METHODS FOR THE DETECTION OF CIRCULATING TUMOR CELLS

• US 20100247492A1
• Filed: 09/03/2009
• Published: 09/30/2010
• Est. Priority Date: 09/05/2008
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Bethel, Kelly, Marrinucci, Dena, Kuhn, Peter, Griffin, John H.
• Granted Patent: US 8,445,225 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/57488 involving compounds identifable in body fluids
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells

Abstract

The present invention provides methods for revealing, detecting, and analyzing circulating tumor cells in the blood of a subject. Revealing detectable circulating tumor cells allows for early stage detection and diagnosis in addition to long term prognosis in subjects with cancer. Additionally, enrichment allows for robust detection and clinically meaningful analysis of low volume samples for use in clinical settings as well as innovative methods for the treatment of cancers.

Claims

1. A method for revealing circulating tumor cells in a sample comprising removing or altering a protein, carbohydrate, cell, or a combination thereof, in physical association with the surface of the circulating tumor cells, thereby unmasking and revealing the circulating tumor cells in the sample.

2. The method of claim 1, wherein the circulating tumor cells remain intact.

3. The method of claim 1, wherein revealing the cells comprises removing all or portions of blood plasma proteins, platelets, or a combination thereof.

4. The method of claim 3, wherein the blood plasma protein is a clotting factor.

5. The method of claim 4, wherein the clotting factor is fibrin.

6. The method of claim 1, wherein the cells are treated enzymatically, mechanically, electrically, electromagnetically, chemically, or any combination thereof.

7. The method of claim 6, wherein the cells are treated enzymatically.

8. The method of claim 7, wherein the enzymatic treatment is by fibrinolysis.

9. The method of claim 8, wherein fibrinolysis is with plasmin.

10. The method of claim 8, wherein enzymatic treatment is by incubation with an animal venom or a toxin.

11. The method of claim 10, wherein the animal venom or toxin is naturally occurring or synthetically derived from a snake or a bat.

12. The method of claim 6, wherein enzymatic treatment is by activation of plasminogen.

13. The method of claim 1, wherein treatment of the cells is performed using an anti-coagulant or a blood thinner.

14. The method of claim 1, wherein the number of revealed circulating tumor cells is increased by a factor of at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 200.

15. The method of claim 1, wherein the sample is about 200 microliters.

16. The method of claim 1, wherein the revealed cells comprise greater than about 7.5 circulating tumor cells per 100 microliters.

17. The method of claim 1, further comprising enriching the sample.

18. The method of claim 17, wherein the sample is enriched immunomagnetically or by filtration.

19. The method of claim 1, further comprising analyzing one or more circulating tumor cells.

20. The method of claim 19, wherein analyzing comprises image analysis, cell number analysis, cell morphology analysis, polymerase chain reaction (PCR) analysis, sequence analysis. DNA analysis, RNA analysis, gene expression profiling, proteome analysis, metabolome analysis, immuno assays, nuclear exclusion analysis.

21. The method of claim 20, wherein the image analysis comprises detection of a cell surface marker.

22. The method of claim 21, wherein the cell surface marker is selected from the group consisting:

EGFR, HER2, ERCC1, CXCR4, EpCAM, E-Cadherin, Mucin-1, Cytokeratin, PSA, PSMA, RRM1, Androgen Receptor, Estrogen Receptor, Progesterone Receptor, IGF1, cMET, EML4, or Leukocyte Associated Receptor (LAR).

23. The method of claim 21, wherein antibodies are used to detect the cell surface markers.

24. The method of claim 23, wherein the antibodies are fluorescently labeled.

25. The method of claim 24, wherein the antibodies are directed to EpCAM, Cytokeratin, or a combination thereof.

26. The method of claim 20, wherein the image analysis is performed by microscopy or flow cytometry.

27. The method of claim 20, wherein the PCR analysis comprises multiplexing using primers specific for genes selected from the group consisting of:

EGFR, HER2, ERCC1, CXCR4, EpCAM, E-Cadherin, Mucin-1, Cytokeratin, PSA, PSMA, RRM1, Androgen Receptor, Estrogen Receptor, Progesterone Receptor, IGF1, cMET, EML4, or Leukocyte Associated Receptor (LAR).

28. The method of claim 20, wherein the nuclear exclusion analysis comprises nuclear exclusion of the Androgen Receptor.

29. The method of claim 19, further comprising providing a diagnosis and prognosis to a subject from which the sample was taken.

30. The method of claim 19, wherein the subject is known to have cancer and is undergoing cancer therapy.

31. The method of claim 30, wherein the therapy is chemotherapy.

32. The method of claim 30, wherein the subject is being administered a candidate agent.

33. The method of claim 30, further comprising determining the responsiveness of the subject to the cancer therapy.

34. A composition comprising a revealed population of circulating tumor cells comprising greater than about 5 circulating tumor cells per 100 microliters.

35. The composition of claim 34, wherein the circulating tumor cells are intact.

36. The composition of claim 34, wherein the revealed population comprises greater than about 7.5 circulating tumor cells per 100 microliters.

37. The composition of claim 34, wherein the revealed population comprises greater than about 10 circulating tumor cells per 100 microliters.

38. A method for diagnosing or prognosing cancer in a subject comprising revealing circulating tumor cells of the subject comprising:

a) removing or degrading proteins, cells, or a combination thereof in physical association with the surface of the circulating tumor cell, thereby unmasking and revealing the circulating tumor cells, and

b) analyzing the revealed circulating tumor cells to determine a diagnosis or prognosis, thereby diagnosing or prognosing cancer in a subject.

39. A method for treating cancer comprising revealing of circulating tumor cells in a subject comprising degrading proteins, cells, or a combination thereof, in physical association with the surface of the circulating tumor cell, thereby unmasking and revealing the circulating tumor cells in the subject.

40. The method of claim 39, wherein revealing is performed ex vivo and the circulating tumor cell is subsequently infused into the subject.

41. The method of claim 39, wherein the subject is coadministered a therapeutic agent.

42. The method of claim 41, wherein the'"'"'agent is a chemotherapeutic agent.

43. The method of claim 39, wherein the revealing is performed in vivo.

44. The method of claim 43, wherein the subject is coadministered a therapeutic agent.

45. The method of claim 44, wherein the agent is a chemotherapeutic agent.

46. A method for determining responsiveness of a subject to a therapeutic regime comprising revealing circulating tumor cells of the subject comprising:

a) removing or degrading proteins, cells, or a combination thereof, in physical association with the surface of the circulating tumor cell, thereby unmasking and revealing the circulating tumor cells, and

b) analyzing the revealed circulating tumor cells, thereby determining the responsiveness of the subject to a therapeutic regime.

47. A method for determining a candidate subject for a clinical trial comprising revealing circulating tumor cells of the subject comprising:

a) removing or degrading proteins, cells, or a combination thereof, in physical association with the surface of the circulating tumor cell, thereby unmasking and revealing the circulating tumor cells, and

b) analyzing the revealed circulating tumor cells, thereby determining a candidate subject for a clinical trial.

48. A method for determining effectiveness of a candidate agent in the treatment of cancer comprising revealing circulating tumor cells of a subject being administered the candidate agent comprising:

a) removing or degrading proteins, cells, or a combination thereof, in physical association with the surface of the circulating tumor cell, thereby unmasking and revealing the circulating tumor cells, and

b) analyzing the revealed circulating tumor cells, thereby determining the effectiveness of the candidate agent in the treatment of cancer.