CA2432361A1 - Methods and reagents for the rapid and efficient isolation of circulating cancer cells - Google Patents
Methods and reagents for the rapid and efficient isolation of circulating cancer cells Download PDFInfo
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- CA2432361A1 CA2432361A1 CA002432361A CA2432361A CA2432361A1 CA 2432361 A1 CA2432361 A1 CA 2432361A1 CA 002432361 A CA002432361 A CA 002432361A CA 2432361 A CA2432361 A CA 2432361A CA 2432361 A1 CA2432361 A1 CA 2432361A1
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
- H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
- H01F1/0036—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties showing low dimensional magnetism, i.e. spin rearrangements due to a restriction of dimensions, e.g. showing giant magnetoresistivity
- H01F1/0045—Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use
- H01F1/0054—Coated nanoparticles, e.g. nanoparticles coated with organic surfactant
Abstract
A highly sensitive assay is disclosed which combines immunomagnetic enrichment with multiparameter flow cytometric and immunocytochemical analysis to detect, enumerate and characterize carcinoma cells in the blood. The assay can detect one epithelial cell or less in 1 ml of blood and has a greater sensitively than conventional PCR or immunohistochemistry by 1-2 orders of magnitude. In addition, the assay facilitates the biological characterization and staging of carcinoma cells.
Claims (83)
1. A method for detecting and enumerating rare cells in a mixed cell population, the presence of said rare cells in said population being indicative of a disease state, comprising:
a) obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing said rare cells;
b) preparing an immunomagnetic sample wherein said biological specimen is mixed with magnetic particles coupled to a biospecific ligand which reacts specifically with the rare cells, to the substantial exclusion of other sample components;
c) contacting said immunomagnetic sample with at least one biospecific reagent which labels said rare cells; and d) analyzing said labeled rare cells to determine the presence and number of any rare cells in said immunomagnetic sample, the greater the number of rare cells present in said sample the greater the severity of said disease state.
a) obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing said rare cells;
b) preparing an immunomagnetic sample wherein said biological specimen is mixed with magnetic particles coupled to a biospecific ligand which reacts specifically with the rare cells, to the substantial exclusion of other sample components;
c) contacting said immunomagnetic sample with at least one biospecific reagent which labels said rare cells; and d) analyzing said labeled rare cells to determine the presence and number of any rare cells in said immunomagnetic sample, the greater the number of rare cells present in said sample the greater the severity of said disease state.
2. A method as claimed in claim 1, wherein as an intermediate step between the preparation of the immunomagnetic sample and contacting said immunomagnetic sample with at least one biospecific reagent, said immunomagnetic sample is subjected to a magnetic field to produce a rare cell enriched cell suspension as the immunomagnetic sample.
3. A method as claimed in claim 3, wherein the volume of said immunomagnetic sample containing said enriched rare cells is reduced.
4. A method as claimed in claim 1, wherein before analysis said immunomagnetic sample is separated into a labeled rare cell-containing fraction and an unlabeled fraction.
5. A method as claimed in claim 4, wherein said magnetic particles are colloidal and separation is effected by subjecting said immunomagnetic sample to a magnetic gradient field.
6. A method as claimed in claim 1, wherein said rare cell is selected from the group consisting of endothelial cells, fetal cells in maternal circulation, bacterial cells, myocardial cells, epithelial cells, and virally infected cells.
7. A method for detecting and enumerating cancer cells in a mixed cell population, comprising:
a) obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing said cancer cells;
b) preparing an immunomagnetic sample wherein said biological specimen is mixed with magnetic particles coupled to a biospecific ligand which reacts specifically with the cancer cells, to the substantial exclusion of other sample components;
c) contacting said immunomagnetic sample with at least one biospecific reagent which labels said cancer cells; and d) analyzing said labeled cancer cells to determine the presence and number of any cancer cells in said immunomagnetic sample, the greater the number of cancer cells present in said sample the greater the severity of said cancer.
a) obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing said cancer cells;
b) preparing an immunomagnetic sample wherein said biological specimen is mixed with magnetic particles coupled to a biospecific ligand which reacts specifically with the cancer cells, to the substantial exclusion of other sample components;
c) contacting said immunomagnetic sample with at least one biospecific reagent which labels said cancer cells; and d) analyzing said labeled cancer cells to determine the presence and number of any cancer cells in said immunomagnetic sample, the greater the number of cancer cells present in said sample the greater the severity of said cancer.
