said prostate cancer.
. A method for prognosing or aiding in the prognosis of prostate cancer in a subject, the method comprising the steps of: a) ing the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the t's macrophage cells, and measuring the levels of at least one or more markers selected from the group consisting of PCNEUTRO 1-200 in a population of the subject's neutrophil cells; b) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject's non-phagocytic cells; and ing the levels of the at least one or more selected TRO markers in a population of the subject's non-phagocytic cells; c) identifying a difference between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and b); and d) identifying a difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps a) and b); wherein the differences fied in c) and d) are indicative of the prognosis of said te cancer in the subject. 16. A method for assessing the efficacy of a treatment for prostate cancer in a subject comprising: a) measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject's macrophage cells before the treatment, and measuring the levels of at least one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject's neutrophil cells before the treatment; b) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject's non-phagocytic cells before the ent; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject's nonphagocytic cells before the treatment; c) identifying a first difference between the ed levels of the at least one or more selected PC-MACRO markers in steps a) and b); and identifying a second difference between the measured levels of the at least one or more ed PC-NEUTRO markers in steps a) and d) measuring the levels of the at least one or more selected PC-MACRO marker in a population of the subject's macrophage cells after the treatment, and measuring the levels of the at least one or more selected PC-NEUTRO marker in a population of the subject's neutrophil cells after the treatment; e) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject's agocytic cells after the treatment; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the t's nonphagocytic cells after the ent; f) identifying a third difference between the measured levels of the at least one or more selected RO markers in steps d) and e); and g) identifying a fourth difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps d) and e); h) identifying a difference between the first and second differences; and i) fying a difference between the third and fourth differences, wherein the differences identified in h) and i) are tive of the efficacy of the treatment for said te cancer in the subject. 17. A method for monitoring the ssion or regression of prostate cancer in a subject comprising: a) measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 in a population of the subject's macrophage cells at a first time point, and measuring the levels of at least one or more s selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject's neutrophil cells at the first time point; b) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject's non-phagocytic cells at the first time point; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject's nonphagocytic cells at the first time point; c) identifying a first difference between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and b); and identifying a second difference n the measured levels of the at least one or more ed PC-NEUTRO markers in steps a) and d) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject's macrophage cells at a second time point, and measuring the levels of the at least one or more ed PC-NEUTRO markers in a population of the subject's neutrophil cells at the second time point; e) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject's non-phagocytic cells at the second time point; and measuring the levels of the at least one or more selected TRO markers in a population of the subject's non-phagocytic cells at the second time point; f) identifying a third difference between the measured levels of the at least one or more selected PC-MACRO markers in steps d) and e); and g) identifying a fourth difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps d) and e); h) identifying a difference between the first and second differences; and i) identifying a difference between the third and fourth differences, wherein the differences identified in h) and i) are indicative of the progression or regression of said prostate cancer in the subject. 18. A method for identifying a compound capable of ameliorating or ng te cancer in a subject comprising: a) measuring the levels of at least one or more markers ed from the group ting of PC-MACRO 1-200 in a population of the subject's macrophage cells before administering the compound to the subject, and measuring the levels of at least one or more markers selected from the group consisting of PC-NEUTRO 1-200 in a population of the subject's neutrophil cells before administering the compound to the subject; b) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject's agocytic cells before administering the compound to the subject; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject's non-phagocytic cells before administering the compound to the subject; c) identifying a first difference between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and b); and identifying a second ence between the measured levels of the at least one or more ed TRO markers in steps a) and d) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject's macrophage cells after administering the compound to the subject, and measuring the levels of the at least one or more selected TRO markers in a population of the subject's neutrophil cells after administering the compound to the t; e) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject's agocytic cells after administering the compound to the subject; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject's non-phagocytic cells after administering the nd to the subject; f) identifying a third difference between the measured levels of the at least one or more selected PC-MACRO markers in steps d) and e); and g) identifying a fourth difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps d) and e); h) identifying a difference between the first and second differences; and i) identifying a difference between the third and fourth differences, wherein the differences identified in h) and i) te that the nd is capable of ameliorating or treating said prostate cancer in the subject. 19. The method of any one of paragraphs 1-18, r comprising measuring at least one standard parameter associated with said te cancer.
. The method of paragraph 19, wherein the standard parameter is selected from the group ting of tumor stage, tumor grade, tumor size, tumor visual characteristics, tumor growth, tumor ess, tumor progression, tumor metastasis tumor distribution within the body, odor, molecular pathology, genomics, or tumor angiograms. 21. The method of any one of paragraphs 13-18, wherein the selected PC-MACRO markers and the selected PC-NEUTRO markers are measured from the same population of nonphagocytic cells in steps b) or e). 22. The method of any one of paragraphs 13-18, wherein the selected PC-MACRO markers and the selected TRO are from different populations of non-phagocytic cells in steps b) or e). 23. The method of any one of paragraphs 1-6 and 13-18, n at least two, three, four, or five markers are selected from PC-MACRO 1-200. 24. The method of any one of paragraphs 1-6 and 13-18, wherein the selected PC-MACRO markers comprise one or more markers selected from the group consisting of P2RY10, 3, CXCR1, DNAJB1, and CHI3L1.
. The method of any one of paragraphs 1-6 and 13-18, wherein the selected PC-MACRO markers are up-regulated or activated in the hage cells compared to the non-phagocytic cells. 26. The method of paragraph 1-6 and 13-18, wherein the selected PC-MACRO markers are up-regulated or activated in the macrophage cells compared to the non-phagocytic cells. 27. The method of any one of paragraphs 1-6 and 13-18, wherein the selected PC-MACRO markers are down-regulated or inhibited in the macrophage cells compared to the nonphagocytic cells. 28. The method of any one of paragraphs 1-6 and 13-18, wherein the selected RO markers are down-regulated or inhibited in the hage cells ed to the nonphagocytic cells. 29. The method of any one of paragraphs 7-18, wherein at least two, three, four, five, six, seven, eight, nine, ten, or eleven markers are selected from PC-NEUTRO 1-200.
. The method of any one of paragraphs 7-18, wherein the selected PC-NEUTRO markers comprise one or more PC-NEUTRO markers selected from the group consisting of EIF3S5, EEEFlAl, RPL23A, RPL14, RPL23A, RPL3, RPS28, and PTMA. 31. The method of any one of paragraphs 7-18, wherein the selected TRO markers comprise one or more markers selected from the group consisting of PC-NEUTRO 1-200 and n the selected PC-NEUTRO markers are down-regulated or inhibited in the neutrophil cells compared to the non-phagocytic cells. 32. The method of any one of paragraphs 7-18, wherein the selected PC-NEUTRO markers are down-regulated or inhibited in the phil cells ed to the non-phagocytic cells. 33. The method of any one of paragraphs 1-6 and 13-18, further comprising lysing the macrophage cells and the non-phagocytic cells before a). 34. The method of any one of aphs 1-6 and 13-18, further comprising extracting the cellular contents from the macrophage cells and the non-phagocytic cells before a).
