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Publication numberUS20040248151 A1
Publication typeApplication
Application numberUS 10/735,118
Publication dateDec 9, 2004
Filing dateDec 11, 2003
Priority dateApr 5, 2002
Also published asUS8512967, US8956822, US9239331, US20110136149, US20130302829, US20150125448, US20160091493
Publication number10735118, 735118, US 2004/0248151 A1, US 2004/248151 A1, US 20040248151 A1, US 20040248151A1, US 2004248151 A1, US 2004248151A1, US-A1-20040248151, US-A1-2004248151, US2004/0248151A1, US2004/248151A1, US20040248151 A1, US20040248151A1, US2004248151 A1, US2004248151A1
InventorsSarah Bacus, Bradley Smith
Original AssigneeVentana Medical Systems, Inc., Cell Signaling Technology
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Method for predicting the response to HER2-directed therapy
US 20040248151 A1
Abstract
This invention provides methods for determining or predicting response to HER2-directed therapy in an individual.
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Claims(83)
What we claim is:
1. A method for identifying a mammalian tumor that responds to a HER2-directed therapy, wherein the mammalian tumor overexpresses HER2, the method comprising the step of assaying a sample obtained from the mammalian tumor to detect a pattern of expression, phosphorylation or both expression and phosphorylation of one or a plurality of polypeptides consisting of:
(a) IGFR polypeptide;
(b) EGFR polypeptide;
(c) NDF polypeptide;
(d) phosphorylated S6 ribosomal polypeptide;
(e) phosphorylated AKT polypeptide; and
(f) phosphorylated ERK polypeptide;
wherein the detected pattern of expression, phosphorylation or both expression and phosphorylation identifies mammalian tumors that respond to a HER2-directed therapy.
2. The method of claim 1, wherein the method comprises the step of assaying a sample obtained from the mammalian tumor to detect a pattern of expression, phosphorylation or both expression and phosphorylation of (a) IGFR polypeptide, and one or a plurality of polypeptides consisting of:
(b) EGFR polypeptide;
(c) NDF polypeptide;
(d) phosphorylated S6 ribosomal polypeptide;
(e) phosphorylated AKT polypeptide; and
(f) phosphorylated ERK polypeptide.
3. The method of claim 1, wherein the detected pattern is decreased expression of IGFR polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
4. The method of claim 1, wherein the detected pattern is normal or increased expression of IGFR polypeptide, accompanied by decreased phosphorylation of AKT polypeptide, decreased phosphorylation of S6 ribosomal polypeptide or both in the mammalian tumor as compared to a non-tumor tissue or cell sample.
5. The method of claim 1, wherein the detected pattern is normal or increased expression of EGFR polypeptide, accompanied by decreased phosphorylation of ERK polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
6. The method of claim 1, wherein the detected pattern is decreased expression of IGFR polypeptide, accompanied by increased phosphorylation of S6 ribosomal polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
7. The method of claim 1, wherein the detected pattern is decreased expression of IGFR polypeptide, accompanied by increased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
8. The method of claim 6, wherein the detected pattern further includes increased phosphorylation of S6 ribosomal polypeptide
9. A method for identifying a mammalian tumor that does not respond to a HER2-directed therapy, wherein the mammalian tumor overexpresses HER2, the method comprising the step of assaying a sample obtained from the mammalian tumor to detect a pattern of expression, phosphorylation or both expression and phosphorylation of one or a plurality of polypeptides consisting of:
(a) IGFR polypeptide;
(b) EGFR polypeptide;
(c) NDF polypeptide;
(d) phosphorylated S6 ribosomal polypeptide;
(e) phosphorylated AKT polypeptide; and
(f) phosphorylated ERK polypeptide;
wherein the detected pattern of expression, phosphorylation or both expression and phosphorylation identifies mammalian tumors that do not respond to HER2-directed therapy.
10. The method of claim 9, wherein the method comprises the step of assaying a sample obtained from the mammalian tumor to detect a pattern of expression, phosphorylation or both expression and phosphorylation of (a) IGFR polypeptide, and one or a plurality of polypeptides consisting of:
(b) EGFR polypeptide;
(c) NDF polypeptide;
(d) phosphorylated S6 ribosomal polypeptide;
(e) phosphorylated AKT polypeptide; and
(f) phosphorylated ERK polypeptide.
11. The method of claim 9, wherein the detected pattern is normal or increased expression of IGFR polypeptide, accompanied by increased phosphorylation of AKT polypeptide, increased phosphorylation of S6 ribosomal polypeptide, or both in the mammalian tumor as compared to a non-tumor tissue or cell sample.
12. The method of claim 9, wherein the detected pattern is decreased expression of EGFR polypeptide and decreased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
13. The method of claim 9, wherein the detected pattern is decreased expression of EGFR polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
14. The method of claim 9, wherein the detected pattern is decreased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
15. The method of claim 9, wherein the detected pattern is decreased expression of EGFR polypeptide and increased phosphorylation of ERK polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
16. The method of claim 9, wherein the detected pattern is normal or increased expression of IGFR polypeptide and decreased expression of NDF in the mammalian tumor as compared to a non-tumor tissue or cell sample.
17. The method of claim 1, wherein the detection of phosphorylation of AKT polypeptide, phosphorylation of S6 ribosomal polypeptide, or both is determined subsequent to contacting the sample obtained from the mammalian tumor with a HER2-directed therapy.
18. The method of claim 4, wherein the detection of phosphorylation of AKT polypeptide, phosphorylation of S6 ribosomal polypeptide, or both is determined subsequent to contacting the sample obtained from the mammalian tumor with a HER2-directed therapy.
19. The method of claim 9, wherein the detection of phosphorylation of AKT polypeptide, phosphorylation of S6 ribosomal polypeptide, or both is determined subsequent to contacting the sample obtained from the mammalian tumor with a HER2-directed therapy.
20. The method of claim 11, wherein the detection of phosphorylation of AKT polypeptide, phosphorylation of S6 ribosomal polypeptide, or both is determined subsequent to contacting the sample obtained from the mammalian tumor with a HER2-directed therapy.
21. The method of claim 1, wherein the HER2-directed therapy comprises rhuMAb HER2 (HERCEPTIN®).
22. The method of claim 9, wherein the HER2-directed therapy comprises rhuMAb HER2 (HERCEPTIN®).
23. The method of claim 1, wherein the sample obtained from the mammalian tumor has been contacted with at least one chemotherapeutic agent.
24. The method of claim 9, wherein the sample obtained from the mammalian tumor has been contacted with at least one chemotherapeutic agent.
25. The method of claim 1, wherein the detected pattern of expression, phosphorylation, or both, of one or a plurality of polypeptides (a) through (f) is determined using a biodetection reagent.
26. The method of claim 25, wherein the biodetection reagent is an antibody.
27. The method of claim 25, wherein the biodetection reagent is a nucleic acid probe.
28. The method of claim 9, wherein the detected pattern of expression, phosphorylation, or both, of one or a plurality of polypeptides (a) through (f) is determined using a biodetection reagent.
29. The method of claim 28, wherein the biodetection reagent is an antibody.
30. The method of claim 28, wherein the biodetection reagent is a nucleic acid probe.
31. The method of claim 1, wherein the detected pattern of phosphorylated AKT polypeptide is determined using an antibody specific for an epitope comprising a phosphorylated serine residue at position 473 in SEQ ID NO: 1.
32. The method of claim 9, wherein the detected pattern of phosphorylated AKT polypeptide is determined using an antibody specific for an epitope comprising a phosphorylated serine residue at position 473 in SEQ ID NO: 1.
33. The method of claim 1, wherein the detected pattern of phosphorylated S6 ribosomal polypeptide is determined using an antibody specific for an epitope comprising a phosphorylated serine residue at position 235 in SEQ ID NO: 2.
34. The method of claim 9, wherein the detected pattern of phosphorylated S6 ribosomal polypeptide is determined using an antibody specific for an epitope comprising a phosphorylated serine residue at position 235 in SEQ ID NO: 2.
35. The method of claim 1, wherein the detected pattern of phosphorylated ERK polypeptide is determined using an antibody specific for an epitope comprising a phosphorylated threonine residue at 202 or a phosphorylated serine residue at position 204 in SEQ ID NO: 3.
36. The method of claim 9, wherein the detected pattern of phosphorylated ERK polypeptide is determined using an antibody specific for an epitope comprising a phosphorylated threonine residue at 202 or a phosphorylated serine residue at position 204 in SEQ ID NO: 3.