8. A method as claimed in claim 7 wherein said magnetic particles are colloidal and before analysis said immunomagnetic sample is separated into a labelled cancer cell-containing fraction and an unlabelled fraction.
9. A method as claimed in claim 7, wherein said colloidal magnetic particles and said at least one biospecific reagent are sequentially mixed with said biological specimen, and, as an intermediate step, said immunomagnetic sample is subjected to a magnetic field to produce a cancer cell enriched cell suspension as the immunomagnetic sample.
10. A method as claimed in claim 9 wherein before analysis said immunomagnetic sample is separated into a labelled cancer cell-containing fraction and an unlabelled traction.
11. A method as claimed in claim 10, wherein said labelled cancer cell-containing fraction is analyzed by a process selected from the group consisting of multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis manual cell analysis and automated cell analysis.
12. A method as claimed in claim 8, wherein said biospecific ligand is a monoclonal antibody specific for at least one cancer cell determinant, and said at least one biospecific reagent comprises at least one additional monoclonal antibody specific for a second cancer cell determinant and a third monoclonal antibody specific for an antigen present on a non tumor-cell, and said method further comprises adding to said labeled cancer cell-containing fraction a cell specific dye to allow exclusion of residual non-nucleated cells and cell debris from analysis.
13. A method as claimed in claim 12, wherein said method is applied to detect and enumerate residual cancer cells in said biological specimen following at least one tumor eradication procedure.
14. A method as claimed in claim 12, wherein said method further comprises the step of assessing the malignant status of said separated cancer cells by immunocytochemical analysis.
15. A method as claimed in claim 12, wherein said biological specimen is obtained from said test subject periodically and assessed for the presence and number of circulating cancer cells as an indicator of progression of said disease state.
16. A method as claimed in claim 12, wherein said biological specimen is obtained from said test subject periodically to assess for the presence and number of circulating cancer cells as an indicator of said patient's response to cancer eradication procedures.
17. A method as claimed in claim 12, wherein said biospecific ligand binds specifically to an epithelial cell adhesion molecule.
18. A method as claimed in claim 12, wherein said one or more biospecific reagent binds specifically to an intracellular cytokeratin.
19. A method as claimed in claim 12, wherein said one or more biospecific reagent is specific for a cytokeratin selected from the group consisting of cytokeratin 7, 8, 18, and 19.
20. A method as claimed in claim 12, wherein said biospecific ligand has binding affinity for epithelial cell adhesion molecule, and said at least one biospecific reagent has affinity for an intracellular cytokeratin selected from the group consisting of cytokeratin 7, 8, 18 and 19.
21. A method as claimed in claim 12, wherein said patient has been diagnosed with a cancer selected from the group consisting of prostate cancer, breast cancer, colon cancer apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell and transitional cell reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, throphoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, leydig cell tumor, papilloma, sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffin, antiokeratoma, angioma sclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcama, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (Kaposi's, and mast-cell), neoplasms (e.g., bone, digestive system, liver, pancreatic, pituitary, testicular, orbital, head and neck, central nervous system, acoustic, pelvic, respiratory tract, and urogenital), neurofibromatosis, and cervical dysplasia.
