Devices and methods for enrichment and alteration of circulating tumor cells and other particles

• US 20070099207A1
• Filed: 06/08/2006
• Published: 05/03/2007
• Est. Priority Date: 04/05/2005
• Status: Abandoned Application

• Current Assignee: Gpb Scientific, LLC, Mehmet Toner
• Original Assignee: Mehmet Toner
• Inventors: Toner, Mehmet, Fuchs, Martin, Krueger, Neil, Huang, Yi-Shuian, Haber, Daniel
• Application Number:
  US11/449,161
• Publication Number:
  US 20070099207A1
• Time in Patent Office: Days
• US Class Current: 435/6.16
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1029 Particle size
• CPC Class Codes: G01N 2015/1087 Particle size
• CPC Class Codes: G01N 2021/0346 Capillary cells; Microcells
• 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 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/574 for cancer

Abstract

The invention features devices and methods for detecting, enriching, and analyzing circulating tumor cells and other particles. The invention further features methods of diagnosing a condition, e.g., cancer, in a subject by analyzing a cellular sample from the subject.

Claims

1. A method for determining the likelihood of effectiveness of an epidermal growth factor receptor (EGFR) targeting treatment in a human patient affected with or at risk for developing cancer comprising:

applying a cellular sample from said patient to a device comprising a channel comprising a structure which directs first cells of one hydrodynamic size in one direction to produce a first output enriched in first cells and one or more second cells in a second direction to produce a second output; and

in cells of said first or second output, detecting the presence or absence of at least one predetermined nucleic acid variant in the EGFR gene, wherein the presence of said at least one nucleic acid variant indicates that the EGFR targeting treatment is likely to be effective.

2. The method of claim 1, wherein said variant is located in the kinase domain of the erbB1 gene of cells of said first output, said variant being the wildtype erbB1 gene.

3. The method of claim 2, wherein the nucleic acid variant increases kinase activity.

4. The method of claim 1, wherein said first cells are cancer cells.

5. The method of claim 3, wherein said cancer is selected from the group consisting of gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, head and neck cancer, lung cancer, non-small cell lung cancer, cancer of the nervous system, kidney cancer, retina cancer, skin cancer, liver cancer, pancreatic cancer, genital-urinary cancer and bladder cancer.

6. The method of claim 4, wherein said cancer is non-small cell lung cancer.

7. The method of claim 2, wherein the variant in the kinase domain of the erbB1 gene affects the conformational structure of the ATP-binding pocket.

8. The method of claim 2, wherein the variant in the kinase domain of erbB1 is in an exon of the erbB1 gene selected from the group consisting of exon 18, 19, 20, and 21.

9. The method of claim 8, wherein the variant is in exon 18, 19 or 21.

10. The method of claim 2, wherein the variant in the kinase domain of the erbB1 gene is an in frame deletion, substitution, or insertion.

11. The method of claim 1, wherein the detection of the presence or absence of said at least one variant comprises amplifying a segment of nucleic acid.

12. The method of claim 2, wherein the detection of the presence or absence of said at least one variant comprises contacting the erbB1 nucleic acid with at least one nucleic acid probe, wherein said at least one probe preferentially hybridizes with a nucleic acid sequence comprising said variant under selective hybridization conditions.

13. The method of claim 2, wherein the detection of the presence or absence of at least one variant comprises performing a polymerase chain reaction (PCR) to amplify nucleic acid comprising the erbB1 coding sequence, and determining the nucleotide sequence of the amplified nucleic acid.

14. The method of claim 1, wherein said structure comprises a two dimensional array of obstacles forming a network of gaps which separate said cells.

15. The method of claim 14, further comprising contacting said first output with a device comprising specific binding moieties which selectively bind epithelial or neoplastic cells.

16. The method of claim 15, wherein said specific binding moieties are antibodies or fragments thereof.

17. The method of claim 16, wherein said antibodies specifically bind Ber-Ep4, EpCam, E-Cadherin, mucin-1, cytokeratin or CD-34.

63. The method of claim 1, wherein said EGFR gene comprises the wild type sequence of SEQ ID NO:

511.

64. The method of claim 1, wherein said variant differs from SEQ ID NO:

511 at one or more nucleotide positions.