37. The method of claim 1, wherein the sample obtained from the mammalian tumor is a paraffin-embedded biopsy sample.
38. The method of claim 9, wherein the sample obtained from the mammalian tumor is a paraffin-embedded biopsy sample.
39. The method of claim 1, wherein the mammalian tumor is identified as overexpressing HER2 using an antibody that binds HER2 polypeptide.
40. The method of claim 9, wherein the mammalian tumor is identified as overexpressing HER2 using an antibody that binds HER2 polypeptide.
41. A method of selecting a subject with cancer for treatment with a molecule targeting HER2, wherein the cancer overexpresses HER2, the method comprising the steps of: (a) determining a pattern of expression, phosphorylation or both expression and phosphorylation in a cell or tissue sample from the subject of one or a plurality of polypeptides consisting of:
(i) IGFR polypeptide;
(ii) EGFR polypeptide;
(iii) NDF polypeptide;
(iv) phosphorylated S6 ribosomal polypeptide;
(v) phosphorylated AKT polypeptide; and
(vi) phosphorylated ERK polypeptide; and
(b) selecting the subject based on the detected pattern of expression, phosphorylation, or both expression and phosphorylation.
42. The method of claim 41, wherein step (a) comprises determining a pattern of expression, phosphorylation or both expression and phosphorylation in a cell or tissue sample from the subject of (a) IGFR polypeptide, and one or a plurality of polypeptides consisting of:
(b) EGFR polypeptide;
(c) NDF polypeptide;
(d) phosphorylated S6 ribosomal polypeptide;
(e) phosphorylated AKT polypeptide; and
(f) phosphorylated ERK polypeptide.
43. The method of claim 41, wherein the subject is selected when the detected pattern is decreased expression of IGFR polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
44. The method of claim 41, wherein the subject is selected when the detected pattern is normal or increased expression of IGFR polypeptide, accompanied by decreased phosphorylation of AKT polypeptide, decreased phosphorylation of S6 ribosomal polypeptide, or both in the mammalian tumor as compared to a non-tumor tissue or cell sample.
45. The method of claim 41, wherein the subject is selected when the detected pattern is normal or increased expression of EGFR polypeptide, accompanied by decreased phosphorylation of ERK polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
46. The method of claim 41, wherein the subject is selected when the detected pattern is decreased expression of IGFR polypeptide, accompanied by increased phosphorylation of S6 ribosomal polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
47. The method of claim 41, wherein the subject is selected when the detected pattern is decreased expression of IGFR polypeptide, accompanied by increased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
48. The method of claim 47, wherein the subject is selected when the detected pattern further includes increased phosphorylation of S6 ribosomal polypeptide
49. The method of claim 41, wherein phosphorylation of AKT polypeptide, phosphorylation of S6 ribosomal polypeptide, or both is determined subsequent to contacting the sample obtained from the mammalian tumor with a HER2-directed therapy.
50. The method of claim 44, wherein phosphorylation of AKT polypeptide, phosphorylation of S6 ribosomal polypeptide, or both is determined subsequent to contacting the sample obtained from the mammalian tumor with a HER2-directed therapy.
51. The method of claim 41, wherein the HER2-directed therapy comprises rhuMAb HER2 (HERCEPTIN®).
52. The method of claim 41, wherein the sample obtained from the mammalian tumor has been contacted with at least one chemotherapeutic.
53. The method of claim 41, wherein the detected pattern of expression, phosphorylation, or both of one or a plurality of polypeptides (i) through (vi) is determined using a biodetection reagent.
54. The method of claim 53, wherein the biodetection reagent is an antibody.
55. The method of claim 53, wherein the biodetection reagent is a nucleic acid probe.
56. The method of claim 41, wherein the detected pattern of phosphorylated AKT polypeptide is determined using an antibody specific for an epitope comprising a phosphorylated serine residue at position 473 in SEQ ID NO: 1.
57. The method of claim 41, wherein the detected pattern of phosphorylated S6 ribosomal polypeptide is determined using an antibody specific for an epitope comprising a phosphorylated serine residue at position 235 in SEQ ID NO: 2.
58. The method of claim 41, wherein the detected pattern of phosphorylated ERK polypeptide is determined using an antibody specific for an epitope comprising a phosphorylated threonine residue at 202 or a phosphorylated serine residue at position 204 in SEQ ID NO: 3.
59. The method of claim 41, wherein the cell or tissue sample from the subject is a paraffin-embedded biopsy sample.
60. The method of claim 41, wherein the mammalian tumor is identified as overexpressing HER2 using an antibody that binds HER2 polypeptide.
61. A method of selecting a subject with cancer to not receive treatment with a molecule targeting HER2, wherein the cancer overexpresses HER2, the method comprising the steps of:
(a) determining of a pattern of expression, phosphorylation, or both expression and phosphorylation in a cell or tissue sample from the subject of one or a plurality of polypeptides consisting of:
(i) IGFR polypeptide;
(ii) EGFR polypeptide;
(iii) NDF polypeptide;
(iv) phosphorylated S6 ribosomal polypeptide;
(v) phosphorylated AKT polypeptide;
(vi) phosphorylated ERK polypeptide; and
(b) selecting the subject based on the detected pattern of expression, phosphorylation, or both expression and phosphorylation.
62. The method of claim 61, wherein step (a) comprises determining a pattern of expression, phosphorylation or both expression and phosphorylation in a cell or tissue sample from the subject of (a) IGFR polypeptide, and one or a plurality of polypeptides consisting of:
(b) EGFR polypeptide;
(c) NDF polypeptide;
(d) phosphorylated S6 ribosomal polypeptide;
(e) phosphorylated AKT polypeptide; and
(f) phosphorylated ERK polypeptide.
63. The method of claim 61, wherein the subject is selected when wherein the detected pattern is normal or increased expression of IGFR polypeptide, accompanied by decreased phosphorylation of AKT polypeptide, decreased phosphorylation of S6 ribosomal polypeptide, or both in the mammalian tumor as compared to a non-tumor tissue or cell sample.
64. The method of claim 61, wherein the subject is selected wherein the detected pattern is decreased expression of EGFR polypeptide and decreased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
65. The method of claim 61, wherein the subject is selected when the detected pattern is decreased expression of EGFR polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
66. The method of claim 61, wherein the subject is selected when the detected pattern is decreased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
67. The method of claim 61, wherein the subject is selected when the detected pattern is decreased expression of EGFR polypeptide and increased phosphorylation of ERK polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample.
68. The method of claim 61, wherein the subject is selected when the detected pattern is normal or increased expression of IGFR polypeptide and decreased expression of NDF in the mammalian tumor as compared to a non-tumor tissue or cell sample.
69. The method of claim 61, wherein phosphorylation of AKT polypeptide, phosphorylation of S6 ribosomal polypeptide, or both is determined subsequent to contacting the sample obtained from the mammalian tumor with a HER2-directed therapy.
70. The method of claim 63, wherein phosphorylation of AKT polypeptide, phosphorylation of S6 ribosomal polypeptide, or both is determined subsequent to contacting the sample obtained from the mammalian tumor with a HER2-directed therapy.
71. The method of claim 61, wherein the HER2-directed therapy comprises rhuMAb HER2 (HERCEPTIN®).
72. The method of claim 61, wherein the sample obtained from the mammalian tumor has been contacted with at least one chemotherapeutic.
73. The method of claim 61, wherein the detected pattern of expression, phosphorylation, or both of one or a plurality of polypeptides (i) through (vi) is determined using a biodetection reagents.
74. The method of claim 73, wherein the biodetection reagent is an antibody.
75. The method of claim 73, wherein the biodetection reagent is a nucleic acid probe.
76. The method of claim 61, wherein the detected pattern of phosphorylated AKT polypeptide is determined using an antibody specific for an epitope comprising a phosphorylated serine residue at position 473 in SEQ ID NO: 1.
77. The method of claim 61, wherein the detected pattern of phosphorylated S6 ribosomal polypeptide is determined using an antibody specific for an epitope comprising a phosphorylated serine residue at position 235 in SEQ ID NO: 2.
78. The method of claim 61, wherein the detected pattern of phosphorylated ERK polypeptide is determined using an antibody specific for an epitope comprising a phosphorylated threonine residue at 202 or a phosphorylated serine residue at position 204 in SEQ ID NO: 3.
79. The method of claim 61, wherein the cell or tissue sample is a paraffin-embedded biopsy sample.
80. The method of claim 61, wherein the mammalian tumor is identified as overexpressing HER2 using an antibody that binds HER2 polypeptide.