22. A method of diagnosing early stage cancer in a test subject, comprising:
a) obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing cancer cells;
b) preparing a cell fraction from said biological specimen, said cell fraction being enriched for cancer cells, if present in said biological specimen; and c) analyzing said enriched fraction for the presence of said cancer cells, the presence of said cancer cells in said specimen indicating the presence of early stage cancer in said test subject.
a) obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing cancer cells;
b) preparing a cell fraction from said biological specimen, said cell fraction being enriched for cancer cells, if present in said biological specimen; and c) analyzing said enriched fraction for the presence of said cancer cells, the presence of said cancer cells in said specimen indicating the presence of early stage cancer in said test subject.
23. A method as claimed in claim 22, wherein preparation of said immunomagnetic cell fraction comprises mixing said biological specimen with magnetic particles coupled to a biospecific ligand which binds specifically to said cancer cells, to the substantial exclusion of other biological specimen components and subjecting the specimen-magnetic particle mixture to a magnetic field to produce a cell suspension enriched in magnetic particle-bound cancer cells as an immunomagnetic sample.
24. A method as claimed in claim 23, wherein before analysis said immunomagnetic sample is contacted with at least one biospecific reagent that labels said cancer cells.
25. A method as claimed in claim 23, wherein the volume of said immunomagnetic sample containing said enriched magnetic particle-bound cancer cells is reduced.
26. A method as claimed in claim 23, wherein said immunomagnetic sample is further separated into a labeled magnetic fraction and an unlabeled fraction by subjecting said immunomagnetic sample to a magnetic gradient field.
27. A method as claimed in claim 23, wherein said magnetic particles are colloidal nanoparticles.
28. A coated, magnetic particle comprising a nanoparticle core of magnetic material, and a base coating material on said magnetic core in an amount sufficient to hinder non-specific binding of biological macromolecules to said magnetic core.
29. A coated, magnetic particle comprising a nanoparticle core of magnetic material, a base coating material that forms a discontinous coating on said magnetic core, providing at least one area of discontinuity which, if accessible, contributes to non-specific binding of said base coated particle to biological macromolecules, and an additional coating material that hinders access to said at least one area of discontinuity by said biological macromolecules.
30. A magnetic particle as claimed in claim 28, wherein said magnetic core material comprises at least one transition metal oxide and said base coating material comprises a protein.
31. A magnetic particle as claimed in claim 30, wherein said additional coating material is one member of a specific binding pair which is coupled to said base coating material.
32. A magnetic particle as claimed in claim 31, wherein said one member of said specific binding pair is coupled to said base coating material through a bifunctional linking compound.
33. A magnetic particle as claimed in claim 31, wherein said specific binding pair is selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin.
34. A magnetic particle as claimed in claim 31, wherein said specific binding pair is biotin-streptavidin and streptavidin is coupled to said base coating material.
35. A magnetic particle as claimed in claim 34, wherein a fraction of the streptavidin which is coupled to said base coating material is bound to biotinylated antibody and the remainder of said streptavidin is unbound.
36. A magnetic particle as claimed in claim 34, wherein a fraction of the streptavidin which is coupled to said base coating material is bound to biotinylated antibody and a substantial portion of said streptavidin remaining is bound to free biotin.
37. A magnetic particle as claimed in claim 34, wherein a fraction of the streptavidin which is coupled to said base coating material is bound to biotinylated antibody and a substantial portion of said streptavidin remaining is bound to a biotin-protein conjugate.
38. A magnetic particle as claimed in claim 34, wherein the protein component of said biotin-protein conjugate comprises bovine serum albumin.
39. A coated magnetic particle, as claimed in claim 29, having between 70-90% magnetic mass.
40. A coated magnetic particle, as claimed in claim 29, wherein a major portion of said particles have a particle size in the range of 90-120 nm.
41. A coated magnetic particle, as claimed in claim 29, wherein a major portion of said particles have a particle size in the range of 120-150 nm.
42. A coated magnetic particle, as claimed in claim 29, wherein a major portion of said particles have a particle size in the range of 90-150 nm.
43. A composition comprising a plurality of magnetic particles as claimed in claim 29, suspended in a biologically compatible aqueous medium.
