Method to determine responsiveness of cancer to epidermal growth factor receptor targeting treatments

• US 20080234264A1
• Filed: 08/20/2007
• Published: 09/25/2008
• Est. Priority Date: 03/31/2004
• Status: Active Grant

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: Dana-Farber Cancer Institute, The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Lynch, Thomas J., Sordella, Raffaella, Paez, Juan Guillermo, Janne, Pasi Antero, Johnston, Bruce E., Haber, Daniel A., Meyerson, Matthew, Sellers, William R., Bell, Daphne Winifred, Settleman, Jeffrey E.
• Granted Patent: US 8,465,916 B2
• Time in Patent Office: Days
• US Class Current: 514/234.5
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G01N 2333/485 Epidermal growth factor [EGF] (urogastrone)
• 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 33/574 for cancer
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

The present invention is directed to a method for determining the responsiveness of cancer to an epidermal growth factor receptor (EGFR) treatment. In a preferred embodiment, the presence of at least one variance in the kinase domain of the erbB1 gene confers sensitivity to the tyrosine kinase inhibitor gefitinib. Thus, a diagnostic assay for these mutations will allow for the administration of gefitinib, erlotinib and other tyrosine kinase inhibitors to those patients most likely to respond to the drug.

Claims

1. A method for determining an increased likelihood of pharmacological effectiveness of treatment by an EGFR tyrosine kinase inhibitor in an individual diagnosed with cancer comprising:

obtaining DNA from a non-small lung cancer tumor sample from the individual; and

determining the presence or absence of at least one nucleotide variance in exon 18, 19, 20 or 21 of the epidermal growth factor receptor (EGFR) gene in the DNA, wherein the presence of at least one nucleotide variance selected from an in-frame deletion of amino acid glutamic acid at codon 746 of SEQ ID NO;

512, an in-frame deletion of amino acid leucine at codon 747 of SEQ ID NO;

512, an in-frame deletion of amino acid arginine at codon 748 of SEQ ID NO;

512, an in-frame deletion of amino acid glutamic acid at codon 749 of SEQ ID NO;

512, an in-frame deletion of amino acid alanine at codon 750 of SEQ ID NO;

512, an insertion after codon 770 and before codon 771 of SEQ ID NO;

512, an amino acid substitution for amino acid leucine at codon 858 of SEQ ID NO;

512, an amino acid substitution for amino acid leucine at codon 861 of SEQ ID NO;

512, and an amino acid substitution for amino acid glycine at codon 719 of SEQ ID NO;

512 indicates that treatment by the EGFR tyrosine kinase inhibitor is likely to be pharmacologically effective in the individual.

2. The method of claim 1, wherein the presence of the at least one nucleotide variance is determined by DNA sequencing of exons 18, 19, 20 and 21 of the EGFR gene.

3. The method of claim 1, further comprising one or more of the steps:

a. amplifying exon 19 of the EGFR gene in said DNA, and determining the presence of the at least one nucleotide variance in exon 19, wherein the presence of an in-frame deletion of at least amino acid glutamic acid at codon 746, leucine at codon 747, arginine at codon 748, glutamic acid at codon 749, or alanine at codon 750 of SEQ ID NO;

512 indicates that treatment by gefitinib or erlotinib are likely to be pharmacologically effective in the individual;

b. amplifying exon 18, and determining the presence of at least one nucleotide variance in exon 18, wherein the presence of at least a substitution in exon 18 comprising a cysteine or a glycine at codon 719 of SEQ ID NO;

512 indicates that treatment by gefitinib or erlotinib is likely to be pharmacologically effective in the individual;

c. amplifying exon 20, and determining the presence of at least one nucleotide variance in exon 20, wherein the presence of at least an amino acid insertion comprising amino acids serine, valine and aspartic acid or amino acids asparagine, proline and glycine after codon 770 and before codon 771 of SEQ ID NO;

512 indicates that treatment by gefitinib or erlotinib is likely to be pharmacologically effective in the individual;

or

d. amplifying exon 21, and determining the presence of at least one nucleotide variance in exon 21, wherein the presence of at least an amino acid substitution comprising an arginine for a leucine at codon 858 of SEQ ID NO;

512 or a glutamine for a leucine at codon 861 of SEQ ID NO;

512 indicates that treatment by gefitinib or erlotinib is likely to be pharmacologically effective in the individual.