81. A kit for characterizing a mammalian tumor's responsiveness to a HER2-directed therapy, the kit comprising:
(a) an antibody that binds IGFR polypeptide, and one or more of the following:
(b) an antibody that binds phosphorylated AKT polypeptide;
(c) an antibody that binds phosphorylated S6 ribosomal polypeptide;
(d) an antibody that binds EGFR polypeptide;
(e) an antibody that binds HER2 polypeptide;
(f) an antibody that binds NDF polypeptide; and
(g) an antibody that binds phosphorylated ERK polypeptide.
82. The kit of claim 81, wherein the antibody of (b) is immunologically specific for AKT polypeptide having a phosphorylated serine residue at position 473 in SEQ ID NO: 1;
antibody of (c) is immunologically specific for S6 ribosomal polypeptide having a phosphorylated serine residue at position 235 in SEQ ID NO: 2; and/or antibody of (f) is immunologically specific for EKT polypeptide having a phosphorylated threonine residue at position 202 and a phosphorylated tyrosine at position 204 in SEQ ID NO: 3.
83. The kit of claim 81, wherein the kit further comprises at least one secondary antibody that binds to an antibody of subpart (a) through (g).
Description
  • [0001]
    This application claims priority to U.S. provisional application Ser. No. 60/432,942, filed Dec. 11, 2002.
  • BACKGROUND OF THE INVENTION
  • [0002]
    1. Field of the Invention
  • [0003]
    This invention relates to methods for predicting the response to HER2-directed therapy in an individual.
  • [0004]
    2. Background of the Invention
  • [0005]
    Cellular growth and differentiation processes involve growth factors that exert their actions through specific receptors expressed in the surfaces of responsive cells. Ligands binding to surface receptors, such as those that carry an intrinsic tyrosine kinase activity, trigger a cascade of events that eventually lead to cellular proliferation and differentiation (Carpenter et al., 1979, Biochem., 48: 193-216; Sachs et al., 1987, Cancer Res., 47: 1981-1986). Receptor tyrosine kinases can be classified into several groups on the basis of sequence similarity and distinct features. One of these groups includes the epidermal growth factor receptor family, which includes erbB-1 (also termed EGFR or HER-1) (Carpenter et al., supra); erbB-2 (HER-2/neu) (Semba et al., 1985, Proc. Natl. Acad. Sci., 82: 6497-6501; Coussens et al., 1985, Science, 230: 1130-1139; Bargmann et al., 1986, Cell, Vol. 45, 649-657); erbB-3 (HER-3) (Kraus et al., 1989, Proc. Natl. Acad. Sci., 86: 9193-9197; Carraway et al., 1994, J. Biol. Chem., 269: 14303-14306), and erbB-4 (HER-4) (Plowman et al., 1993, Nature, 366: 473-475; Tzahar et al., 1994, J. Biol. Chem., 269: 25226-25233).
  • [0006]
    Most tumors of epithelial origin express multiple erbB (HER) receptors and co-express one or more EGF-related ligands suggesting that autocrine-receptor activation plays a role in tumor cellular proliferation. Because these ligands activate different erbB/HER receptors, it is possible that multiple erbB receptor combinations might be active in a tumor, a characteristic that could influence its response to an erbB-targeted therapeutic. ErbB receptors form homodimers and heterodimers that can be stimulated by various ligands leading to downstream signaling events, the extent and nature of which depend on the combination of specific dimers and ligands. For example, HER2/neu appears to be the preferred heterodimerization partner with other members of the epidermal growth factor receptor family, but ultimately the dimers formed are determined by the ligand and the erbB receptors expressed in the cell. Not only may the ligand select the dimerization partners, but it may also influence the time course of membrane translocation, activation, and internalization of the receptor. For example, NDF/Heregulin can stimulate tyrosine phosphorylation of erbB-2 through heterodimerization with either receptors erbB-3 or erbB-4 (Peles et al., 1992, Cell 69, 205-216, Peles et al., 1993, EMBO J. 3, 961-71, Holmes et al., 1992, Science 256, 1205-1210; Tzahar et al., 1994, Biol. Chem., 269, 25226-25233; Plowman et al., 1993, Nature 366, 473-475; Pinkas-Kramarski et al., 1994, Proc. Natl. Acad. Sci. USA, 91, 9387-9391; Pinkas-Kramarski et al., 1996, J. Biol. Chem., 271, 19029-19032; Pinkas-Kramarski et al., 1998, Oncogene, 16, 1249-1258). Depending on the cell line studied, NDF/Heregulin can either elicit a growth arrest and differentiation phenotype, resulting in morphological changes, induction of lipids, and expression of intracellular adhesion molecule-1; or it can induce a mitogenic response (Holmes et al., 1992, Science, 256:1205-1210; Peles et al., 1992, Cell, 69:205-216; Bacus et al., 1993, Cancer Res. 53:5251-5261).
  • [0007]
    Downstream signaling after ligand binding may be determined by the set of docking proteins that may bind to the activated receptors. For example, activation of erbB receptor heterodimers is coupled to and stimulates downstream MAPK-Erk1/2 and PI3K-AKT growth and survival pathways, whose deregulation in cancer has been linked to disease progression and refractoriness to therapy (Tzahar et al., 1996, Mol. Cell. Biol. 16, 5276-5287; Fukazawa et al., 1996, J. Biol. Chem. 271, 14554-14559, Olayioye et al., 1998, Mol. Cell. Biol. 18, 5042-5051; Lange et al., 1998, J. Biol. Chem. 273, 31308-31316; Hackel et al., 1999, Curr. Opin. Cell Biol. 11, 184-189). HER-3 is a major docking site for phosphoinositide-3-kinase (PI3K). In addition, NDF/Heregulin stimulation causes activation of the PI3K pathway and phosphorylation of AKT (Altiok et al., 1999, J. Bio. Chem. 274, 32274-32278; Liu et al., 1999, Biochem. Biophys. Res. Comm. 261 897-903; Xing et al., 2000, Nature, Med. 6 189-195). These observations implicate PI3K/AKT in the signaling cascade that results from HER-3 heterodimerization with overexpressed HER-2/neu receptors in breast cancer cells; activation of PI3K/AKT promotes cell survival and enhanced tumor aggressiveness (Bacus et al., 2002, Oncogene 21, 3532-3540). In addition, AKT2 was reported to be activated and overexpressed in HER-2/neu-overexpressing breast cancers (Id.).
  • [0008]
    erbB-2/HER-2 is overexpressed in 20 to 30% of all breast cancers, and its overexpression is associated with poor prognosis, suggesting that it could be used as a target for anti-tumor agents (Slamon et al., 1987; Hudziak et al., 1989; Tagliabue et al., 1991). In erbB-2-overexpressing breast cancer cells, treatment with antibodies specific to HER-2/erbB-2 in combination with chemotherapeutic agents (such as cisplatin, doxoubicin, and taxol) elicits a higher cytotoxic response than treatment with chemotherapy alone (Hancock et al., 1991; Arteaga et al., 1994; Pietras et al., 1994). One possible mechanism by which HER-2/erbB-2 antibodies might enhance cytotoxicity to chemotherapeutic agents is through the modulation of the HER-2/erbB-2 protein expression (Bacus et al., 1992 & 1993; Stancovski et al., 1991; Klapper et al., 1997 & 2000), or by interfering with DNA repair (Arteaga et al., 1994 & 2001; Pietras et al., 1994).
  • [0009]
    Because of the effect of anti-HER-2/erbB-2 antibodies on cellular growth, a number of approaches have been used to therapeutically target HER-2/erbB-2- or EGFR-overexpressing cancers. For clinical use, one approach is to interfere with the kinase activity of the receptor by using inhibitors that block the nucleotide binding site of HER-2/neu or EGFR (Bruns et al., 2000; Christensen et al, 2001, Erlichman et al., 2001, Herbst et al., 2002; Hidalgo et al, 2001; Moasser et al, 2001; Fujimura et al., 2002; Normanno et al., 2002). A second approach is using ansamycins to influence the stability of HER2/neu receptors (Munster et al., 2002; Basso et al, 2002). Another approach is the use of antibodies directed to various erbB receptors, specifically EGFR or HER-2/neu (Alaoui-Jamali et al., 1997; Albanell et al., 2001(a); Baselga et al., 1994 & 2002; Mendelsohn, 1990). Analysis of various antibodies to HER-2/neu led to the identification of the murine monoclonal, 4D5. This antibody recognizes an extracellular epitope (amino acids 529 to 627) in the cysteine-rich II domain that resides very close to the transmembrane region. Treatment of breast cancer cells with 4D5 partially blocks NDF/heregulin activation of HER-2-HER-3 complexes, as measured by receptor phosphorylation assays. To allow for chronic human administration, murine 4D5 was fully humanized to generate trastuzumab/HERCEPTIN® (Sliwkowski et al., 1999; Ye et al., 1999; Carter et al, 1992; Fujimoto-Ouchi et al, 2002; Vogel, et al., 2001 & 2002).