44. A coated magnetic particle comprising a nanoparticle core material of a magnetic transition metal oxide, a protein base coating material and an additional coating material coupled to said base coating material through a biofunctional linking compound, said additional coating material being one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin.
45. A coated magnetic particle as claimed in claim 44, wherein said core material is magnetite, said base coating material is bovine serum albumin, said specific binding pair is biotin-streptavidin and streptavidin is coupled to said base coating material.
46. A coated magnetic particle as claimed in claim 45, wherein a fraction of the streptavidin which is coupled to said base coating material is bound to biotinylated antibody and a substantial portion of said streptavidin remaining is bound to a biotin-bovine serum albumin conjugate.
47. A composition comprising a plurality of magnetic particles as claimed in claim 46, suspended in a biologically compatible aqueous medium.
48. A test kit for screening a patient sample for the presence of circulating rare cells comprising:
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and an antibody that binds specifically to a first characteristic determinant of said rare cell, said antibody being coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a second characteristic determinant of said rare cell; and c) a cell specific dye for excluding sample components other than said rare cells from analysis.
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and an antibody that binds specifically to a first characteristic determinant of said rare cell, said antibody being coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a second characteristic determinant of said rare cell; and c) a cell specific dye for excluding sample components other than said rare cells from analysis.
49. A kit as claimed in claim 48, said kit further containing an antibody which has binding affinity for non-target cells, a biological buffer, a permeabilization buffer, a protocol and optionally, an information sheet.
50. A kit as claimed in claim 48, wherein said rare cells are selected from the group consisting of endothelial cells, fetal cells in maternal circulation, bacterial cells, myocardial cells, epithelial cells, and virally infected cells.
51. A kit for screening a patient sample for the presence of circulating tumor cells, comprising:
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a cancer cell determinant; and c) cell specific dye for excluding sample components other than said tumor cells from analysis.
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a cancer cell determinant; and c) cell specific dye for excluding sample components other than said tumor cells from analysis.
52. A kit as claimed in claim 51, said kit further containing an antibody which has binding affinity for non-tumor cells, a biological buffer, a permeabilization buffer, a protocol and optionally, an information sheet.
53. A kit as claimed in claim 51 for screening patients for breast cancer, wherein said at least one antibody having binding specificity for a cancer cell determinant specifically binds a breast cancer cell determinant, said determinant being selected from the group of determinants consisting of MUC-1, estrogen, progesterone receptor, cathepsin D, p53, urokinase type plasminogen activator, epidermal growth factor, epidermal growth factor receptor, BRCA1, BRCA2, CA27.29, CA15.5, prostate specific antigen, plasminogen activator inhibitor and Her2-neu.
54. A kit as claimed in claim 51 for screening patients for prostate cancer, wherein said at least one antibody having binding specificity for a cancer cell determinant specifically binds a prostate cancer cell determinant, said determinant being selected from the group of determinants consisting of prostate specific antigen, prostatic acid phosphatase, thymosin b-15, p53, HPC1 basic prostate gene, creatine kinase and prostate specific membrane antigen.
55. A kit as claimed in claim 51 for screening patients for colon cancer, wherein said at least one antibody having binding specificity for a cancer cell determinant specifically binds a colon cancer cell determinant, said determinant being selected from the group of determinants consisting of carcinoembryonic antigen, C protein, APC gene, p53 and matrix metalloproteinase (MMP-9).
56. A kit as claimed in claim 51 for screening patients with bladder cancer, wherein said at least one antibody having binding specificity for a cancer cell determinant specifically binds a bladder cancer cell determinant, said determinant being selected from the group of determinants consisting of nuclear matrix protein (NMP22),Bard Bladder tumor antigen (BTA), and fibrin degradation product (FDP).
57. A test kit as claimed in claim 51, wherein said at least one antibody comprises a panel of antibodies each having binding specificity for a different cancer cell characteristic determinant.