. The method of any one of paragraphs 7-18, r comprising lysing the neutrophil cells and the non-phagocytic cells before a). 36. The method of any one of paragraphs 7-18, further comprising extracting the cellular contents from the neutrophil cells and the non-phagocytic cells before a). 37. The method of paragraph 34, wherein the cellular contents of the macrophage cells comprise viable diseased cells, dead diseased cells, apoptotic diseased cells, circulating tumor cells, infectious agents, fetal cells, trophoblasts, or fragments thereof. 38. The method of aph 36, wherein the cellular contents of the phil cells comprise viable diseased cells, dead diseased cells, apoptotic diseased cells, circulating tumor cells, ious agents, fetal cells, trophoblasts, or fragments thereof. 39. The method of paragraph 34, wherein the selected one or more markers are present in the cellular contents of the macrophage cells. 40. The method of paragraph 34, n the selected one or more markers are not present in the cellular contents of the agocytic cells. 41. The method of any one of paragraphs 1-6 and 13-18, wherein the macrophage cells express the one or more selected PC-MACRO markers. 42. The method of paragraph 36, wherein the selected one or more markers are present in the cellular contents of the phil cells. 43. The method of paragraph 36, wherein the selected one or more markers are not present in the cellular contents of the agocytic cells. 44. The method of any one of paragraphs 7-18, wherein the neutrophil cells express the one or more selected PC-NEUTRO markers. 45. The method of any one of paragraphs 1-18, wherein the non-phagocytic cells are T cells, B cells, null cells, basophils, or mixtures thereof. 46. The method of any one of aphs 1-6 and 13-18, wherein the macrophage cells are ed from a bodily fluid sample, tissues, or cells of the subject. 47. The method of any one of paragraphs 7-18, wherein the neutrophil cells are isolated from a bodily fluid sample, tissues, or cells of the subject. 48. The method of any one of paragraphs 1-18, wherein the agocytic cells are isolated from a bodily fluid sample, tissues, or cells of the subject. 49. The method of any one of paragraphs 46-48, wherein the bodily fluid sample is blood, urine, stool, saliva, lymph fluid, ospinal fluid, synovial fluid, cystic fluid, ascites, pleural effusion, fluid obtained from a pregnant woman in the first trimester, fluid obtained from a pregnant woman in the second trimester, fluid obtained from a pregnant woman in the third trimester, maternal blood, amniotic fluid, chorionic villus sample, fluid from a preimplantation embryo, maternal urine, maternal saliva, placental sample, fetal blood, lavage and cervical vaginal fluid, interstitial fluid, or ocular fluid. 50. The method of any one of paragraphs 1-6 and 13-18, wherein the macrophage cells are isolated using antibodies, using a ligand that binds to a molecular receptor expressed on the plasma nes of white blood cells, or by flow cytometry, fluorescence activated cell sorting, tion, nt-based centrifugation, elution, micro fluidics, magnetic separation technique, fluorescent-magnetic separation technique, nanostructure, m dots, high throughput microscope-based platforms, or a combination f. 51. The method of any one of paragraphs 7-18, wherein the neutrophil cells are isolated using antibodies, using a ligand that binds to a molecular receptor sed on the plasma membranes of white blood cells, or by flow cytometry, fluorescence activated cell sorting, filtration, nt-based fugation, elution, micro fluidics, magnetic separation technique, fluorescent-magnetic separation technique, nanostructure, quantum dots, high throughput microscope-based platforms, or a ation thereof. 52. The method of any one of paragraphs 1-18, wherein the non-phagocytic cells are isolated using antibodies, using a ligand that binds to a molecular receptor expressed on the plasma membranes of white blood cells, or by flow cytometry, fluorescence activated cell g, filtration, gradient-based centrifugation, n, micro fluidics, magnetic separation technique, fluorescent-magnetic separation que, nanostructure, quantum dots, high throughput microscope-based platforms, or a combination thereof. 53. The method of any one of paragraphs 1-6 and 13-18, wherein the macrophage cells are isolated using a product secreted by the macrophage cells. 54. The method of any one of paragraphs 7-18, wherein the neutrophil cells are isolated by using a product secreted by the neutrophil cells. 55. The method of any one the paragraphs 1-6 and 13-18, wherein the macrophage cells are isolated by using a cell surface target on the surface of macrophage cells. 56. The method of any one of paragraphs 7-18, wherein the neutrophil cells are isolated by using a cell surface target on the surface of neutrophil cells. 57. The method of paragraph 55, wherein the target is sed by the macrophage cells. 58. The method of paragraph 55, wherein the target is not expressed by the macrophage cells. 59. The method of paragraph 56, n the target is expressed by the neutrophil cells. 60. The method of paragraph 56, wherein the target is not expressed by the neutrophil cells. 61. The method of any one of paragraphs 55-60, wherein the target is a marker of said prostate cancer. 62. The method of any one of paragraphs 1-18, n the measured levels are gene expression levels. 63. The method of any one of paragraphs 1-18, wherein the measured levels are protein expression levels. 64. The method of any one of the paragraph 1-18, wherein the levels or activities are measured by a qualitative assay, a tative assay, or a combination f. 65. The method of paragraph 64, wherein the quantitative assay uses sequencing, direct sequencing, RNA sequencing, whole transcriptome shotgun sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex cing, cycle sequencing, single-base extension sequencing, solid-phase cing, roughput sequencing, massively parallel signature sequencing, emulsion PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read cing, single-molecule sequencing, sequencing-by-synthesis, ime sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD® sequencing, MSPET cing, mass spectrometry, matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surfaceenhanced laser deorption/ionization-time of flight (SELDI-TOF) mass spectrometry, quadrupole-time of flight (Q-TOF) mass spectrometry, heric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry , polymerase chain reaction (PCR) analysis, quantitative PCR, real-time PCR, fluorescence assay, colorimetric assay, chemiluminescent assay, or a ation thereof. 66. The method of paragraph 62, wherein the gene expression levels are measured by polymerase chain on (PCR) analysis, sequencing analysis, electrophoretic analysis, restriction nt length polymorphism (RFLP) analysis, Northern blot analysis, quantitative PCR, reverse-transcriptase-PCR analysis (RT-PCR), -specific oligonucleotide hybridization is, comparative genomic hybridization, heteroduplex mobility assay (HMA), single strand mational polymorphism (SSCP), denaturing nt gel electrophisis (DGGE), R Aase mismatch analysis, mass spectrometry, tandem mass spectrometry, matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass ometry, surface-enhanced laser ion/ionization-time of flight (SELDI-TOF) mass spectrometry, quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure onization mass spectrometry (APPIMS ), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption/ionization- Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, ary ion mass spectrometry (SIMS), e n resonance, Southern blot analysis, in situ hybridization, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), microarray, comparative genomic hybridization, yping, multiplex ligation-dependent probe amplification (MLPA), Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF), microscopy, methylation specific PCR (MSP) assay, Hpall tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay, radioactive acetate labeling assays, colorimetric DNA ation assay, chromatin immunoprecipitation combined with microarray (ChlP-on-chip) assay, restriction landmark c scanning, Methylated DNA immunoprecipitation (MeDIP), molecular break light assay for DNA adenine methyltransferase activity, chromatographic separation, methylation-sensitive restriction enzyme analysis, bisulfite-driven conversion of non-methylated cytosine to uracil, methylbinding PCR analysis, or a combination thereof. 