  • [0010]
    A number of monoclonal antibodies and small molecule, tyrosine kinase inhibitors targeting EGFR or erbB-2 have been developed. For example, HERCEPTIN® is approved for treating the 25% of women whose breast cancers overexpress erbB-2 protein or demonstrate erbB-2 gene amplification (Cobleigh et al., 1999, J. Clin. Oncol. 17, 2639-2648). In addition, several EGFR-targeted therapies are currently under clinical investigation (Mendelsohn & Baselga, 2000, Oncogene 19, 6550-6565; Xia et al., 2002, Oncogene 21, 6255-6263).
  • [0011]
    The development of successful oncological drugs has followed a well-established evaluation process including phases I, II, and III clinical trial. Phase I studies aim to determine the maximally tolerated dose of the drug, its optimal schedule of administration and the dose-limiting toxicities. Historically, cytotoxic cancer therapies have been developed based on maximum tolerated doses (MTD), treating patients without understanding the tumor profile for likely responders. Hence, patients were often subjected to toxic therapies with limited therapeutic benefit. Recently, elucidating tumor growth and survival pathways has led to the development of tumor-targeted therapies. For such targeted therapeutics that are not expected to produce serious adverse side effects, relying on a MTD may not be suitable. More relevant may be the determination of the optimal dose and schedule that is sufficient to inhibit cellular signaling in patient samples. Biological assays for signaling biomarkers are needed to establish such a protocol.
  • [0012]
    Preclinically, most erbB-receptor targeted therapies primarily exert cytostatic anti-tumor effects, necessitating their chronic administration in clinical practice. Identification of biologically effective doses (BED), the dose or dose range that maximally inhibits the intended target, beyond which dose escalation is likely to add toxicity without benefit, is therefore essential. Moreover, many of these agents will be used in combination with cytotoxic therapies, where added toxicity may not be tolerable, further supporting BED-based dosing. “Targeted-therapy” implies that populations of likely responders exists, and can be identified.
  • [0013]
    In view of the severe and deleterious consequences of administering an inappropriate or ineffective therapy to a human cancer patient, there exists a need in the art for predicting the response to cancer therapy in an individual. Further, there is a need to develop diagnostics that are capable of predicting patient response for the successful development and clinical acceptance of new HER-2 targeted therapeutics similar to HERCEPTIN®.
  • SUMMARY OF THE INVENTION
  • [0014]
    This invention provides methods for predicting a response of an individual to a HER2-directed therapy.
  • [0015]
    In a first aspect, the invention provides methods for identifying a mammalian tumor that responds to a HER2-directed therapy, wherein the mammalian tumor overexpresses HER2, the method comprising the step of assaying a sample obtained from the mammalian tumor to detect a pattern of expression, phosphorylation or both expression and phosphorylation of one or a plurality of polypeptides consisting of:
  • [0016]
    (a) IGFR polypeptide;
  • [0017]
    (b) EGFR polypeptide;
  • [0018]
    (c) NDF polypeptide;
  • [0019]
    (d) phosphorylated S6 ribosomal polypeptide;
  • [0020]
    (e) phosphorylated AKT polypeptide; and
  • [0021]
    (f) phosphorylated ERK polypeptide;
  • [0022]
    wherein the particular combination of polypeptides and pattern of expression, phosphorylation or both expression and phosphorylation identifies mammalian tumors that respond to a HER2-directed therapy.
  • [0023]
    In certain embodiments, the pattern that identifies a mammalian tumor as responding is decreased expression of IGFR polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In other embodiments, the detected pattern is normal or increased expression of IGFR polypeptide, accompanied by decreased phosphorylation of AKT polypeptide, decreased phosphorylation of S6 ribosomal polypeptide or both in the mammalian tumor as compared to a non-tumor tissue or cell sample. In further embodiments, the detected pattern is normal or increased expression of EGFR polypeptide, accompanied by decreased phosphorylation of ERK polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In additional embodiments, the detected pattern is decreased expression of IGFR polypeptide, accompanied by increased phosphorylation of S6 ribosomal polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In other embodiments, the detected pattern is decreased expression of IGFR polypeptide, accompanied by increased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample; where further the detected pattern can include increased phosphorylation of S6 ribosomal polypeptide.
  • [0024]
    In a second aspect, the invention provides methods for identifying a mammalian tumor that does not respond to a HER2-directed therapy, wherein the mammalian tumor overexpresses HER2, the method comprising the step of assaying a sample obtained from the mammalian tumor to detect a pattern of expression, phosphorylation or both expression and phosphorylation of one or a plurality of polypeptides consisting of:
  • [0025]
    (a) IGFR polypeptide;
  • [0026]
    (b) EGFR polypeptide;
  • [0027]
    (c) NDF polypeptide;
  • [0028]
    (d) phosphorylated S6 ribosomal polypeptide;
  • [0029]
    (e) phosphorylated AKT polypeptide; and
  • [0030]
    (f) phosphorylated ERK polypeptide;
  • [0031]
    wherein the particular combination of polypeptides and pattern of expression, phosphorylation or both expression and phosphorylation identifies mammalian tumors that do not respond to a HER2-directed therapy.
  • [0032]
    In certain embodiments, the pattern that identifies a mammalian tumor as not responding is normal or increased expression of IGFR polypeptide, accompanied by increased phosphorylation of AKT polypeptide, increased phosphorylation of S6 ribosomal polypeptide, or both in the mammalian tumor as compared to a non-tumor tissue or cell sample. In other embodiments, the detected pattern is decreased expression of EGFR polypeptide and increased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In further embodiments, the detected pattern is decreased expression of EGFR polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In other embodiments, the detected pattern is decreased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In additional embodiments, the detected pattern is decreased expression of EGFR polypeptide and increased phosphorylation of ERK polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In further embodiments, the detected pattern is normal or increased expression of IGFR polypeptide and decreased expression of NDF in the mammalian tumor as compared to a non-tumor tissue or cell sample.
  • [0033]
    In a third aspect, the invention provides methods of selecting a subject with cancer for treatment with a molecule targeting HER2, wherein the cancer overexpresses HER2, the methods comprising the steps of:
  • [0034]
    (a) determining the pattern of expression, phosphorylation or both expression and phosphorylation in a cell or tissue sample from the subject of one or a plurality of polypeptides consisting of:
  • [0035]
    (i) IGFR polypeptide;
  • [0036]
    (ii) EGFR polypeptide;
  • [0037]
    (iii) NDF polypeptide;
  • [0038]
    (iv) phosphorylated S6 ribosomal polypeptide;
  • [0039]
    (v) phosphorylated AKT polypeptide; and
  • [0040]
    (vi) phosphorylated ERK polypeptide; and
  • [0041]
    (b) selecting the subject based on the detected pattern of expression, phosphorylation, or both expression and phosphorylation. The particular combination of polypeptides and pattern of expression, phosphorylation or both expression and phosphorylation is used to select the subjects with cancer for treatment with a molecule targeting HER2.
  • [0042]
    In certain embodiments, the detected pattern for selecting a subject for treatment with a molecule targeting HER2 is decreased expression of IGFR polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In other embodiments, the detected pattern is normal or increased expression of IGFR polypeptide, accompanied by decreased phosphorylation of AKT polypeptide, decreased phosphorylation of S6 ribosomal polypeptide or both in the mammalian tumor as compared to a non-tumor tissue or cell sample. In further embodiments, the detected pattern is normal or increased expression of EGFR polypeptide, accompanied by decreased phosphorylation of ERK polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In additional embodiments, the detected pattern is decreased expression of IGFR polypeptide, accompanied by increased phosphorylation of S6 ribosomal polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In other embodiments, the detected pattern is decreased expression of IGFR polypeptide, accompanied by increased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample; where further the detected pattern can include increased phosphorylation of S6 ribosomal polypeptide.