58. A kit for screening a patient sample for the presence of circulating breast cancer cells, comprising:
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a breast cancer cell determinant; and c) cell specific dye for excluding sample components other than said breast cancer cells from analysis.
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a breast cancer cell determinant; and c) cell specific dye for excluding sample components other than said breast cancer cells from analysis.
59. A kit for screening a patient sample for the presence of circulating prostate cancer cells, comprising:
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a prostate cancer cell determinant; and c) cell specific dye for excluding sample components other than said prostate cancer cells from analysis.
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a prostate cancer cell determinant; and c) cell specific dye for excluding sample components other than said prostate cancer cells from analysis.
60. A kit for screening a patient sample for the presence of circulating colon cancer cells, comprising:
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a colon cancer cell determinant; and c) cell specific dye for excluding sample components other than said colon cancer cells from analysis.
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a colon cancer cell determinant; and c) cell specific dye for excluding sample components other than said colon cancer cells from analysis.
61. A kit for screening a patient sample for the presence of circulating bladder cancer cells, comprising:
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a bladder cancer cell determinant; and c) cell specific dye for excluding sample components other than said bladder cancer cells from analysis.
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a bladder cancer cell determinant; and c) cell specific dye for excluding sample components other than said bladder cancer cells from analysis.
62. A test kit for monitoring a patient for the recurrence of cancer, said test kit comprising:
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) an antibody having binding specificity for an intracellular cytokeratin marker;
c) an antibody having binding specificity for a predetermined cancer specific determinant which is different from said intracellular cytokeratin marker;
and d) a cell specific dye for excluding sample components other than said cancer cells from analysis.
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) an antibody having binding specificity for an intracellular cytokeratin marker;
c) an antibody having binding specificity for a predetermined cancer specific determinant which is different from said intracellular cytokeratin marker;
and d) a cell specific dye for excluding sample components other than said cancer cells from analysis.
63. A kit as claimed in claim 62, said kit further containing an antibody having specificity for non-tumor cells, a biological buffer, a permeabilization buffer, a protocol and optionally, an information sheet.
64. A kit as claimed in claim 62 for monitoring a patient with breast cancer, wherein said antibody having binding specificity for a predetermined cancer specific determinant binds specifically to a breast cancer cell determinant, said determinant being selected from the group of determinants consisting of MUC-1, estrogen, progesterone receptor, cathepsin D, p53, urokinase type plasminogen activator, epidermal growth factor, epidermal growth factor receptor, BRCA1, BRCA2, CA27.29, CA15.5, prostate specific antigen, plasminogen activator inhibitor and Her2-neu.
65. A kit as claimed in claim 62 for monitoring patients with prostate cancer, wherein said antibody having binding specificity for a predetermined cancer specific determinant binds specifically to a prostate cancer cell determinant, said determinant being selected from the group of determinants consisting of prostate specific antigen, prostatic acid phosphatase, thymosin b-15, p53, HPC1 basic prostate gene, creatine kinase and prostate specific membrane antigen.
66. A kit as claimed in claim 62 for monitoring patients with colon cancer, wherein said antibody having binding specificity for a predetermined cancer specific determinant binds specifically to a colon cancer cell determinant, said determinant being selected from the group of determinants consisting of carcinoembryonic antigen, Cand protein, APC gene, p53 and matrix metalloproteinase (MMP-9).
67. A kit as claimed in claim 62 for monitoring patients with bladder cancer, wherein said antibody having binding specificity for a predetermined cancer specific determinant binds to a bladder cancer cell determinant, said determinant being selected from the group of determinants consisting of nuclear matrix protein (NMP22), Bard Bladder tumor antigen (BTA) and fibrin degradation product (FDP).