6. The method of claim 3, wherein the presence of the at least one nucleotide variance is determined by DNA sequencing, allele-specific amplification, mismatch cleavage analysis, single strand conformation polymorphism, denaturing gradient gel electrophoresis or temperature gradient gel electrophoresis analysis of the amplified exon.

23. The method of claim 1 wherein the EGFR tyrosine kinase inhibitor is gefitinib or erlotinib.

24. The method of claim 1 wherein the cancer is non-small cell lung cancer.

25. The method of claim 2, wherein the EGFR tyrosine kinase inhibitor is an anilinoquinazoline.

26. The method of claim 25, wherein the anilinoquinazoline is gefitinib or erlotinib.

37. The method of claim 25, 33, 34 and 36, wherein the EGFR targeting treatment is a tyrosine kinase inhibitor.

39. The method of claim 25, 33, 34 and 36, wherein the EGFR tyrosine kinase inhibitor is selected from the group consisting of gefitinib and erlotinib.

4-5. -5. (canceled)

7-22. -22. (canceled)

27. 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:

detecting the presence or absence of at least one nucleic acid variance in the kinase domain of the erbB1 gene of said patient relative to the wildtype erbB1 gene, wherein the presence of the at least one nucleic acid variance indicates that the EGFR targeting treatment is likely to be effective.

28. The method of claim 27, wherein the variance in the kinase domain of the erbB1 gene effects the conformational structure of the ATP-binding pocket.

29. The method of claim 27, wherein the variance in the kinase domain of erbB1 is in an exon of the erbB1 gene selected from the group consisting of exon 18, 19, 20 or 21.

30. The method of claim 29, wherein the variance in the kinase domain of the erbB1 gene is at least an in frame deletion in exon 19 of the erbB1 gene that comprises a deletion of at least amino acids leucine, arginine, glutamic acid and alanine, at codons 747, 748, 749, and 750 of SEQ. ID. NO. 512 or a deletion consisting of 2235 to 2249, 2240 to 2251 and 2240 to 2257 of SEQ ID NO:

511, or a substitution in exon 18 of a thymine for a guanine at nucleotide 2155 of SEQ ID NO;

511 or a substitution in exon 21 comprising a substitution of guanine for a thymine at nucleotide 2573 of SEQ ID NO;

511 or an adenine for a thymine at nucleotide 2582 of SEQ ID NO;

511.

31. The method of claim 30, wherein the substitution is in exon 21 and comprises at least one amino acid.

32. The method of claim 27, wherein the detection of the presence or absence of said at least one variance 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 variance under selective hybridization conditions.

46. A method of treating a patient having an EGFR mediated disease, comprising administering to said patient a pharmaceutical composition comprising the compound of claim 32.

47. The method of claim 46, wherein the EGFR mediated disease is cancer.

48. The method of claim 47, wherein the 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.

59. The method of claim 27, wherein the nucleic acid variance of the erbB1 gene is selected from the group consisting of:

a substitution of a thymine for a guanine or a serine for a guanine at nucleotide 2155 of SEQ ID NO;

511;

a deletion of nucleotides 2235 to 2249, 2240 to 2251, 2240 to 2257, 2236 to 2250, 2254 to 2277, or 2236 to 2244 of SEQ ID NO;

511; and

a substitution of a guanine for a thymine at nucleotide 2573 or an adenine for a thymine at nucleotide 2582 of SEQ ID NO;

511.