  • [0043]
    In a fourth aspect, the invention provides methods of selecting a subject with cancer to not receive treatment with a molecule targeting HER2, wherein the cancer overexpresses HER2, the methods comprising the steps of:
  • [0044]
    (a) determining the pattern of expression, phosphorylation or both expression and phosphorylation in a cell or tissue sample from the subject of one or a plurality of polypeptides consisting of:
  • [0045]
    (i) IGFR polypeptide;
  • [0046]
    (ii) EGFR polypeptide;
  • [0047]
    (iii) NDF polypeptide;
  • [0048]
    (iv) phosphorylated S6 ribosomal polypeptide;
  • [0049]
    (v) phosphorylated AKT polypeptide; and
  • [0050]
    (vi) phosphorylated ERK polypeptide; and
  • [0051]
    (b) selecting the subject based on the detected pattern of expression, phosphorylation, or both expression and phosphorylation. The particular combination of polypeptides and pattern of expression, phosphorylation or both expression and phosphorylation is used to select the subjects with cancer to not receive treatment with a molecule targeting HER2.
  • [0052]
    In certain embodiments, the detected pattern for selecting a subject not to receive treatment with a molecule targeting HER2 is normal or increased expression of IGFR polypeptide, accompanied by increased phosphorylation of AKT polypeptide, increased phosphorylation of S6 ribosomal polypeptide, or both in the mammalian tumor as compared to a non-tumor tissue or cell sample. In other embodiments, the detected pattern is decreased expression of EGFR polypeptide and increased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In further embodiments, the detected pattern is decreased expression of EGFR polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In other embodiments, the detected pattern is decreased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In additional embodiments, the detected pattern is decreased expression of EGFR polypeptide and increased phosphorylation of ERK polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample. In further embodiments, the detected pattern is normal or increased expression of IGFR polypeptide and decreased expression of NDF in the mammalian tumor as compared to a non-tumor tissue or cell sample.
  • [0053]
    In various aspects of the invention, including those mentioned above, the detection of phosphorylation of AKT polypeptide, phosphorylation of S6 ribosomal polypeptide, or both can determined subsequent to contacting the sample obtained from the mammalian tumor with a HER2-directed therapy. Further, the HER2-directed therapy can be or comprise rhuMAb HER2 (HERCEPTIN®). In addition, the sample can be contacted with at least one chemotherapeutic agent. Further, the detected pattern of expression, phosphorylation, or both, of one or a plurality of polypeptides (a) through (f) can be determined using a biodetection reagent. The biodetection reagent can be an antibody or a nucleic acid probe. Further, the detected pattern of phosphorylated AKT polypeptide can be determined using an antibody specific for an epitope comprising a phosphorylated serine residue at position 473, the detected pattern of phosphorylated S6 ribosomal polypeptide can be determined using an antibody specific for an epitope comprising a phosphorylated serine residue at position 235, and/or the detected pattern of phosphorylated ERK polypeptide can be determined using an antibody specific for an epitope comprising a phosphorylated threonine residue at position 202 and a phosphorylated tyrosine residue at position 204. Further, the sample obtained from the mammalian tumor can be a paraffin-embedded biopsy sample. Also, the mammalian tumor can be identified as overexpressing HER2 using an antibody that binds HER2 polypeptide.
  • [0054]
    In a fifth embodiment, the invention provides kits for characterizing a mammalian tumor's responsiveness to a HER2-directed therapy, the kit comprising:
  • [0055]
    (a) an antibody that binds IGFR polypeptide, and one or more of the following:
  • [0056]
    (b) an antibody that binds phosphorylated AKT polypeptide;
  • [0057]
    (c) an antibody that binds phosphorylated S6 ribosomal polypeptide;
  • [0058]
    (d) an antibody that binds EGFR polypeptide;
  • [0059]
    (e) an antibody that binds HER2 polypeptide;
  • [0060]
    (f) an antibody that binds NDF polypeptide; and
  • [0061]
    (g) an antibody that binds phosphorylated ERK polypeptide.
  • [0062]
    In certain embodiments, the antibody of (b) is immunologically specific for AKT polypeptide having a phosphorylated serine residue at position 473; antibody of (c) is immunologically specific for S6 ribosomal polypeptide having a phosphorylated serine residue at position 235; and/or the antibody of (f) is immunologically specific for EKT polypeptide having a phosphorylated threonine residue at position 202 and a phosphorylated tyrosine at position 204. In other embodiments, the kit further comprises at least one secondary antibody that binds to an antibody of subpart (a) through (g).
  • [0063]
    Specific embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.
  • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • [0064]
    This invention provides methods for predicting response in cancer subjects to cancer therapy, including human cancer patients. In addition, this invention provides predictive biomarkers to identify the cancer patients for whom the administering a therapeutic agent will be most effective, including a therapeutic agent for treating breast cancer. Specifically, this invention provides predictive biomarkers for assessing the efficacy of therapeutic agents targeted to Her2/neu, including such agents such as HERCEPTIN®.
  • [0065]
    In contrast to traditional anticancer methods, where chemotherapeutic drug treatment is undertaken as an adjunct to and after surgical intervention, neoadjuvant (or primary) chemotherapy consists of administering drugs as an initial treatment in certain cancer patients. One advantage of such an approach is that, for primary tumors of more than 3 cm, it permits the later or concomitant use of conservative surgical procedures (as opposed to, e.g., radical mastectomy in breast cancer patients) for the majority of patients, due to the tumor shrinking effect of the chemotherapy. Another advantage is that for many cancers, a partial and/or complete response is achieved in about two-thirds of all patients. Finally, because the majority of patients are responsive after two to three cycles of chemotherapeutic treatment, it is possible to monitor the in vivo efficacy of the chemotherapeutic regimen employed, in order to identify patients whose tumors are non-responsive to chemotherapeutic treatment. Timely identification of non-responsive tumors allows the clinician to limit a cancer patient's exposure to unnecessary side-effects of treatment and to institute alternative treatments. Unfortunately, methods present in the art, including histological examination, are insufficient for such a timely and accurate identification. The present invention provides methods for developing more informed and effective regimes of therapy that can be administered to cancer patients with an increased likelihood of an effective outcome (i.e., reduction or elimination of the tumor).
  • [0066]
    A cancer diagnosis, both an initial diagnosis of disease and subsequent monitoring of the disease course (before, during, or after treatment) is conventionally confirmed through histological examination of cell or tissue samples removed from a patient. Clinical pathologists need to be able to accurately determine whether such samples are benign or malignant and to classify the aggressiveness of tumor samples deemed to be malignant, because these determinations often form the basis for selecting a suitable course of patient treatment. Similarly, the pathologist needs to be able to detect the extent to which a cancer has grown or gone into remission, particularly as a result of or consequent to treatment, most particularly treatment with chemotherapeutic or biological agents.
  • [0067]
    Histological examination traditionally entails tissue-staining procedures that permit morphological features of a sample to be readily observed under a light microscope. A pathologist, after examining the stained sample, typically makes a qualitative determination of whether the tumor sample is malignant. It is difficult, however, to ascertain a tumor's aggressiveness merely through histological examination of the sample, because a tumor's aggressiveness is often a result of the biochemistry of the cells within the tumor, such as protein expression or suppression and protein phosphorylation, which may or may not be reflected by the morphology of the sample. Therefore, it is important to be able to assess the biochemistry of the cells within a tumor sample. Further, it is desirable to be able to observe and quantitate both gene expression and protein phosphorylation of tumor-related genes or proteins, or more specifically cellular components of tumor-related signaling pathways.
  • [0068]
    Cancer therapy can be based on molecular profiling of tumors rather than simply their histology or site of the disease. Elucidating the biological effects of targeted therapies in tumor tissue and correlating these effects with clinical response helps identify the predominant growth and survival pathways operative in tumors, thereby establishing a pattern of likely responders and conversely providing a rational for designing strategies to overcome resistance. Successful diagnostic targeting of a growth factor receptor must determine if tumor growth or survival is being driven by the targeted receptor or receptor family, by other receptors not targeted by the therapy, and whether downstream signaling suggests that another oncogenic pathway is involved.
