Methods and reagents for the rapid and efficient isolation of circulating cancer cells

• US 20030129676A1
• Filed: 10/11/2002
• Published: 07/10/2003
• Est. Priority Date: 02/12/1998
• Status: Active Grant

• Current Assignee: Menarini Silicon Biosystems S.P.A
• Original Assignee: Veridex LLC (Johnson & Johnson)
• Inventors: Rao, Galla Chandra, Uhr, Jonathan W., Terstappen, Leon W.M.M., Liberti, Paul A., Racila, Emilian V.
• Granted Patent: US 7,332,288 B2
• Time in Patent Office: Days
• US Class Current: 435/7.21
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B82Y 25/00 Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: G01N 33/57484 involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells
• CPC Class Codes: H01F 1/0054 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 sensitivity 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

1. A method for determining the severity of a disease in a test subject by means of detecting and enumerating rare cells in a mixed cell population obtained from a biological specimen of said test subject, the presence of said rare cells in said cell population being indicative of a disease state, said method comprises:

a) obtaining said biological specimen from said test subject, said specimen comprises said 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 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 labeling 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 labeling 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 2, wherein said intermediate step further includes reducing the volume of said immunomagnetic sample containing said enriched rare cells.

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.

44. 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;

c) a cell specific dye for excluding sample components other than said rare cells from analysis; and

d) a protocol for using the method of claim 1.

45. A kit as claimed in claim 44, said kit further containing an antibody which has binding affinity for non-target cells, a biological buffer, a permeabilization buffer, and optionally, an information sheet.

46. A kit as claimed in claim 44, 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.

7. A method for determining the severity of cancer in a test subject by means of detecting and enumerating cancer cells in a mixed cell population obtained from a biological specimen of said test subject, said method comprises:

a) obtaining said biological specimen from said test subject, said specimen comprises said 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 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 labeling 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 8, wherein said colloidal magnetic particles and said at least one labeling 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 fraction.

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 ligand is a monoclonal antibody specific for at least one cancer cell determinant, and said at least one labeling reagent comprises at least one additional monoclonal antibody specific for a second cancer cell determinant, and said method further comprises adding to said labeled cancer cell-containing fraction a third monoclonal antibody specific for an antigen present on a non-tumor cell and 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.

16. A method as claimed in claim 12, wherein said biological specimen is obtained from said test subject periodically after a cancer eradication procedure to assess for the presence and number of circulating cancer cells as an indicator of said test subject'"'"'s response to said cancer eradication procedure.

17. A method as claimed in claim 12, wherein said ligand binds specifically to an epithelial cell adhesion molecule.

18. A method as claimed in claim 12, wherein said one or more labeling reagent binds specifically to an intracellular cytokeratin.

19. A method as claimed in claim 12, wherein said one or more labeling 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 ligand has binding affinity for epithelial cell adhesion molecule, and said at least one labeling 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 test subject has been diagnosed with a cancer selected from the group consisting of prostate cancer, breast cancer, and colon cancer.

22. A method as claimed in claim 12, wherein said test subject has been diagnosed with a carcinoma selected from the group consisting of 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, Kaposi'"'"'s sarcoma, and mast-cell sarcoma.

23. A method as claimed in claim 12, wherein said test subject has been diagnosed with a neoplasm selected from the group consisting of bone, digestive system, liver, pancreatic, pituitary, testicular, orbital, head and neck, central nervous system, acoustic, pelvic, respiratory tract, urogenital neoplasm, neurofibromatosis, and cervical dysplasia.

47. A kit for screening a patient sample for the presence of circulating cancer cells, comprising:

a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and an anti-epithelial cell adhesion molecule antibody coupled, directly or indirectly, to said base coating material;

b) at least one antibody having binding specificity for a cancer cell determinant;

c) cell specific dye for excluding sample components other than said cancer cells from analysis; and

d) a protocol for using the method of claim 7.

48. A kit as claimed in claim 47, said kit further containing an antibody which has binding affinity for non-tumor cells, a biological buffer, a permeabilization buffer, and optionally, an information sheet.

49. A kit as claimed in claim 47, wherein said at least one antibody having binding specificity for a cancer cell determinant is selected from the group consisting of antibody having specificity for a breast cancer cell determinant, antibody having specificity for a prostate cancer cell determinant, antibody having specificity for a colon cancer cell determinant and antibody having specificity for a bladder cancer cell determinant.