65. The method of claim 1, wherein the polypeptide encoded by said EGFR gene comprises the wild type sequence of SEQ ID NO:

512.

66. The method of claim 1, wherein the polypeptide encoded by said variant differs from SEQ ID NO:

512 at one or more amino acid positions.

67. The method of claim 1, wherein said variant is selected from Table 3.

18. A method for determining the likelihood of effectiveness of an EGFR targeting treatment in a patient comprising:

applying a cellular sample from said patient to a device comprising a channel comprising a structure which directs first cells of one hydrodynamic size in one direction to produce a first output enriched in first cells and one or more second cells in a second direction to produce a second output;

stimulating said cells with an EGFR ligand; and

determining the kinase activity of the erbB1 gene-encoded kinase in said first cells, wherein an increase in kinase activity, compared to a control, indicates that the EGFR targeting treatment is likely to be effective.

19. The method of claim 18, wherein said first cells are cancer cells.

20. The method of claim 19, wherein said cancer is selected from the group consisting of gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, head and neck cancer, lung cancer, non-small cell lung cancer, cancer of the nervous system, kidney cancer, retina cancer, skin cancer, liver cancer, pancreatic cancer, genital-urinary cancer, and bladder cancer.

21. The method of claim 19, wherein said cancer is non-small cell lung cancer.

22. The method of claim 18, wherein said structure comprises a two dimensional array of obstacles forming a network of gaps which separate said cells.

23. The method of claim 22, further comprising contacting said first output with a device comprising specific binding moieties which selectively bind epithelial or neoplastic cells.

24. The method of claim 23, wherein said specific binding moieties are antibodies or fragments thereof.

25. The method of claim 24, wherein said antibodies specifically bind Ber-Ep4, EpCam, E-Cadherin, mucin-1, cytokeratin or CD-34.

26. The method of claim 18, wherein the EGFR targeting treatment is a tyrosine kinase inhibitor.

27. The method of claim 26, wherein the tyrosine kinase inhibitor is an artilinoquinazoline.

28. The method of claim 27, wherein the anilinoquinazoline is a synthetic anilinoquinazoline.

29. The method of claim 28, wherein the synthetic amlinoquinazoline is selected from the group consisting of gefitinib and erlotinib.

30. A method for determining the likelihood of effectiveness of an epidermal growth factor receptor (EGFR) targeting treatment in a human patient affected, with or at risk for developing cancer comprising:

applying a cellular sample from said patient to a device comprising a channel comprising a structure which directs first cells of one hydrodynamic size in one direction to produce a first output enriched in first cells and one or more second cells in a second direction to produce a second output;

in said first cells, detecting the presence or absence of at least one nucleic acid variant in exon 18, 19, 20, or 21 by performing a polymerase chain reaction (PCR) to amplify a portion of exon 18, 19, 20, or 21; and

determining the nucleotide sequence of the amplified nucleic acid by sequencing at least one portion of the amplified exon 18, 19, 20, or 21, wherein the presence of at least one nucleotide variant in exon 18, 19, 20, or 21 compared to a wildtype erbB1 control indicates that the EGFR targeting treatment is likely to be effective.

31. The method of claim 30, wherein said structure comprises a two dimensional array of obstacles forming a network of gaps which separate said cells.

32. The method of claim 31, further comprising contacting said first output with a device comprising specific binding moieties which selectively bind epithelial or neoplastic cells.

33. The method of claim 32, wherein said specific binding moieties are antibodies or fragments thereof.

34. The method of claim 33, wherein said antibodies specifically bind Ber-Ep4, EpCam, E-Cadherin, mucin-1, cytokeratin or CD-34.

35. A kit comprising:

a device comprising a channel comprising a structure which directs first cells of one hydrodynamic size in one direction to produce a first output enriched in first cells and one or more second cells in a second direction to produce a second output; and

reagents for detecting the presence or absence of at least one nucleic acid mutation in the EGFR gene.

36. The kit of claim 35, wherein said reagents comprise at least one degenerate primer pair designed to anneal to nucleic acid and products and reagents required to carry out PCR amplification.

37. The kit of claim 35, wherein said structure comprises a two dimensional array of obstacles forming a network of gaps which separate said cells.

38. The kit of claim 37, further comprising a device adapted to receive said first output, which device comprises specific binding moieties which selectively bind epithelial or neoplastic cells.