  • [0069]
    For subjects considered for treatment with HERCEPTIN®, it is necessary to consider additional biomarkers beyond the presence of the target HER-2/neu, at least because the status of the EGFR and erbB ligands NDF and TGF-α affect HERCEPTIN® therapy response in breast cancer patients. Therefore, considering HER2/neu expression alone does not necessarily predict overall erbB oncogenic activity or potential response to erbB inhibitors. In addition, previous studies have shown that not all tumor cells respond to inhibition of ErbB receptors, despite exhibiting aberrant EGFR and/or HER2/neu expression. Examples include the MKN7 and BT474 erbB receptor-overexpressing carcinoma cell lines: BT474 cells respond to HERCEPTIN® but MKN7 cells do not (Motoyama, et al., Cancer Research, 62, 3151-3158 (2002)). In addition, the proliferation block induced as a consequence of decreased EGFR or HER2 receptor activity, such as by the activity of an erbB-inhibitor, may be overcome by the presence of EGF-related ligands such as EGF or NDF/Heregulin (Id). This phenomenon can be attenuated using a bispecific ErbB-1/ErbB-2 inhibitor, thus supporting increased antitumor efficacy through simultaneous inhibition of multiple ErbB receptors.
  • [0070]
    In addition, in many cancers NDF/Heregulin or TGF-α expression produces an autocrine loop of HER-2/EGFR heterodimerization. Downregulation of HER-2/neu expression is an important way of inhibiting signals generated by these heterodimers. Downregulation can be accomplished by treatment with HERCEPTIN®, which produces receptor endocytosis. Furthermore, high levels of phosphorylated ERK (or pAKT) can be a negative predictor for positive treatment outcome, when observed in conjunction with the expression of EGFR and the presence of NDF, suggesting the existence of other pathways that might promote proliferation of the tumor cellular growth. High pERK is also associated with resistance to HERCEPTIN® through downregulation of p27; this may implicate other signals (such as estrogen receptor's cross activation of the MAPK and AKT pathways) that may contribute to high pERK and thus contribute to proliferation of the tumor cells growth. In addition, phosphorylated AKT has been shown to be an important part of the response to HERCEPTIN®, as high pAKT-positive patients had poor response to HERCEPTIN®. High-phosphorylated AKT has been shown to be associated with high expression of insulin like growth factor receptors (IGFR-1) as well as PDGFR and results in resistance to HERCEPTIN®. Interestingly, data from clinical trials have shown that using a dual inhibitor (i.e., specific for HER-1/neu and HER-2/neu) has clinical efficacy in patients when treatment induced downregulation of pERK and pAKT, but not in patients in which pERK and pAKT levels didn't diminish after treatment. Thus, in those patients who overexpressed HER-1 and HER-2, as well as pERK and pAKT, antitumor activity was dependent on HER-1 and HER-2 receptor activation and a clinical response was observed. In contrast, in patients for whom pERK and pAKT activity remained high after treatment with a dual inhibitor, clinical response didn't occur. Combination therapies can have clinical significance. A combination of the ErbB-1-directed monoclonal antibodies mAb 225 and mAb 4D5 inhibited proliferation of an ovarian tumor cell line more strongly than either mAb alone (Ye et al., 1999, Oncogene 18: 731-8). In addition to ErbB-targeted mAbs, a number of different ErbB-1/ErbB-2-bispecific inhibitors, also referred to as dual EGFR/erbB-2 kinase inhibitors, have been described recently, such as GW572016 and PKI166, that are currently in clinical trials (Motoyama et al., 2002, Cancer Research 62: 3151-3158). Therefore, response to HERCEPTIN® is affected by the expression of multiple erbB receptors and their ligands in tumors.
  • [0071]
    Thus, HER-2/neu overexpression alone is not the only predictor of response to molecules such as HERCEPTIN®). HER-2/neu is an orphan, ligandless receptor in need of its partners EGFR (HER-1) and HER-3 in order to exert its activity. HER-1 and HER-3 heterodimerization with HER-2 is enhanced by the presence of EGF or NDF (Tzahar et al., 1996, EMBO J. 15: 254-64, Graus-Porta, 1997, EMBO J, 16 1647-55), and thus HER-2 activity is dependant on HER-1 or HER-3. Other receptors may also transactivate the erbB receptors. These receptors may be mediating tumorigenesis through signaling to downstream proliferative and survival pathways. For example, the IGFR receptor may mediate patient response to breast cancer therapies targeting HER2/neu. High IGFR expression combined with high S6 ribosomal protein phosphorylation correlates with poor patient response regardless of erb-B expression, indicating that IGFR acts directly to activate signaling downstream of erb-B receptors rather than through transactivation of erb-B receptors. Cell line studies also have suggested a role for IGFR in patient response. HERCEPTIN® resistance has been suggested to occur though activation of IGFR (Lu et al., 2001, J National Cancer Institute 93: 1852). In addition, co-targeting IGFR as well as HER2/neu has been shown to produce synergistic inhibition of growth in breast cancer cells (Camirand et al., 2002, Med Sci Monit. 8: (12): BR521-6). Therefore, analysis of IGFR expression and downstream signaling can be critical for an accurate assessment of potential HERCEPTIN® response in breast cancer patients.
  • [0072]
    Thus, there is no one marker of downstream signaling protein activation that would integrate multiple upstream signals and predict response. However, analysis of p-ERK and p-AKT has been found to be predictive in patients over-expressing EGFR. Therefore, in the presence of active erbB receptors, high ERK and AKT signaling indicates that HERCEPTIN® therapy is less likely to be effective. AKT activation has been shown to result in HERCEPTIN® resistance in breast cancer cell lines (Yakes, et al., 2002, Cancer Res. 62: 4132-41; Clark et al., 2202, Mol. Cancer Ther. 1: 707-17). In addition, analysis of S6 ribosomal protein phosphorylation greatly increased the predictive power of IGFR expression. In patients with high S6 phosphorylation, positive response ranged from 8% to 67% based upon IGFR expression. Approximately 30% of patients with low S6 phosphorylation responded, regardless of IGFR expression. These results were also reflected in an analysis of clinical samples, in which only those patients that lacked active IGFR signaling responded to HERCEPTIN® therapy. IGF signaling in breast cancer occurs through AKT activation (Oh et al., 2002, Neoplasia 4: 204-17; Dufourny et al., 1997, J. Biol. Chem. 272: 31163-71), which leads to S6 ribosomal protein phosphorylation. Hence, S6 phosphorylation can be indicative of active IGF signaling in those tumors over-expressing IGFR.
  • [0073]
    Analysis of down-stream signaling and patient response is complicated when chemotherapy and radiotherapy therapy is included in addition to HERCEPTIN® treatment. AKT and MAP kinase pathway activation, for example, are known to play a role in response to DNA-damaging agents (Clark et al., 2002, Mol. Cancer Ther. 1: 707-17; Bacus et al., 2001, Oncogene 20: 147-155). Consideration of downstream signaling in patients undergoing a combination of therapies provides additional predictive information not obtained solely from analysis of receptor or ligand expression levels. Analysis of patients treated with HERCEPTIN® as a single agent therapy can be used to determine which of the identified biomarkers mediated the response to Herceptin®, as opposed to the biomarkers that mediate the response to the other therapies. Nonetheless, the identified biomarkers are useful, among other things, for designing diagnostics for breast cancer patients undergoing the common HERCEPTIN® combination therapies.
  • [0074]
    Further, up-regulation of the AKT/mTOR pathway by Heregulin/NDF is an important predictor for response. pAKT has been associated with high levels of Cyclin E and low levels of the cyclin inhibitor p27.
  • [0075]
    Before administration of HER2-targeted therapies, a panel of diagnostics of each tumor is used according to the methods of this invention to find the best candidate for each therapy. According to the methods of this invention, treatment by a HER2-targeted therapy, such as HERCEPTIN®, is effective when a patient's tumor growth depends on a cellular pathway such as AKT/mTOR that is driven by the erbB receptors and not by other tyrosine kinases, such as Insulin-like Growth Factor Receptors (IGFR). When high levels of activation of these downstream signals occur independent of erbB receptors, HERCEPTIN® treatment is not effective. Use of the methods of this invention permits a clinician to choose a more effective combination of targeted therapies for cancer patients.
  • [0076]
    The HER2-directed therapies of the present invention can include, for example, rhuMAb HER2, otherwise known as HERCEPTIN®. The samples obtained from the mammalian tumor can be contacted with at least one chemotherapeutic agent, for example cisplaint, doxorubicin, or taxol.