50. A kit as claimed in claim 49 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.

51. A kit as claimed in claim 49 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.

52. A kit as claimed in claim 49 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).

53. A kit as claimed in claim 49 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).

54. A test kit as claimed in claim 47, wherein said at least one antibody comprises a panel of antibodies each having binding specificity for a different cancer cell determinant.

55. 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 an anti-epithelial cell adhesion molecule antibody coupled, directly or indirectly, to said base coating material;

b) at least one antibody having binding specificity for a breast cancer cell determinant;

c) cell specific dye for excluding sample components other than said breast cancer cells from analysis; and

d) a protocol for using the method of claim 7.

56. 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 an anti-epithelial cell adhesion molecule antibody coupled, directly or indirectly, to said base coating material;

b) at least one antibody having binding specificity for a prostate cancer cell determinant;

c) cell specific dye for excluding sample components other than said prostate cancer cells from analysis; and

d) a protocol for using the method of claim 7.

57. 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 an anti-epithelial cell adhesion molecule antibody coupled, directly or indirectly, to said base coating material;

b) at least one antibody having binding specificity for a colon cancer cell determinant;

c) cell specific dye for excluding sample components other than said colon cancer cells from analysis; and

d) a protocol for using the method of claim 7.

58. 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 an anti-epithelial cell adhesion molecule antibody coupled, directly or indirectly, to said base coating material;

b) at least one antibody having binding specificity for a bladder cancer cell determinant;

c) cell specific dye for excluding sample components other than said bladder cancer cells from analysis; and

d) a protocol for using the method of claims 7.

59. 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 an anti-epithelial cell adhesion molecule antibody 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;

d) a cell specific dye for excluding sample components other than said cancer cells from analysis; and

e) a protocol for using the method of claim 7.

60. A kit as claimed in claim 59, said kit further containing an antibody having specificity for non-tumor cells, a biological buffer, a permeabilization buffer, and optionally, an information sheet.

61. A kit as claimed in claim 59 for monitoring a patient with breast cancer, wherein said antibody having binding specificity for a 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.

62. A kit as claimed in claim 59 for monitoring patients with prostate cancer, wherein said antibody having binding specificity for a 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.

63. A kit as claimed in claim 59 for monitoring patients with colon cancer, wherein said antibody having binding specificity for a 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).

64. A kit as claimed in claim 59 for monitoring patients with bladder cancer, wherein said antibody having binding specificity for a 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 as claimed in claim 8, wherein said biological specimen is peripheral blood.

24. 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.

26. A magnetic particle as claimed in claim 24, wherein said magnetic core material comprises at least one transition metal oxide and said base coating material comprises a protein.

27. A magnetic particle as claimed in claim 26, wherein said additional coating material is one member of a specific binding pair which is coupled to said base coating material.

28. A magnetic particle as claimed in claim 27, wherein said one member of said specific binding pair is coupled to said base coating material through a bifunctional linking compound.

29. A magnetic particle as claimed in claim 27, 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.

30. A magnetic particle as claimed in claim 27, wherein said specific binding pair is biotin-streptavidin and streptavidin is coupled to said base coating material.

31. A magnetic particle as claimed in claim 30, 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.

32. A magnetic particle as claimed in claim 30, 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.

33. A magnetic particle as claimed in claim 30, 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.

34. A magnetic particle as claimed in claim 30, wherein the protein component of said biotin-protein conjugate comprises bovine serum albumin.

25. 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.

35. A coated magnetic particle, as claimed in claim 25, having between 70-90% magnetic mass.

36. A coated magnetic particle, as claimed in claim 25, wherein a major portion of said particles have a particle size in the range of 90-120 nm.

37. A coated magnetic particle, as claimed in claim 25, wherein a major portion of said particles have a particle size in the range of 120-150 nm.

38. A coated magnetic particle, as claimed in claim 25, wherein a major portion of said particles have a particle size in the range of 90-150 nm.

39. A composition comprising a plurality of magnetic particles as claimed in claim 25, suspended in a biologically compatible aqueous medium.

40. 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.

41. A coated magnetic particle as claimed in claim 40, 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.

42. A coated magnetic particle as claimed in claim 41, 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.

43. A composition comprising a plurality of magnetic particles as claimed in claim 42, suspended in a biologically compatible aqueous medium.

65. 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.

66. 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.

67. 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.

69. 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.