68. A method for determining likelihood of cancer recurrence in a human subject previously treated for cancer, comprising:
a) obtaining a blood sample from said subject;
b) determining the number of epithelial cells in said sample;
c) comparing the number of epithelial cells with a statistically determined number of epithelial cells from a group of tumor-free patient controls; and d) assigning a likelihood of cancer recurrence when the number of epithelial cells exceeds a pre-determined value based on statistical averages of circulating epithelial cells from healthy subjects compared with statistical averages of circulating epithelial cells from cancer patients.
a) obtaining a blood sample from said subject;
b) determining the number of epithelial cells in said sample;
c) comparing the number of epithelial cells with a statistically determined number of epithelial cells from a group of tumor-free patient controls; and d) assigning a likelihood of cancer recurrence when the number of epithelial cells exceeds a pre-determined value based on statistical averages of circulating epithelial cells from healthy subjects compared with statistical averages of circulating epithelial cells from cancer patients.
69. The method of claim 68 further comprising identifying the tissue origin of the epithelial cells.
70. The method of claim 69 wherein tissue origin of the epithelial cells is identified by cell surface binding with selected tumor specific antibodies.
71. The method of claim 68 wherein tumor recurrence is determined to be likely when epithelial cell number in blood is about 5 to about 15 epithelial cells per 20 ml of blood.
72. A method of distinguishing an organ confined carcinoma from a carcinoma with metastatic properties, comprising:
a) obtaining a blood sample from a test subject;
b) determining the number of circulating epithelial cells in said sample;
c) identifying the tissue origin of the epithelial cells;
d) comparing the number of circulating tissue specific epithelial cells determined for said test subject with a statistically determined average number of circulating tissue specific epithelial cells from a group of healthy subjects, a group of organ-confined carcinoma patients and a group of patients with metastatic carcinoma; and e) identifying said human test subject cells as metastatic when the number is within the statistically determined average for the group of patients with metastatic carcinoma.
a) obtaining a blood sample from a test subject;
b) determining the number of circulating epithelial cells in said sample;
c) identifying the tissue origin of the epithelial cells;
d) comparing the number of circulating tissue specific epithelial cells determined for said test subject with a statistically determined average number of circulating tissue specific epithelial cells from a group of healthy subjects, a group of organ-confined carcinoma patients and a group of patients with metastatic carcinoma; and e) identifying said human test subject cells as metastatic when the number is within the statistically determined average for the group of patients with metastatic carcinoma.
73. The method of claim 72 wherein the carcinoma is a carcinoma selected from the group consisting of prostate cancer, breast cancer, colon cancer apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell and transitional cell reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, throphoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, leydig cell tumor, papilloma, sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffin, antiokeratoma, angioma sclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (Kaposi's, and mast-cell), neoplasms (e.g., bone, digestive system, liver, pancreatic, pituitary, testicular, orbital, head and neck, central nervous system, acoustic, pelvic, respiratory tract, and urogenital), neurofibromatosis, and cervical dysplasia.
74. A method as claimed in claim 72 wherein the carcinoma is a breast carcinoma.
75. A method as claimed in claim 74 wherein a number of epithelial cells in the human test subject's blood greater than the average number in a group of organ-confined breast carcinoma patients is indicative of metastatic breast carcinoma.
76. A method as claimed in claim 74 wherein a number of epithelial cells in the human test subject's blood which is higher than average number in a group of healthy subjects but less than the average number in a group of metastatic breast carcinoma patients is indicative of organ-confined breast carcinoma.
77. A method of following remission status in a human cancer patient undergoing cancer therapy treatment, comprising:
a) obtaining a blood sample from a human cancer patient;
b) determining the number of circulating epithelial cells in said sample prior to and periodically subsequent to said cancer therapy treatment wherein an increase over time in the number of epithelial cells following treatment compared to the number of epithelial cells just after treatment is indicative of relapse and wherein maintenance of epithelial cell levels comparable to levels just after treatment is indicative of continuing remission.
a) obtaining a blood sample from a human cancer patient;
b) determining the number of circulating epithelial cells in said sample prior to and periodically subsequent to said cancer therapy treatment wherein an increase over time in the number of epithelial cells following treatment compared to the number of epithelial cells just after treatment is indicative of relapse and wherein maintenance of epithelial cell levels comparable to levels just after treatment is indicative of continuing remission.