67. The method of paragraph 62, wherein the gene expression levels are measured by a sequencing technique selected from the group consisting of direct sequencing, RNA sequencing, whole transcriptome shotgun sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, quencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base ion sequencing, solid-phase cing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term cing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-bysynthesis , real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD® cing, MS-PET sequencing, mass ometry, and a combination f. 68. The method of paragraph 63, wherein the protein expression levels are measured by an immunohistochemistry assay, an enzyme-linked immunosorbent assay (ELISA), in situ ization, chromatography, liquid chromatography, size exclusion chromatography, high performance liquid chromatography (HPLC), gas chromatography, mass spectrometry, tandem mass ometry, matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser deorption/ionization-time of flight (SELDI-TOF) mass spectrometry, pole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPIMS ), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption/ionization- Fourier transform-ion ron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry , radioimmunoassays, microscopy, microfluidic chip-based assays, surface plasmon resonance, sequencing, n blotting assay, or a combination thereof. 69. The method of any one the paragraphs 1-68, wherein the subject is a mammal. 70. The method of paragraph 69, wherein the subject is a human. 71. The method of any one the paragraphs 1-18, wherein the difference is greater than a 1- fold difference. 72. The method of paragraph 71, wherein the difference is at least 1.05 -fold, 1.1-fold, 1.2- fold, 1.3-fold, 1.4-fold, ld, 2-fold, 2.5-fold, 3-fold, 4-fold, , 6-fold, 7-fold, 8-fold, 9- fold, or 10-fold difference. 73. A kit for measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200, comprising ts for specifically measuring the levels of the ed PC-MACRO . 74. A kit for measuring the levels of at least one or more markers selected from the group consisting of PC-NEUTRO 1-200, comprising reagents for specifically measuring the levels of the selected PC-NEUTRO marker. 75. A kit for measuring the levels of at least one or more markers selected from the group consisting of PC-MACRO 1-200 and at least one or more markers selected from the group consisting of PC-NEUTRO 1-200, comprising reagents for specifically measuring the levels of the selected PC-MACRO marker and reagents for specifically measuring the levels of the selected PC-NEUTRO marker. 76. The kit of paragraph 73 or 75, wherein the selected RO markers se one or more markers ed from the group consisting of P2RY10, TNFAIP3, CXCR1, , and CHI3L1. 77. The kit of paragraph 74 or 75, wherein the selected PC-NEUTRO s comprise one or more markers selected from the group consisting of EIF3S5, 1, RPL23A, RPL14, RPL23A, RPL3, RPS28, and PTMA. 78. The kit of any one of paragraphs 73-77, wherein the reagents comprise one or more antibodies or fragments thereof, oligonucleotides, or aptamers. 79. A method of treating or preventing prostate cancer in a subject comprising administering to said subject an agent that tes the activity or expression of at least one or more markers selected from the group consisting of PC-MACRO 1-200. 80. A method of treating or preventing prostate cancer in a subject comprising administering to said subject an agent that modulates the activity or expression of at least one or more markers selected from the group consisting of PC-NEUTRO 1-200. 81. The method of paragraph 79 or 80, wherein the agent is a small le modulator, siRNA, or an antibody or fragment thereof.
In a first aspect, the invention relates to a method for diagnosing or aiding in the diagnosis of prostate cancer, or for assessing the risk of ping prostate cancer, or for prognosing or aiding in the prognosis of prostate cancer in a subject, the method comprising the in vitro steps of: a) measuring the levels of one or more PC-MACRO s ed from the group consisting of P2RY10, TNFAIP3, CXCR1, DNAJB1, CHI3L1, KIAA1324, CEACAM1, TIPARP, ALPL, SLC38A1, PBXIP1, , TNF, ZDHHC18, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, MAPK13, ROPN1L, TSC22D3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, GPR183, NR4A1, CCRN4L, ICOSLG, N4BP1, SDCBP, SMCHD1, GZF1, SNAI1, MME, MMP9, KIAA0040, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, FAM129A, C12orf35, SLC12A6, SMCHD1, BCL6, ZNF200, TMEM45B, SEC14L2, IFITM1, IL18RAP, GNG10, TNFRSF10C, IRF1, EGLN1, LBR, HCG27, ANKRD13A, OSM, HIST1H3A, MBD4, NSFL1C, ELOVL5, SLC44A2, PIK3R1, SYNE2, , JUNB, RHOG, ABCG1, VEGFA, FRAT1, UBE2J1, C19orf2, PDE4D, MS4A1, MMP25, FAS, UBN1, TMEM154, SNRK, CD83, FAM8A1, HIST2H2BA, , FAM65B, BAZ1A, and ZNFX in a population of the t’s hage cells; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells; and c) identifying a difference between the measured levels of the one or more selected PCMACRO markers in steps a) and b), wherein the identified difference tes that the subject has said prostate cancer, or that the subject has a risk of developing said prostate cancer, or that the identified difference is indicative of the prognosis of said prostate cancer.
In a second aspect, the invention relates to a method for assessing the efficacy of a treatment for prostate cancer or for monitoring the progression or regression of prostate cancer, or for identifying a compound capable of ameliorating or treating prostate cancer in a subject comprising the in vitro steps of: a) measuring the levels of one or more PC-MACRO markers ed from the group consisting of P2RY10, TNFAIP3, CXCR1, DNAJB1, CHI3L1, KIAA1324, CEACAM1, TIPARP, ALPL, SLC38A1, PBXIP1, DUSP16, TNF, ZDHHC18, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, , ROPN1L, 3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, GPR183, NR4A1, CCRN4L, ICOSLG, N4BP1, SDCBP, SMCHD1, GZF1, SNAI1, MME, MMP9, KIAA0040, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, A, C12orf35, SLC12A6, SMCHD1, BCL6, ZNF200, TMEM45B, SEC14L2, IFITM1, IL18RAP, GNG10, TNFRSF10C, IRF1, EGLN1, LBR, HCG27, ANKRD13A, OSM, HIST1H3A, MBD4, NSFL1C, ELOVL5, SLC44A2, PIK3R1, SYNE2, TRANK1, JUNB, RHOG, ABCG1, VEGFA, FRAT1, UBE2J1, C19orf2, PDE4D, MS4A1, MMP25, FAS, UBN1, TMEM154, SNRK, CD83, FAM8A1, HIST2H2BA, MANSC1, , BAZ1A, and ZNFX in a tion of the subject’s macrophage cells at a first time point, wherein the first time point is before the ent, or a first time point in the ring process, or before the administration of the compound to the subject; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells at the first time point, or before the administration of the compound to the subject; c) fying a first difference between the measured levels of the one or more selected PC-MACRO markers in steps a) and b); d) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s macrophage cells at a second time point, wherein the second time point is after the treatment, or a second time point in the monitoring process, or after the administration of the compound to the subject; e) measuring the levels of the one or more selected RO markers in a population of the subject’s non-phagocytic cells at the second time point, or after the administration of the compound to the subject; f) identifying a second difference between the measured levels of the one or more selected PC-MACRO s in steps d) and e); and g) identifying a difference between the first difference and the second ence, wherein the difference identified in g) is indicative of the cy of the treatment for said prostate cancer, or the progression or regression of said prostate cancer, or indicates that the compound is capable of ameliorating or ng said prostate cancer in the t.