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

determining the kinase activity of the erbB1 gene in a biological sample from said patient, wherein an increase in kinase activity following stimulation with an EGFR ligand, compared to a control, indicates that the EGFR targeting treatment is likely to be effective.

35. The method of claim 33 and 34, wherein the EGFR targeting treatment is a tyrosine kinase inhibitor.

34. A method of treating a patient affected with or at risk for developing cancer, comprising detecting the presence or absence of at least one nucleic acid variance in the kinase domain of the erbB1 gene of the patient, wherein the patient is administered an EGFR targeting treatment if the presence of the said at least one nucleic acid variance is detected.

40. The method of claim 34 and 36, wherein said cancer is non-small cell lung cancer.

41. The method of claim 34 and 36, wherein the variance in the kinase domain of the erbB1 gene effects the conformation of the ATP-binding pocket.

42. The method of claim 34 and 36, wherein the nucleic acid variance is:

a) an in frame deletion in exon 19 of the erbB1 gene comprising;

a deletion of nucleic acids which encode at least amino acids leucine, arginine, glutamic acid and alanine, at codons 747, 748, 749, and 750;

or

a deletion of nucleotides selected from the group consisting of 2235 to 2249, 2240 to 2251, and 2240 to 2257 of SEQ ID NO;

511;

or

b) a substitution in exon 21 comprising a substitution from the group consisting of a guanine for a thymine at nucleotide 2573 of SEQ ID NO;

511, and an adenine for a thymine at nucleotide 2582 of SEQ ID NO;

511;

or

c) a substitution in exon 18 of a thymine for a guanine at nucleotide 2155 of SEQ ID NO;

511.

36. A method of treating a patient affected with or at risk for developing cancer, comprising:

a. detecting the presence or absence of at least one nucleic acid variance in exon 18, 19, 20, or 21;

b. determining whether a variance exists; and

c. administering an EGFR targeting treatment to the patient if the presence of the said at least one nucleic acid variance is detected.

38. The method of claim 36 wherein determining step b) is by sequencing and/or PCR.

43. A kit comprising:

a. a probe comprising;

at least one degenerate primer pair designed to anneal to nucleic acid regions bordering or within exon 18, 19, 20 or 21 of the EGFR kinase domain;

or

an antibody designed to bind to the ATP binding site of the EGFR kinase domain protein;

b. products and reagents required to carry out PCR amplification; and

c. instructions.

44. The kit of claim 43, wherein the probe is at least one degenerate primer pair that comprises the sequence primers selected from the group consisting of SEQ ID NOS:

505-508, and SEQ ID NOS. 646-673 with SEQ ID NO. 645 on the 5′

end of all forward primers and SEQ ID NO. 674 on the 5′

end of all reverse primers.

45. A compound that inhibits the catalytic kinase activity of a variant EGFR as identified by:

a) contacting the compound with a variant EGFR; and

b) detecting a reduced kinase activity of the variant EGFR when contacted with the compound, to thereby identify the compound as inhibitory;

wherein the compound is selected from the group consisting of an antibody, antibody fragment, small molecule, peptide, protein, antisense nucleic acid, ribozyme, PNA, siRNA, oligonucleotide aptamer, and peptide aptamer.

49. A method for predicting the acquisition of secondary mutations in the kinase domain of the erbB1 gene comprising:

a. contacting a cell having a variant form of the erbB1 gene with a sub-lethal dose of a tyrosine kinase inhibitor,

b. selecting cells that are resistant to a growth arrest effect of the tyrosine kinase inhibitor; and

c. analyzing the erbB1 nucleic acid from said resistant cells for the presence of secondary mutations in the erbB1 kinase domain.

50. The method of claim 49, wherein the cell is in vitro.

51. The method of claim 49, wherein the cell is obtained from a transgenic animal.

52. The method of claim 51, wherein the transgenic animal is a mouse.

53. The method of claim 51, wherein the cell is obtained from a tumor biopsy.

54. The method of claim 49, further comprising first contacting the cells with an effective amount of a mutagenizing agent.