78. The method of claim 77 wherein the cancer is selected from the group of cancers consisting of prostate cancer, breast cancer, colon cancer apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell and transitional cell reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, throphoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, leydig cell tumor, papilloma, sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffin, antiokeratoma, angioma sclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (Kaposi's, and mast-cell), neoplasms (e.g., bone, digestive system, liver, pancreatic, pituitary, testicular, orbital, head and neck, central nervous system, acoustic, pelvic, respiratory tract, and urogenital), neurofibromatosis, and cervical dysplasia.
79. A fraction of peripheral blood enriched for circulating neoplastic cells, said enrichment being at least 2,500 fold with reference to the sample of peripheral blood from which said fraction was obtained.
80. A fraction of peripheral blood enriched for circulating neoplastic cells, said enrichment being at least 5,000 fold with reference to the sample of peripheral blood from which said fraction was obtained.
81. A fraction of peripheral blood enriched for circulating neoplastic cells, said enrichment being at least 10,000 fold with reference to the sample of peripheral blood from which said fraction was obtained.
82. A method as claimed in claim 8, wherein said biological specimen is peripheral blood.
83. A method for increasing numbers of circulating epithelial cells in a blood sample, comprising massaging a tissue suspected of being cancerous prior to obtaining said blood sample.
Applications Claiming Priority (7)
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|---|---|---|---|
| US7453598P | 1998-02-12 | 1998-02-12 | |
| US60/074,535 | 1998-02-12 | ||
| US11027998P | 1998-11-30 | 1998-11-30 | |
| US11020298P | 1998-11-30 | 1998-11-30 | |
| US60/110,279 | 1998-11-30 | ||
| US60/110,202 | 1998-11-30 | ||
| CA002320418A CA2320418C (en) | 1998-02-12 | 1999-02-12 | Methods and reagents for the rapid and efficient isolation of circulating cancer cells |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002320418A Division CA2320418C (en) | 1998-02-12 | 1999-02-12 | Methods and reagents for the rapid and efficient isolation of circulating cancer cells |
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| Publication Number | Publication Date |
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| CA2432361A1 true CA2432361A1 (en) | 1999-08-19 |
| CA2432361C CA2432361C (en) | 2011-10-11 |
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| CA2432363A Expired - Lifetime CA2432363C (en) | 1998-02-12 | 1999-02-12 | Methods and reagents for the rapid and efficient isolation of circulating cancer cells |
| CA2432361A Expired - Lifetime CA2432361C (en) | 1998-02-12 | 1999-02-12 | Methods and reagents for the rapid and efficient isolation of circulating cancer cells |
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| CA2432363A Expired - Lifetime CA2432363C (en) | 1998-02-12 | 1999-02-12 | Methods and reagents for the rapid and efficient isolation of circulating cancer cells |
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Cited By (1)
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| WO2015127101A1 (en) * | 2014-02-19 | 2015-08-27 | The Trustees Of Columbia University In The City Of New York | Method and composition for diagnosis of aggressive prostate cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110501208B (en) * | 2018-05-17 | 2023-06-27 | 国家纳米科学中心 | Folic acid functionalized streptavidin modified magnetic nanoparticle, preparation method and application thereof |
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1999
- 1999-02-12 CA CA2432363A patent/CA2432363C/en not_active Expired - Lifetime
- 1999-02-12 CA CA2432361A patent/CA2432361C/en not_active Expired - Lifetime
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2015127101A1 (en) * | 2014-02-19 | 2015-08-27 | The Trustees Of Columbia University In The City Of New York | Method and composition for diagnosis of aggressive prostate cancer |
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| Publication number | Publication date |
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| CA2432363A1 (en) | 1999-08-19 |
| CA2432361C (en) | 2011-10-11 |
| CA2432363C (en) | 2010-04-20 |
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