In a third aspect, the invention relates to a method for diagnosing or aiding in the sis of prostate cancer, or for assessing the risk of developing prostate cancer, or for prognosing or aiding in the prognosis of te cancer in a subject, the method comprising the in vitro steps of: a) measuring the levels of at least one or more PC-MACRO markers selected from the group consisting of P2RY10, TNFAIP3, CXCR1, DNAJB1, CHI3L1, KIAA1324, CEACAM1, TIPARP, ALPL, SLC38A1, PBXIP1, DUSP16, TNF, 8, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, MAPK13, ROPN1L, TSC22D3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, GPR183, NR4A1, CCRN4L, ICOSLG, N4BP1, SDCBP, SMCHD1, GZF1, SNAI1, MME, MMP9, KIAA0040, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, FAM129A, C12orf35, SLC12A6, SMCHD1, BCL6, ZNF200, B, SEC14L2, IFITM1, IL18RAP, GNG10, TNFRSF10C, IRF1, EGLN1, LBR, HCG27, ANKRD13A, OSM, HIST1H3A, MBD4, NSFL1C, ELOVL5, SLC44A2, PIK3R1, SYNE2, TRANK1, JUNB, RHOG, ABCG1, VEGFA, FRAT1, UBE2J1, C19orf2, PDE4D, MS4A1, MMP25, FAS, UBN1, TMEM154, SNRK, CD83, FAM8A1, HIST2H2BA, MANSC1, , BAZ1A, and ZNFX in a population of the subject’s macrophage cells, measuring the levels of at least one or more PC-NEUTRO markers selected from the group consisting of , RPL23A, RPL14, PTMA, RPS13, RPL10, RPL3, RPL5, RPS28, RPL8, , RPL18A, RPS28, RPL15, RPS2, GLTSCR2, RPS8, RPL18, RPL7, SLC25A6, RPS15, RPL35, RPL6, EEF1G, TCF12, SNAPC3, , RPS17, ILF2, RPL9, YBX1, RPL, RPL35A, RPL27, RPL12, RPS4X, RPL39, DNAJC15, RPLP0, RPL13AP5, TMSB10, RPS2, MPV17, CCDC6, CD52, RPLP1, ZMYM4, ATXN10, NDUFA12, PLAC8, ANXA6, RPL7A, HLA-DPB1, TRIM44, VPS45, RPL13AP20, RPL18A, RPS3A, HLA-DPB1, APEX1, RPS14, RPS20, HLA-DPB1, RCC2, ALG3, RPL38, HIST1H1E, TCERG1, SNRPA, CYB5B, C13orf2, IL10RA, RSL1D1, and NPIP in a population of the subject’s neutrophil cells; b) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells; and ing the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells; c) identifying a difference between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and b); and d) identifying a difference between the measured levels or activities of the at least one or more selected PC-NEUTRO s in steps a) and b); wherein the differences identified in c) and d) indicate that the t has said prostate cancer, or that the subject has a risk of developing said prostate cancer, or that the identified differences are indicative of the prognosis of said prostate cancer.
In a fourth aspect, the invention s to a method for assessing the cy of a treatment for prostate cancer, or for monitoring the ssion or regression of te cancer, or for identifying a compound capable of ameliorating or treating prostate cancer in a subject comprising the in vitro steps of: a) measuring the levels of at least one or more PC-MACRO markers selected from the group consisting of P2RY10, TNFAIP3, CXCR1, DNAJB1, CHI3L1, 24, CEACAM1, , ALPL, SLC38A1, , DUSP16, TNF, ZDHHC18, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, MAPK13, ROPN1L, TSC22D3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, GPR183, NR4A1, CCRN4L, ICOSLG, N4BP1, SDCBP, SMCHD1, GZF1, SNAI1, MME, MMP9, KIAA0040, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, FAM129A, C12orf35, SLC12A6, SMCHD1, BCL6, ZNF200, TMEM45B, SEC14L2, IFITM1, IL18RAP, GNG10, TNFRSF10C, IRF1, EGLN1, LBR, HCG27, ANKRD13A, OSM, HIST1H3A, MBD4, , ELOVL5, SLC44A2, PIK3R1, SYNE2, TRANK1, JUNB, RHOG, ABCG1, VEGFA, FRAT1, UBE2J1, C19orf2, PDE4D, MS4A1, MMP25, FAS, UBN1, TMEM154, SNRK, CD83, FAM8A1, HIST2H2BA, MANSC1, FAM65B, BAZ1A, and ZNFX in a tion of the subject’s macrophage cells at a first time point, wherein the first time point is before the treatment, or a first time point in the monitoring process, or before the administration of the compound to the subject, and measuring the levels of at least one or more PC-NEUTRO s ed from the group consisting of EEF1A1, RPL23A, RPL14, PTMA, RPS13, RPL10, RPL3, RPL5, RPS28, RPL8, RPL13A, RPL18A, RPS28, RPL15, RPS2, GLTSCR2, RPS8, RPL18, RPL7, SLC25A6, RPS15, RPL35, RPL6, EEF1G, TCF12, SNAPC3, GNB2L1, RPS17, ILF2, RPL9, YBX1, RPL, RPL35A, RPL27, RPL12, RPS4X, RPL39, DNAJC15, RPLP0, P5, TMSB10, RPS2, MPV17, CCDC6, CD52, RPLP1, ZMYM4, ATXN10, NDUFA12, PLAC8, ANXA6, RPL7A, HLA-DPB1, TRIM44, VPS45, RPL13AP20, RPL18A, RPS3A, HLA-DPB1, APEX1, RPS14, RPS20, B1, RCC2, ALG3, RPL38, HIST1H1E, TCERG1, SNRPA, CYB5B, C13orf2, IL10RA, RSL1D1, and NPIP in a population of the subject’s neutrophil cells at the first time point or before the administration of the compound; b) measuring the levels of the at least one or more selected RO markers in a population of the subject’s non-phagocytic cells at the first time point or before the administration of the nd; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells at the first time point or before the administration of the compound; c) identifying a first difference between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and b); and identifying a second difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps a) and b); d) ing the levels of the at least one or more selected PC-MACRO markers in a population of the subject’s macrophage cells at a second time point, wherein the second time point is after the treatment, or a second time point in the monitoring process, or after the administration of the compound to the subject, and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s neutrophil cells at the second time point, or after the stration of the compound to the subject; e) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject’s agocytic cells at the second time point, or after the administration of the compound to the subject; and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells at the second time point, or after the administration of the compound to the subject; f) identifying a third difference between the measured levels of the at least one or more selected PC-MACRO markers in steps d) and e); and g) identifying a fourth difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps d) and e); h) identifying a difference between the first and second differences; and i) identifying a difference between the third and fourth differences, wherein the differences identified in h) and i) are indicative of the efficacy of the treatment for said prostate cancer, or the progression or regression of said prostate cancer, or te that the compound is capable of ameliorating or treating said prostate cancer in the subject.
In a fifth aspect, the invention relates to a kit when used for measuring in vitro the levels of at least two or more RO markers ed from the group ting of P2RY10, TNFAIP3, CXCR1, DNAJB1, CHI3L1, KIAA1324, CEACAM1, TIPARP, ALPL, SLC38A1, , DUSP16, TNF, ZDHHC18, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, MAPK13, ROPN1L, TSC22D3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, GPR183, NR4A1, CCRN4L, ICOSLG, N4BP1, SDCBP, SMCHD1, GZF1, SNAI1, MME, MMP9, 40, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, FAM129A, C12orf35, SLC12A6, SMCHD1, BCL6, ZNF200, TMEM45B, SEC14L2, IFITM1, IL18RAP, GNG10, TNFRSF10C, IRF1, EGLN1, LBR, HCG27, 3A, OSM, HIST1H3A, MBD4, NSFL1C, ELOVL5, SLC44A2, PIK3R1, SYNE2, TRANK1, JUNB, RHOG, ABCG1, VEGFA, FRAT1, UBE2J1, C19orf2, PDE4D, MS4A1, MMP25, FAS, UBN1, 4, SNRK, CD83, FAM8A1, 2BA, MANSC1, FAM65B, BAZ1A, and ZNFX, comprising reagents for specifically measuring the levels of the selected PC-MACRO marker.