  • [0077]
    Automated (computer-aided) image analysis systems known in the art can augment visual examination of tumor samples. In a representative embodiment, the cell or tissue sample is exposed to detectably-labeled reagents specific for a particular biological marker, and the magnified image of the cell is then processed by a computer that receives the image from a charge-coupled device (CCD) or camera such as a television camera. Such a system can be used, for example, to detect and measure expression and activation levels of EGFR, HER2, HER3, pERK, NDF, TGF-α, IGFR, pS6, and pAKT in a sample, or any additional diagnostic biomarkers. Thus, the methods of the invention provide more accurate cancer diagnosis and better characterization of gene expression in histologically identified cancer cells, most particularly with regard to expression of tumor marker genes or genes known to be expressed in particular cancer types and subtypes (e.g., having different degrees of malignancy). This information permits a more informed and effective regimen of therapy to be administered, because drugs with clinical efficacy for certain tumor types or subtypes can be administered to patients whose cells are so identified.
  • [0078]
    Another drawback of conventional anticancer therapies is that the efficacy of specific chemotherapeutic agents in treating a particular cancer in an individual human patient is unpredictable. In view of this unpredictability, the art is unable to determine, prior to starting therapy, whether one or more selected agents would be active as anti-tumor agents or to render an accurate prognosis of course of treatment in an individual patient. This is especially important because a particular clinical cancer may present the clinician with a choice of treatment regimens, without any current way of assessing which regimen will be most efficacious for a particular individual. It is an advantage of the methods of this invention that they are able to better assess the expected efficacy of a proposed therapeutic agent (or combination of agents) in an individual patient. The claimed methods are advantageous for the additional reasons that they are both time- and cost-effective in assessing the efficacy of chemotherapeutic regimens and are minimally traumatic to cancer patients.
  • [0079]
    Methods of this invention can be used to identify a mammalian tumor that responds to HER-2 directed therapies. Further, methods of this invention can be used to select a subject with cancer for treatment with a molecule targeting HER. Moreover, methods of this invention can be used to identify a mammalian tumor that does not respond to HER-2 directed therapies. Further, methods of this invention can be used to select a subject with cancer to not receive treatment with a molecule targeting HER2.
  • [0080]
    Patterns of expression and phosphorylation of polypeptides are detected and quantified using methods of the present invention. More particularly, patterns of expression and phosphorylation of polypeptides that are cellular components of a tumor-related signaling pathway are detected and quantified using methods of the present invention. For example, the patterns of expression and phosphorylation of polypeptides can be detected using biodetection reagents specific for the polypeptides. For example, the biodetection reagents can be antibodies. Alternatively, the biodetection reagents can be nucleic acid probes. A nucleic acid probe is defined to be a collection of one or more nucleic acid fragments whose hybridization to a sample can be detected. The probe may be unlabeled or labeled so that its binding to the target or sample can be detected. The probe is produced from a source of nucleic acids from one or more particular (preselected) portions of the genome, e.g., one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products. The nucleic acid probe may also be isolated nucleic acids immobilized on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides), as in an array. The probe may be a member of an array of nucleic acids as described, for instance, in WO 96/17958. Techniques capable of producing high density arrays can also be used for this purpose (see, e.g., Fodor (1991) Science 767-773; Johnston (1998) Curr. Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23: 120-124; U.S. Pat. No. 5,143,854). One of skill will recognize that the precise sequence of the particular probes can be modified to a certain degree to produce probes that are “substantially identical,” but retain the ability to specifically bind to (i.e., hybridize specifically to) the same targets or samples as the probe from which they were derived. The term “nucleic acid” refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides that have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. The term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved for the purposes desired. The term also encompasses nucleic-acid-like structures with synthetic backbones. One of skill in the art would recognize how to use a nucleic acid probes for screening of cancer cells in a sample by reference, for example, to U.S. Pat. No. 6,326,148, directed to screening of colon carcinoma cells.
  • [0081]
    Polypeptides associated with breast cancer can be quantified by image analysis using a suitable primary antibody against biomarkers, including but not limited to EGFR, HER-2, HER-3, IGFR, NDF, TGF-α, p-ERK, pS6, or p-AKT, detected directly or using an appropriate secondary antibody (such as rabbit anti-mouse IgG when using mouse primary antibodies) and/or a tertiary avidin (or Strepavidin) biotin complex (“ABC”).
  • [0082]
    Examples of reagents useful in the practice of the methods of the invention as exemplified herein include antibodies specific for HER2/neu, including but not limited to the mouse monoclonal antibody CB11, obtained from Ventana Medical Systems, Inc. (VMSI, Tucson, Ariz.). In addition, reagents useful in the practice of the methods of the invention include antibodies specific for phosphorylated AKT, including but not limited to antibodies specific for a phosphorylated serine residue of position 473, wherein the sequence of AKT is represented by SEQ ID NO: 1 (Table 8). Further, reagents useful in the practice of the methods of the invention include antibodies specific for phosphorylated S6, including but not limited to antibodies specific for a phosphorylated serine residue of position 235, wherein the sequence of S6 is represented by SEQ ID NO:2 (Table 8). Also, reagents useful in the practice of the methods of the invention include antibodies specific for phosphorylated ERK, including but not limited to antibodies specific for a phosphorylated threonine residue at position 202 and a phosphorylated tyrosine residue of position 204, wherein the sequence of ERK is represented by SEQ ID NO:3 (Table 8).
  • [0083]
    Further, the pattern of expression, phosphorylation, or both expression and phosphorylation of the predictive polypeptides can be compared to a non-tumor tissue or cell sample. The non-tumor tissue or cell sample can be obtained from a non-tumor tissue or cell sample from the same individual, or alternatively, a non-tumor tissue or cell sample from a different individual. A detected pattern for a polypeptide is referred to as decreased in the mammalian tumor, tissue, or cell sample, if there is less polypeptide detected as compared to the a non-tumor tissue or cell sample. A detected pattern for a polypeptide is referred to as increased in the mammalian tumor, tissue, or cell sample, if there is more polypeptide detected as compared to the a non-tumor tissue or cell sample. A detected pattern for a polypeptide is referred to as normal in the mammalian tumor, tissue, or cell sample, if there is the same, or approximately the same, polypeptide detected as compared to the a non-tumor tissue or cell sample.
  • [0084]
    The methods of this invention for identifying mammalian tumors that respond, or that do not respond, to a HER2-directed therapy comprise the step of assaying a sample obtained from the mammalian tumor to detect a pattern of expression, phosphorylation or both of one or a plurality of polypeptides consisting of: (a) IGFR polypeptide; (b) EGFR polypeptide; (c) NDF polypeptide; (d) phosphorylated S6 ribosomal polypeptide; (e) phosphorylated AKT polypeptide; (f) phosphorylated EKT polypeptide. The combination of polypeptides and pattern of expression, phosphorylation, or both expression and phosphorylation identifies mammalian tumors that respond, or that do not respond, to a HER2-directed therapy. The methods can include the detection of a pattern of expression, phosphorylation or both of one, two, three, four, five, or all six of these polypeptides. Further, the methods can, but need not, include other steps, including steps such as the detection of a pattern of expression, phosphorylation or both of different polypeptides.
  • [0085]
    The methods of this invention for selecting a subject with cancer for treatment, or to not receive treatment, with a molecule targeting HER2, such as, but not limited to treatment with HERCEPTIN®, comprise the step of determining the pattern of expression, phosphorylation or both in a cell or tissue sample from the subject of one or a plurality of polypeptides consisting of: (a) IGFR polypeptide; (b) EGFR polypeptide; (c) NDF polypeptide; (d) phosphorylated S6 ribosomal polypeptide; (e) phosphorylated AKT polypeptide; (f) phosphorylated EKT polypeptide. The combination of polypeptides and pattern of expression, phosphorylation, or both expression and phosphorylation is used to select a subject with cancer for treatment, or to not receive treatment, with a molecule targeting HER2. The methods can include the detection of a pattern of expression, phosphorylation or both of one, two, three, four, five, or all six of these polypeptides. Further, the methods can, but need not, include other steps, including steps such as the detection of a pattern of expression, phosphorylation or both of different polypeptides.
  • [0086]
    For example, the pattern that identifies a mammalian tumor as responding or that can be used to select a subject with cancer for treatment with a molecule targeted to HER2 is decreased expression of IGFR polypeptide as compared to a non-tumor tissue or cell sample. Alternatively, the detected pattern is normal or increased expression of IGFR polypeptide, accompanied by decreased phosphorylation of AKT polypeptide, decreased phosphorylation of S6 ribosomal polypeptide or both as compared to a non-tumor tissue or cell sample. Another potential detected pattern is normal or increased expression of EGFR polypeptide, accompanied by decreased phosphorylation of ERK polypeptide as compared to a non-tumor tissue or cell sample. Further detected patterns include decreased expression of IGFR polypeptide, accompanied by increased phosphorylation of S6 ribosomal polypeptide as compared to a non-tumor tissue or cell sample. In other embodiments, the detected pattern is decreased expression of IGFR polypeptide, accompanied by increased expression of NDF polypeptide in the mammalian tumor as compared to a non-tumor tissue or cell sample; where further the detected pattern can include increased phosphorylation of S6 ribosomal polypeptide. These identified patterns are understood to be non-limiting.