39. The kit of claim 38, wherein said specific binding moieties are antibodies or fragments thereof.

40. The kit of claim 39, wherein said antibodies specifically bind Ber-Ep4, EpCam, E-Cadherin, mucin-1, cytokeratin or CD-34.

41. The kit of claim 35, wherein said reagents comprise at least one probe capable of binding to the ATP-binding pocket of the EGFR kinase domain protein.

42. The kit of claim 35 or 41, wherein said reagents comprise an antibody, antibody fragment of chimeric antibody.

43. The kit of claim 42, wherein said reagents further comprise a detectable label.

47. The method of claim 36, further comprising contacting said first output with a device comprising specific binding moieties which selectively bind epithelial or neoplastic cells.

48. The method of claim 47, wherein said specific binding moieties are antibodies or fragments thereof.

49. The method of claim 48, wherein said antibodies specifically bind Ber-Ep4, EpCam, E-Cadherin, mucin-1, cytokeratin or CD-34.

44. A method for predicting the acquisition of secondary mutations in the EGFR gene of a cancer cell from a patient comprising:

contacting a cellular sample from said patient with a device comprising a channel comprising a structure which directs first cells of one hydrodynamic size in one direction to produce a first output enriched in first cells and one or more second cells in a second direction to produce a second output;

contacting said first cells with a sublethal dose of a tyrosine kinase inhibitor;

selecting cells that are resistant to the effect of the tyrosine inhibitor; and

analyzing the nucleic acid from said resistant cells for the presence of secondary mutations.

45. The method of claim 44, wherein said first cells have a variant of the erbB1 gene.

46. The method of claim 44, wherein said structure comprises a two dimensional array of obstacles forming a network of gaps which separate said cells.

50. The method of claim 44, wherein the cell is obtained from a tumor biopsy.

51. The method of claim 44, further comprising first contacting said first cells with an effective amount of a mutagenizing agent.

52. The method of claim 51, wherein the mutagenizing agent is selected from the group consisting of ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), phocarbaxine hydrocfciloride (Prc), methyl methanesulfonate (MeMS), chlorambucil (Chi), melphalan, porcarbazine hydrochloride, cyclophosphamide (Cp), diethyl sulfate (Et2SO4), acrylamide monomer (AA), triethylene melamin (TEM), nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N′

-nitro-Nitrosoguanidine (MNNG), 7,12 dimethylbenz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, and ethyl methanesulforate (EtMs).

53. A method for determining the likelihood of effectiveness of an epidermal growth factor receptor (EGFR) targeting treatment in a patient affected with or at risk for developing cancer comprising:

contacting a biological sample from a patient with a device comprising a channel comprising a structure which directs first cells of one hydrodynamic size in one direction to produce a first output enriched in first cells and one or more second cells in a second direction to produce a second output, wherein said first cells are epithelial or neoplastic cells; and

determining whether Akt, STAT5, or STAT3 are activated in said first cells from said first output, wherein activated Akt, STATS, or STATS indicates that said EGFR targeting treatment is likely to be effective.

54. The method of claim 53, wherein the biological sample is a biopsy or an aspirate.

55. The method of claim 53, wherein activated Akt, STAT3, or STATS is phosphorylated.

56. The method of claim 53, wherein the activated Akt, STATS, or STATS is determined immunologically.

57. The method of claim 56, wherein the immunological detection methods are selected from the group consisting of immunohistochemistry, immunocytochemistry, FACS scanning, immunoblotting, radioimmunoassays, western blotting, immunoprecipitation, or enzyme-linked immunoadsorbant assays (ELISA).

58. The method of claim 57, wherein the immunological detection method is immunohistochemistry or immunocytochemistry using anti-phospho Akt, antiphospho STATS or anti-phospho STATS antibodies.

59. The method of claim 53, wherein said structure comprises a two dimensional array of obstacles forming a network of gaps which separate said cells.

60. The method of claim 59, further comprising contacting said first output with a device comprising specific binding moieties which selectively bind epithelial or neoplastic cells.

61. The method of claim 60, wherein said specific binding moieties are antibodies or fragments thereof.

62. The method of claim 61, wherein said antibodies specifically bind Ber-Ep4, EpCam, E-Cadherin, mucin-1, cytokeratin, or CD-34.