In a sixth aspect, the invention relates to a kit when used for measuring in vitro the levels of at least one or more PC-MACRO markers ed from the group consisting of , TNFAIP3, CXCR1, DNAJB1, , KIAA1324, CEACAM1, TIPARP, ALPL, SLC38A1, , DUSP16, TNF, ZDHHC18, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, MAPK13, ROPN1L, TSC22D3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, GPR183, NR4A1, CCRN4L, ICOSLG, N4BP1, SDCBP, SMCHD1, GZF1, SNAI1, MME, MMP9, KIAA0040, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, FAM129A, C12orf35, SLC12A6, SMCHD1, BCL6, ZNF200, TMEM45B, SEC14L2, IFITM1, IL18RAP, GNG10, 10C, IRF1, EGLN1, LBR, HCG27, ANKRD13A, OSM, HIST1H3A, MBD4, NSFL1C, ELOVL5, SLC44A2, PIK3R1, SYNE2, TRANK1, JUNB, RHOG, ABCG1, VEGFA, FRAT1, UBE2J1, C19orf2, PDE4D, MS4A1, MMP25, FAS, UBN1, TMEM154, SNRK, CD83, , 2BA, MANSC1, FAM65B, BAZ1A, and ZNFX and at least one or more PC-NEUTRO markers ed from the group consisting of EEF1A1, RPL23A, RPL14, PTMA, RPS13, RPL10, RPL3, RPL5, RPS28, RPL8, RPL13A, , RPS28, RPL15, RPS2, GLTSCR2, RPS8, RPL18, RPL7, SLC25A6, RPS15, RPL35, RPL6, EEF1G, TCF12, SNAPC3, GNB2L1, RPS17, ILF2, RPL9, YBX1, RPL, RPL35A, RPL27, RPL12, RPS4X, RPL39, DNAJC15, RPLP0, RPL13AP5, TMSB10, RPS2, MPV17, CCDC6, CD52, RPLP1, ZMYM4, ATXN10, NDUFA12, PLAC8, ANXA6, RPL7A, HLADPB1 , TRIM44, VPS45, RPL13AP20, RPL18A, RPS3A, HLA-DPB1, APEX1, RPS14, RPS20, HLA-DPB1, RCC2, ALG3, RPL38, HIST1H1E, TCERG1, SNRPA, CYB5B, C13orf2, IL10RA, RSL1D1, and NPIP, comprising reagents for specifically measuring the levels of the selected PC-MACRO marker and ts for specifically measuring the levels of the selected PC-NEUTRO marker.
In a seventh aspect, the invention relates to use of an agent that modulates the activity or expression of at least one or more PC-MACRO markers selected from the group consisting of , TNFAIP3, CXCR1, DNAJB1, CHI3L1, 24, CEACAM1, TIPARP, ALPL, SLC38A1, PBXIP1, DUSP16, TNF, ZDHHC18, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, MAPK13, ROPN1L, TSC22D3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, GPR183, NR4A1, CCRN4L, ICOSLG, N4BP1, SDCBP, SMCHD1, GZF1, SNAI1, MME, MMP9, KIAA0040, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, FAM129A, 35, SLC12A6, , BCL6, ZNF200, TMEM45B, SEC14L2, IFITM1, IL18RAP, GNG10, TNFRSF10C, IRF1, EGLN1, LBR, HCG27, ANKRD13A, OSM, HIST1H3A, MBD4, NSFL1C, ELOVL5, SLC44A2, PIK3R1, SYNE2, TRANK1, JUNB, RHOG, ABCG1, VEGFA, FRAT1, UBE2J1, C19orf2, PDE4D, MS4A1, MMP25, FAS, UBN1, TMEM154, SNRK, CD83, FAM8A1, HIST2H2BA, MANSC1, FAM65B, BAZ1A, and ZNFX, in the manufacture of a medicament for treating or preventing prostate cancer in a subject.
The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to e any other r or any other integers, unless in the context or usage an exclusive interpretation of the term is required.
Still further embodiments are within the scope of the following claims.
Claims (36)
1. A method for diagnosing or aiding in the diagnosis of prostate cancer, or for assessing the risk of developing prostate cancer, or for sing or aiding in the prognosis of prostate cancer in a subject, the method comprising the in vitro steps of: a) measuring the levels of one or more PC-MACRO markers selected from the group consisting of P2RY10, TNFAIP3, CXCR1, DNAJB1, CHI3L1, 24, CEACAM1, TIPARP, ALPL, SLC38A1, PBXIP1, DUSP16, TNF, ZDHHC18, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, MAPK13, ROPN1L, TSC22D3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, , NR4A1, CCRN4L, ICOSLG, N4BP1, SDCBP, , GZF1, SNAI1, MME, MMP9, KIAA0040, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, FAM129A, C12orf35, SLC12A6, SMCHD1, BCL6, ZNF200, TMEM45B, SEC14L2, IFITM1, P, GNG10, TNFRSF10C, IRF1, EGLN1, LBR, HCG27, ANKRD13A, OSM, HIST1H3A, MBD4, NSFL1C, ELOVL5, SLC44A2, PIK3R1, SYNE2, TRANK1, JUNB, RHOG, ABCG1, VEGFA, FRAT1, , 2, PDE4D, MS4A1, MMP25, FAS, UBN1, TMEM154, SNRK, CD83, FAM8A1, HIST2H2BA, MANSC1, FAM65B, BAZ1A, and ZNFX in a population of the t’s macrophage cells; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells; and c) identifying a difference between the measured levels of the one or more selected PCMACRO markers in steps a) and b), wherein the identified difference indicates that the subject has said prostate cancer, or that the t has a risk of developing said prostate cancer, or that the identified difference is indicative of the prognosis of said prostate cancer.
2. A method for assessing the efficacy of a treatment for te cancer or for ring the progression or regression of prostate cancer, or for fying a compound capable of ameliorating or treating prostate cancer in a subject comprising the in vitro steps of: a) measuring the levels of one or more PC-MACRO markers selected from the group consisting of P2RY10, TNFAIP3, CXCR1, DNAJB1, CHI3L1, KIAA1324, 1, TIPARP, ALPL, SLC38A1, PBXIP1, DUSP16, TNF, ZDHHC18, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, MAPK13, ROPN1L, TSC22D3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, GPR183, NR4A1, CCRN4L, ICOSLG, N4BP1, SDCBP, SMCHD1, GZF1, SNAI1, MME, MMP9, KIAA0040, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, A, C12orf35, SLC12A6, SMCHD1, BCL6, ZNF200, TMEM45B, SEC14L2, IFITM1, IL18RAP, GNG10, 10C, IRF1, EGLN1, LBR, HCG27, ANKRD13A, OSM, HIST1H3A, MBD4, NSFL1C, ELOVL5, SLC44A2, PIK3R1, SYNE2, TRANK1, JUNB, RHOG, ABCG1, VEGFA, FRAT1, UBE2J1, C19orf2, PDE4D, MS4A1, MMP25, FAS, UBN1, TMEM154, SNRK, CD83, , HIST2H2BA, MANSC1, FAM65B, BAZ1A, and ZNFX in a population of the subject’s macrophage cells at a first time point, wherein the first time point is before the treatment, or a first time point in the ring process, or before the administration of the compound to the subject; b) measuring the levels of the one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells at the first time point, or before the administration of the compound to the subject; c) identifying a first difference between the measured levels of the one or more selected PC-MACRO markers in steps a) and b); d) measuring the levels of the one or more selected PC-MACRO markers in a population of the t’s macrophage cells at a second time point, wherein the second time point is after the treatment, or a second time point in the monitoring process, or after the administration of the compound to the subject; e) ing the levels of the one or more ed PC-MACRO markers in a population of the subject’s agocytic cells at the second time point, or after the administration of the compound to the subject; f) identifying a second difference n the measured levels of the one or more selected PC-MACRO markers in steps d) and e); and g) identifying a difference between the first difference and the second difference, wherein the difference identified in g) is indicative of the efficacy of the treatment for said prostate cancer, or the progression or regression of said prostate cancer, or indicates that the compound is capable of ameliorating or treating said te cancer in the t.