55. The method of claim 54, 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 hydrochloride (Prc), methyl methanesulfonate (MeMS), chlorambucil (Chl), 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).

56. The method of claim 49, further comprising propagating a variant form of the EGFR gene in a DNA repair-deficient bacterial strain before introducing it into a cell.

57. A method for selecting a compound comprising contacting the compound with a variant epidermal growth factor receptor (EGFR) having a secondary mutation in the kinase domain and detecting the resulted kinase activity, wherein a compound is selected that inhibits the kinase activity of the variant EGFR.

58. The method of claim 57, wherein the secondary mutation results in a resistance to gefitinib or erlotinib.

60. 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:

a. obtaining a biological sample from said patient; and

b. determining whether Akt, STAT5, or STAT3 are activated in said patient, wherein activated Akt, STAT5, or STAT3 indicates that said EGFR targeting treatment is likely to be effective.

61. The method of claim 60, wherein the biological sample is a biopsy or an aspirate.

62. The method of claim 60, wherein activated Akt, STAT3, or STAT5 is phosphorylated.

63. The method of claim 60, wherein the activated Akt, STAT5, or STAT3 is determined immunologically.

64. A probe which specifically binds under selective binding conditions to a nucleic acid sequence comprising at least one variance in the erbB1 gene, wherein the variance is a mutation in the kinase domain of erbB1 that confers a structural change in the ATP-binding pocket.

65. The probe of claim 64, wherein said variance is in an exon of the erbB1 gene selected from the group consisting of exon 18, 19, 20 or 21.

66. The probe of claim 64, wherein said probe comprises a nucleic acid sequence 500 nucleotide bases or less in length.

67. The probe of claim 64, wherein said probe comprises peptide nucleic acid (PNA).

68. The probe of claim 64, further comprising a detectable label.

69. The probe of claim 64, wherein said probe comprises at least 10 consecutive nucleic acids selected from the group consisting of at least nucleic acids 15-25 of SEQ ID NO:

495, at least nucleic acids 20-30 of SEQ ID NO;

497, or at least nucleic acids 20-30 of SEQ ID NO;

499, and compliments thereof.

70. An isolated nucleic acid comprising SEQ ID NO:

495 or a fragment of at least 18 consecutive nucleic acids thereof.

71. An isolated nucleic acid comprising SEQ ID NO:

511, wherein nucleotides 2235 to 2249 are deleted.

72. An isolated nucleic acid comprising SEQ ID NO:

497.

73. An isolated nucleic acid comprising SEQ ID NO:

511, wherein nucleotides 2240 to 2251 are deleted.

74. An isolated nucleic acid comprising SEQ ID NO:

499.

75. An isolated nucleic acid comprising SEQ ID NO:

511, wherein nucleotides 2240 to 2257 are deleted.

76. An isolated nucleic acid comprising SEQ ID NO:

502.

77. An isolated nucleic acid comprising SEQ ID NO:

511, wherein the guanine at nucleotide 2573 is substituted for a thymine.

78. An isolated nucleic acid comprising SEQ ID NO:

504.

79. An isolated nucleic acid comprising SEQ ID NO:

511, wherein the adenine at nucleotide 2582 is substituted for a thymine.

80. An isolated nucleic acid of SEQ ID NO:

511, wherein a thymine at nucleotide 2155 is substituted for a guanine.

81. An isolated protein having amino acid sequence of SEQ ID NO:

512, wherein amino acids selected from the group consisting of the 746-750, 747-751, and 747 to 753 are deleted.

82. An isolated protein having amino acid sequence of SEQ ID NO:

512 containing a substitution selected from the group consisting of Leucine at amino acid 858 substituted with an Arginine, Leucine at amino acid 861 substituted with a Glutamine, and Glycine at amino acid 719 substituted with a Cysteine.