3. A method for diagnosing or aiding in the sis of prostate cancer, or for assessing the risk of developing prostate cancer, or for prognosing or aiding in the prognosis of prostate cancer in a subject, the method comprising the in vitro steps of: a) measuring the levels of at least one or more PC-MACRO markers selected from the group consisting of P2RY10, TNFAIP3, CXCR1, DNAJB1, CHI3L1, KIAA1324, CEACAM1, TIPARP, ALPL, SLC38A1, , DUSP16, TNF, ZDHHC18, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, MAPK13, ROPN1L, TSC22D3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, GPR183, NR4A1, CCRN4L, ICOSLG, N4BP1, SDCBP, SMCHD1, GZF1, SNAI1, MME, MMP9, 40, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, FAM129A, C12orf35, SLC12A6, SMCHD1, BCL6, ZNF200, TMEM45B, SEC14L2, IFITM1, IL18RAP, GNG10, TNFRSF10C, IRF1, EGLN1, LBR, HCG27, ANKRD13A, OSM, HIST1H3A, MBD4, NSFL1C, ELOVL5, SLC44A2, PIK3R1, SYNE2, TRANK1, JUNB, RHOG, ABCG1, VEGFA, FRAT1, UBE2J1, C19orf2, PDE4D, MS4A1, MMP25, FAS, UBN1, TMEM154, SNRK, CD83, FAM8A1, HIST2H2BA, MANSC1, , BAZ1A, and ZNFX in a population of the subject’s macrophage cells, measuring the levels of at least one or more PC-NEUTRO markers selected from the group consisting of EEF1A1, RPL23A, RPL14, PTMA, RPS13, RPL10, RPL3, RPL5, RPS28, RPL8, RPL13A, RPL18A, RPS28, RPL15, RPS2, GLTSCR2, RPS8, RPL18, RPL7, SLC25A6, RPS15, RPL35, RPL6, EEF1G, TCF12, , GNB2L1, RPS17, ILF2, RPL9, YBX1, RPL, RPL35A, RPL27, RPL12, RPS4X, RPL39, DNAJC15, RPLP0, RPL13AP5, TMSB10, RPS2, MPV17, CCDC6, CD52, RPLP1, ZMYM4, ATXN10, NDUFA12, PLAC8, ANXA6, RPL7A, B1, TRIM44, VPS45, RPL13AP20, RPL18A, RPS3A, HLA-DPB1, APEX1, RPS14, RPS20, HLA-DPB1, RCC2, ALG3, RPL38, HIST1H1E, , SNRPA, CYB5B, C13orf2, IL10RA, RSL1D1, and NPIP in a population of the subject’s neutrophil cells; b) measuring the levels of the at least one or more selected PC-MACRO s in a population of the subject’s non-phagocytic cells; and ing the levels of the at least one or more selected PC-NEUTRO markers in a population of the t’s non-phagocytic cells; c) identifying a difference between the measured levels of the at least one or more selected RO markers in steps a) and b); and d) identifying a difference between the measured levels or activities of the at least one or more selected PC-NEUTRO s in steps a) and b); wherein the differences fied in c) and d) indicate that the subject has said prostate cancer, or that the subject has a risk of developing said prostate cancer, or that the identified differences are indicative of the prognosis of said prostate cancer.
4. A method for assessing the efficacy of a treatment for prostate cancer, or for monitoring the progression or regression of prostate cancer, or for identifying a compound capable of ameliorating or treating prostate cancer in a subject comprising the in vitro steps of: a) measuring the levels of at least one or more PC-MACRO markers selected from the group consisting of , TNFAIP3, CXCR1, DNAJB1, CHI3L1, KIAA1324, CEACAM1, TIPARP, ALPL, SLC38A1, PBXIP1, DUSP16, TNF, ZDHHC18, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, MAPK13, ROPN1L, TSC22D3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, GPR183, NR4A1, CCRN4L, ICOSLG, N4BP1, SDCBP, SMCHD1, GZF1, SNAI1, MME, MMP9, KIAA0040, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, FAM129A, C12orf35, SLC12A6, SMCHD1, BCL6, ZNF200, TMEM45B, SEC14L2, , IL18RAP, GNG10, TNFRSF10C, IRF1, EGLN1, LBR, HCG27, ANKRD13A, OSM, HIST1H3A, MBD4, NSFL1C, ELOVL5, 2, PIK3R1, SYNE2, TRANK1, JUNB, RHOG, ABCG1, VEGFA, FRAT1, UBE2J1, C19orf2, PDE4D, MS4A1, MMP25, FAS, UBN1, TMEM154, SNRK, CD83, FAM8A1, HIST2H2BA, MANSC1, FAM65B, BAZ1A, and ZNFX in a population of the subject’s macrophage cells at a first time point, wherein the first time point is before the treatment, or a first time point in the ring process, or before the administration of the nd to the subject, and measuring the levels of at least one or more PC-NEUTRO markers selected from the group consisting of EEF1A1, RPL23A, RPL14, PTMA, RPS13, RPL10, RPL3, RPL5, RPS28, RPL8, RPL13A, RPL18A, RPS28, RPL15, RPS2, GLTSCR2, RPS8, RPL18, RPL7, SLC25A6, RPS15, RPL35, RPL6, EEF1G, TCF12, , GNB2L1, RPS17, ILF2, RPL9, YBX1, RPL, RPL35A, RPL27, RPL12, RPS4X, RPL39, DNAJC15, RPLP0, RPL13AP5, TMSB10, RPS2, MPV17, CCDC6, CD52, RPLP1, ZMYM4, ATXN10, NDUFA12, PLAC8, ANXA6, RPL7A, HLA-DPB1, TRIM44, VPS45, RPL13AP20, RPL18A, RPS3A, HLA-DPB1, APEX1, RPS14, RPS20, HLA-DPB1, RCC2, ALG3, RPL38, HIST1H1E, TCERG1, SNRPA, CYB5B, C13orf2, IL10RA, RSL1D1, and NPIP in a population of the subject’s phil cells at the first time point or before the stration of the compound; b) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells at the first time point or before the administration of the compound; and ing the levels of the at least one or more ed PC-NEUTRO markers in a population of the subject’s non-phagocytic cells at the first time point or before the administration of the compound; c) identifying a first difference between the measured levels of the at least one or more selected PC-MACRO markers in steps a) and b); and fying a second difference between the measured levels of the at least one or more selected PC-NEUTRO markers in steps a) and b); d) measuring the levels of the at least one or more selected PC-MACRO markers in a population of the subject’s macrophage cells at a second time point, wherein the second time point is after the treatment, or a second time point in the monitoring process, or after the administration of the compound to the subject, and measuring the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s neutrophil cells at the second time point, or after the administration of the compound to the subject; e) ing the levels of the at least one or more selected PC-MACRO markers in a population of the subject’s non-phagocytic cells at the second time point, or after the administration of the compound to the subject; and ing the levels of the at least one or more selected PC-NEUTRO markers in a population of the subject’s non-phagocytic cells at the second time point, or after the administration of the compound to the subject; f) identifying a third difference between the ed levels of the at least one or more selected PC-MACRO markers in steps d) and e); and g) fying a fourth difference between the measured levels of the at least one or more selected TRO markers in steps d) and e); h) identifying a difference between the first and second differences; and i) identifying a difference between the third and fourth differences, wherein the differences identified in h) and i) are indicative of the efficacy of the ent for said prostate cancer, or the progression or regression of said prostate cancer, or indicate that the nd is capable of ameliorating or treating said prostate cancer in the subject.
5. The method of any one of claims 1-4, wherein at least one standard parameter associated with said prostate cancer has been measured, n the standard parameter is selected from the group consisting of tumor stage, tumor grade, tumor size, tumor visual teristics, tumor growth, tumor ess, tumor progression, tumor metastasis tumor distribution within the body, odor, molecular pathology, genomics, or tumor angiograms.
6. The method of claim 3 or claim 4, wherein the selected PC-MACRO markers and the selected PC-NEUTRO markers are measured from the same population of non-phagocytic cells in steps b) or e), or wherein the selected PC-MACRO markers and the selected PC-NEUTRO are from different populations of non-phagocytic cells in steps b) or e).
7. The method of any one of claims 1-4, wherein the selected PC-MACRO markers are upregulated or activated in the macrophage cells compared to the non-phagocytic cells.
8. The method of any one of claims 1-4, wherein the selected RO markers are down-regulated or inhibited in the macrophage cells compared to the non-phagocytic cells.
9. The method of claim 3 or claim 4, wherein the selected PC-NEUTRO s are down-regulated or inhibited in the neutrophil cells compared to the non-phagocytic cells.
10. The method of any one of claims 1-4, r comprising lysing the macrophage cells and the non-phagocytic cells and extracting the cellular contents from said macrophage cells and non-phagocytic cells before a).
11. The method of claim 3 or claim 4, further sing lysing the neutrophil cells and the agocytic cells and extracting the cellular contents from said phil cells and nonphagocytic cells before a).
12. The method of claim 10, wherein the cellular ts of the macrophage cells se viable diseased cells, dead diseased cells, apoptotic diseased cells, circulating tumor cells, infectious agents, fetal cells, trophoblasts, or fragments thereof.
13. The method of claim 11, wherein the cellular contents of the neutrophil cells comprise viable diseased cells, dead diseased cells, apoptotic diseased cells, circulating tumor cells, infectious agents, fetal cells, trophoblasts, or fragments thereof.
14. The method of claim 10, wherein the ed one or more markers are present in the cellular contents of the macrophage cells and not present in the cellular contents of the non-phagocytic cells.
15. The method of any one of claims 1-4, wherein the macrophage cells s the one or more selected PC-MACRO markers.
16. The method of claim 11, wherein the selected one or more s are present in the cellular ts of the neutrophil cells and not present in the cellular contents of the non-phagocytic cells.
17. The method of claim 3 or claim 4, wherein the neutrophil cells express the one or more selected PC-NEUTRO markers.
18. The method of any one of claims 1-4, wherein the non-phagocytic cells are T cells, B cells, null cells, basophils, or mixtures thereof.
19. The method of any one of claims 1-4, wherein the macrophage cells are isolated using antibodies, using a ligand that binds to a molecular receptor expressed on the plasma membranes of white blood cells, or by flow cytometry, fluorescence activated cell sorting, filtration, gradient-based centrifugation, elution, microfluidics, magnetic separation que, fluorescent-magnetic separation technique, nanostructure, quantum dots, high throughput cope-based platforms, or a combination thereof.
20. The method of claim 3 or claim 4, n the phil cells are isolated using antibodies, using a ligand that binds to a molecular or expressed on the plasma membranes of white blood cells, or by flow cytometry, fluorescence activated cell sorting, filtration, gradient-based centrifugation, elution, microfluidics, magnetic separation que, fluorescent-magnetic separation que, nanostructure, quantum dots, high throughput microscope-based platforms, or a combination thereof.
21. The method of any one of claims 1-4, wherein the non-phagocytic cells are isolated using antibodies, using a ligand that binds to a molecular receptor expressed on the plasma membranes of white blood cells, or by flow cytometry, fluorescence activated cell sorting, filtration, gradient-based centrifugation, elution, microfluidics, magnetic separation technique, fluorescent-magnetic separation technique, nanostructure, quantum dots, high throughput microscope-based platforms, or a combination thereof.
22. The method of any one of claims 1-4, wherein the macrophage cells are ed using at least one of the ing: (1) a product secreted by the macrophage cells, or (2) a cell surface target on the surface of macrophage cells.
23. The method of claim 3 or claim 4, wherein the neutrophil cells are isolated by using at least one of the following: (1) a t secreted by the neutrophil cells, or (2) a cell surface target on the surface of neutrophil cells.
24. The method of claim 22 or claim 23, wherein the target is a marker of said prostate cancer.
25. The method of any one of claims 1-4, wherein the measured levels are at least one of gene expression levels or protein expression levels.
26. The method of any one of claims 1-4, wherein the levels or activities are measured by a qualitative assay, a quantitative assay, or a combination thereof.
27. The method of claim 26, n the quantitative assay uses sequencing, direct sequencing, RNA sequencing, whole riptome n sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by ization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, highthroughput cing, massively parallel signature sequencing, emulsion PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by on, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore cing, 454 sequencing, Solexa Genome er sequencing, Sequencing by Oligonucleotide Ligation and Detection (SOLiD, Life Technologies), MS-PET sequencing, mass spectrometry, matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry, quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric re photoionization mass spectrometry (APPIMS ), Fourier transform mass spectrometry (FTMS), -assisted laser desorption/ionization- r transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), polymerase chain reaction (PCR) analysis, quantitative PCR, realtime PCR, scence assay, colorimetric assay, chemiluminescent assay, or a combination thereof.
28. The method of claim 25, wherein the gene sion levels are measured by polymerase chain reaction (PCR) analysis, sequencing analysis, electrophoretic analysis, restriction fragment length polymorphism (RFLP) analysis, rn blot analysis, quantitative PCR, reverse-transcriptase-PCR analysis (RT-PCR), allele-specific oligonucleotide hybridization analysis, ative genomic hybridization, duplex mobility assay (HMA), single strand conformational polymorphism (SSCP), denaturing gradient gel electrophisis , RNAase ch analysis, mass spectrometry, tandem mass spectrometry, matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser deorption/ionization-time of flight (SELDI-TOF) mass ometry, quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry S ), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption/ionization- Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), surface plasmon resonance, Southern blot analysis, in situ hybridization, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), microarray, comparative genomic hybridization, karyotyping, lex ligation-dependent probe amplification , Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF), microscopy, methylation specific PCR (MSP) assay, HpaII tiny nt Enrichment by on-mediated PCR (HELP) assay, radioactive acetate labeling assays, colorimetric DNA acetylation assay, chromatin immunoprecipitation combined with microarray (ChIP-on-chip) assay, restriction landmark genomic scanning, ated DNA immunoprecipitation (MeDIP), molecular break light assay for DNA adenine methyltransferase activity, chromatographic separation, methylation-sensitive restriction enzyme analysis, bisulfite-driven conversion of non-methylated cytosine to uracil, methylbinding PCR analysis, or a combination thereof.
29. The method of claim 25, wherein the gene expression levels are measured by a cing technique selected from the group consisting of direct sequencing, RNA sequencing, whole transcriptome shotgun sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, sequencing by reversible dye terminator, paired-end sequencing, erm sequencing, exonuclease sequencing, cing by ligation, short-read sequencing, -molecule sequencing, sequencing-bysynthesis , real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, Sequencing by Oligonucleotide Ligation and Detection (SOLiD, Life Technologies), MS-PET sequencing, mass spectrometry, and a combination thereof.
30. The method of claim 25, wherein the protein expression levels are measured by an histochemistry assay, an enzyme-linked immunosorbent assay ), in situ hybridization, chromatography, liquid chromatography, size exclusion chromatography, high performance liquid tography (HPLC), gas chromatography, mass spectrometry, tandem mass spectrometry, matrix ed laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, e-enhanced laser deorption/ionization-time of flight (SELDI-TOF) mass spectrometry, quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass ometry (APPIMS ), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption/ionization- Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), radioimmunoassays, microscopy, microfluidic ased assays, surface plasmon resonance, sequencing, Western blotting assay, or a ation thereof.
31. The method of any one of claims 1-30, wherein the subject is a mammal.
32. The method of claim 31, wherein the subject is a human.
33. The method of any one of claims 1-4, wherein the difference is greater than a 1-fold difference.
34. The method of claim 33, wherein the ence is at least 1.05-fold, 1.1-fold, 1.2-fold, 1.3-fold, ld, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold difference.
35. A kit when used for measuring in vitro the levels of at least two or more PC-MACRO markers selected from the group consisting of P2RY10, TNFAIP3, CXCR1, DNAJB1, CHI3L1, KIAA1324, CEACAM1, TIPARP, ALPL, SLC38A1, PBXIP1, , TNF, ZDHHC18, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, MAPK13, ROPN1L, TSC22D3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, , NR4A1, , ICOSLG, N4BP1, SDCBP, SMCHD1, GZF1, SNAI1, MME, MMP9, KIAA0040, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, FAM129A, C12orf35, SLC12A6, , BCL6, ZNF200, TMEM45B, SEC14L2, IFITM1, IL18RAP, GNG10, 10C, IRF1, EGLN1, LBR, HCG27, ANKRD13A, OSM, HIST1H3A, MBD4, NSFL1C, ELOVL5, SLC44A2, PIK3R1, SYNE2, TRANK1, JUNB, RHOG, ABCG1, VEGFA, FRAT1, UBE2J1, C19orf2, PDE4D, MS4A1, MMP25, FAS, UBN1, TMEM154, SNRK, CD83, FAM8A1, HIST2H2BA, MANSC1, FAM65B, BAZ1A, and ZNFX, comprising reagents for ically measuring the levels of the ed RO marker.
36. A kit when used for measuring in vitro the levels of at least one or more PC-MACRO markers selected from the group consisting of P2RY10, TNFAIP3, CXCR1, DNAJB1, CHI3L1, KIAA1324, CEACAM1, TIPARP, ALPL, SLC38A1, PBXIP1, DUSP16, TNF, ZDHHC18, DUSP5, PI3, PHOSPHO1, MPZL3, CXCR2, FAIM3, MAPK13, ROPN1L, TSC22D3, GPR97, FAM46C, HBEGF, EREG, SNAP, RAB20, GPR183, NR4A1, CCRN4L, ICOSLG, N4BP1, SDCBP, SMCHD1, GZF1, SNAI1, MME, MMP9, KIAA0040, KLF2, ZEB1, NR4A3, ATF4, XPO6, SLC6, LRP10, PELI, OLR1, CYP4F, FAM129A, C12orf35, SLC12A6, SMCHD1, BCL6, ZNF200, B, SEC14L2, IFITM1, IL18RAP, GNG10, TNFRSF10C, IRF1, EGLN1, LBR, HCG27, ANKRD13A, OSM, HIST1H3A, MBD4, NSFL1C, ELOVL5, 2, PIK3R1, SYNE2, TRANK1, JUNB, RHOG, ABCG1, VEGFA, FRAT1, UBE2J1, C19orf2, PDE4D, MS4A1, MMP25, FAS, UBN1, TMEM154, SNRK, CD83, FAM8A1, HIST2H2BA, MANSC1, FAM65B, BAZ1A, and ZNFX and at least one or more PC-NEUTRO markers selected from the group consisting of EEF1A1, RPL23A, RPL14, PTMA, RPS13, RPL10, RPL3, RPL5, RPS28, RPL8, , RPL18A, RPS28, RPL15, RPS2, GLTSCR2, RPS8, RPL18, RPL7, SLC25A6, RPS15, RPL35, RPL6, EEF1G, TCF12, SNAPC3, GNB2L1, RPS17, ILF2, RPL9, YBX1, RPL, RPL35A, RPL27, RPL12, RPS4X, RPL39, DNAJC15, RPLP0, RPL13AP5, TMSB10, RPS2, MPV17, CCDC6, CD52, RPLP1, ZMYM4, ATXN10, 2, PLAC8, ANXA6, RPL7A, HLA-DPB1, TRIM44, VPS45, RPL13AP20, , RPS3A, HLA-DPB1, APEX1, RPS14, RPS20, HLA-DPB1, RCC2, ALG3, RPL38, HIST1H1E, TCERG1, SNRPA, CYB5B, C13orf2, IL10RA, RSL1D1, and NPIP, comprising reagents for specifically ing the levels of the selected PC-MACRO marker and reagents for specifically measuring the levels of the selected PC-NEUTRO marker. Harry Stylli Colleen Kelly By the patent attorneys for the applicants SPRUSON & FERGUSON 2% w 38mm .3 w H $33 Em.“ wobgou 3mm m . .3 cmm?w ES hwucmu 33$ cmmma SE xmbmg?wd 9mg mwmwmwm mgommm wmmwmwm mmwmmmg?m?u mac mcmcwm? .3 mam?“ mcmm w mawcmmm 353$ 6% 39an am m x32 “$33an w Hum .3 mm- m? magma: S». mwmmw sum 4 Ome<§=0m “Him. war; Ow?bmz GE @342 Om‘w?mzsom asmawow mw?maumnm hmugmw m 30mm.“ “E?wammm © «Ham ““““““““““““““““““““““““““““““““““““““““““““““““““““““““““ “£3333 .3 ???i m mmmw m ?ue—x S w... E, 3 pm. i0 LD‘J'O'FPJ ”mmtmgca m§ummm§ $0.0 ?ed g mmmug?mgummm? mmed N?ed mmgwm w N. m 5 .3 mmmgg?umE $3332 gamrmmgau $3QO mengubmugmu mm «mmng “m8 $8. gamma c033§$§ murm?mmm WEEQU m?wmmmm EchU mm ca?ma?m?t mm D: & mm qucmu 33$me mmEEc m m?wmma .3 gamma usmgmw m?mwma m ?g mmmmma? 5% gmmmmc Nansen“ z: mam. 3&3 ?mummuwgm mm gmwm “E E?mcmmm E?mcmmm mmmm “338 m 93% Nm? mm.NQN mmmMQN 9.5% mmmhgq‘ mw?tmm “E “E Mg Eamammm Eamammm a ”E .ma m ?g mm“ mam mam mmmgmg? $3me 3me vammmg ?gmmmcmm Ema?mcmm ”Wmmm ”Wmmm mmamgau mgmm 8mm mmcmmm m :m $9558 mg?mmm mmabcau magma mcm?gmd mcmESmm? ?mmwmmé “3“, gm,» Eswmcmmm mmiw 2m .N .N ?g 83%:me :m .N ?g 108034-0016-WO1 -
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