  • [0087]
    For example, the pattern that identifies a mammalian tumor as not responding or that can be used to select a subject with cancer to not receive treatment with a molecule targeted to HER2 is normal or increased expression of IGFR polypeptide, accompanied by increased phosphorylation of AKT polypeptide, increased phosphorylation of S6 ribosomal polypeptide, or both as compared to a non-tumor tissue or cell sample. Alternatively, the detected pattern is decreased expression of EGFR polypeptide and increased expression of NDF polypeptide as compared to a non-tumor tissue or cell sample. Or, the detected pattern is decreased expression of EGFR polypeptide as compared to a non-tumor tissue or cell sample. Further, the detected pattern is decreased expression of NDF polypeptide as compared to a non-tumor tissue or cell sample. Or, the detected pattern is decreased expression of EGFR polypeptide and increased phosphorylation of ERK polypeptide as compared to a non-tumor tissue or cell sample. Further, the detected pattern is normal or increased expression of IGFR polypeptide and decreased expression of NDF as compared to a non-tumor tissue or cell sample. These identified patterns are understood to be non-limiting.
  • [0088]
    In practicing the methods of this invention, staining procedures can be carried out by a person, such as a technician in the laboratory. Alternatively, the staining procedures can be carried out using automated systems. In either case, staining procedures for use according to the methods of this invention are preformed according to standard techniques and protocols well-established in the art.
  • [0089]
    By “cell or tissue sample” is meant biological samples comprising cells, most preferably tumor cells, that are isolated from body samples, such as, but not limited to, smears, sputum, biopsies, secretions, cerebrospinal fluid, bile, blood, lymph fluid, urine and faeces, or tissue which has been removed from organs, such as breast, lung, intestine, skin, cervix, prostate, and stomach. For example, a tissue samples can comprise a region of functionally related cells or adjacent cells.
  • [0090]
    The amount of target protein is advantageously quantified by measuring the average optical density of the stained antigens. Concomitantly, the proportion or percentage of total tissue area stained can be readily calculated, for example as the area stained above a control level (such as an antibody threshold level) in the second image. Following visualization of nuclei containing biomarkers, the percentage or amount of such cells in tissue derived from patients after treatment are compared to the percentage or amount of such cells in untreated tissue. For purposes of the invention, “determining” a pattern of expression, phosphorylation, or both expression and phosphorylation o polypeptides is understood broadly to mean merely obtaining the information on such polypeptide(s), either through direct examination or indirectly from, for example, a contract diagnostic service.
  • [0091]
    Breast cancer tissue sections taken from patients treated with HERCEPTIN® and chemotherapy are analyzed, according to the methods of this invention by immunohistochemistry for expression, phosphorylation, or expression and phosphorylation of erb-B ligands, receptors, downstream signaling proteins or any positive treatment response predictive combination thereof. These measurements can be accomplished, for example, by using tissue microarrays. Tissue microarrays are advantageously used in the methods of the invention, being well-validated method to rapidly screen multiple tissue samples under uniform staining and scoring conditions. (Hoos et al., 2001, Am J Pathol. 158: 1245-51). Scoring of the stained arrays can be accomplished by an automated system that accurately quantified the staining observed. The results of this analysis identify biomarkers that best predict patient outcome following treatment, such as HERCEPTIN® therapies. Patient “probability of response” ranging from 0 to 100 percent can be predicted based upon the expression, phosphorylation or both of a small set of ligands, receptors, signaling proteins or predictive combination thereof. Additional samples from breast cancer patients can be analyzed, either as an alternative to or in addition to tissue microarray results. For example, analysis of samples from breast cancer patients can confirm the conclusions from the tissue arrays, if the patient's responses correlate with a specific pattern of receptor expression and/or downstream signaling.
  • [0092]
    The invention provides, in part, kits for carrying out the methods of the invention. For example, the method provides kits for characterizing a mammalian tumor's responsiveness to a HER2-directed therapy comprising an antibody that binds IGFR polypeptide, and one or more of the following: an antibody that binds phosphorylated AKT polypeptide; an antibody that binds phosphorylated S6 ribosomal polypeptide; an antibody that binds EGFR polypeptide; an antibody that binds HER2 polypeptide; an antibody that binds NDF polypeptide; and an antibody that binds phosphorylated ERK polypeptide. In addition to an antibody that binds IGFR polypeptide, the kit can include one, two, three, four, or all five of the following: an antibody that binds phosphorylated AKT polypeptide; an antibody that binds phosphorylated S6 ribosomal polypeptide; an antibody that binds EGFR polypeptide; an antibody that binds HER2 polypeptide; an antibody that binds NDF polypeptide; and an antibody that binds phosphorylated ERK polypeptide. Further, the kit can include additional components other then the above-identified antibodies, including but not limited to additional antibodies. Such kits may be used, for example, by a clinician or physician as an aid to selecting an appropriate therapy for a particular patient, for example, a breast cancer patient under consideration for HER2-directed therapy.
  • [0093]
    Particularly useful embodiments of the present invention and the advantages thereof can be understood by referring to Examples 1-5. These Examples are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
  • EXAMPLE 1 Staining Procedure for Biomarkers
  • [0094]
    Human tumor tissue sections were stained for predictive biomarkers according to the methods of the invention as follows. 10% Neutral Buffered Formalin Paraffin blocks were sectioned at 4 microns and the sections placed onto coated slides. EGFR and HER2 immunostaining was performed by using the pre-diluted EGFR and HER2 antibodies from Ventana Medical Instruments, Inc. (VMSI, Tucson, Ariz.). HER3, Heregulin (NDF), and IGFR antibodies were obtained from NeoMarkers (Fremont, Calif.). TGF-α antibodies were obtained from Oncogene Sciences (San Diego, Calif.). EGFR, HER2/neu, HER3, IGFR, Heregulin, and TGF-α were immunostained using the “BenchMark” (VMSI) with I-VIEW (VMSI) detection chemistry. Antibodies specific for p-ERK (1:100), p-AKT (1:75), and phospho-S6 ribosomal protein were obtained from Cell Signaling Technology (Beverly, Mass.), and immunostained using a labeled streptavidin peroxidase technique. (Vector Elite ABC Kit, Burlingame, Calif.). Prior to staining, slides for p-S6 ribosomal protein, p-ERK and p-AKT were antigen retrieved using 0.1 M citrate buffer, pH 6.0 in the “decloaker” (Biocare Corp.) and the sections incubated overnight with the primary antibodies at 4° C. The next day, the slides were placed onto the Autostainer (Dako Corp.) and the “LSAB2” kit (Dako) was employed as the detection chemistry. DAB (Dako) was used as the chromogen. After immunostaining, all slides were counterstained manually with 4% ethyl green (Sigma).
  • EXAMPLE 2 Procedure for Western Blot Analysis
  • [0095]
    Western blot analysis for detecting expression of predictive markers was performed as follows. Cells were lysed in ice-cold buffer (50 mM Tris-HCl (pH 7.5), 137 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.2% Triton X-100, 10% glycerol, 0.1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 20 mM β-glycerophosphate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 2 μM leupeptin, and 2 μg/ml aprotinin). Protein concentration was determined with a BioRad Protein Assay Kit (BioRad Laboratories, Hercules, Calif.). Equal amounts of protein, typically 15 μg protein per lane, were separated by gel electrophoresis, for example using pre-cast 4-12% Bis-Tris NuPage gradient gels (Invitrogen) or 7.5% or 4-15% gradient SDS-PAGE under reducing conditions, and transferred to membranes, such as HyBond-C nitrocellulose (Amersham Life Science) or Immobilon-P membranes. Membranes were blocked and then incubated with primary antibodies, for example antibodies against p-AKT and p-ERK (Cell Signaling Technology). Antibody incubation was performed overnight at 4° C. in Tris-buffered saline containing 3% bovine serum albumin/0.1% Tween 20. Signal was detected by chemiluminescence (PerkinElmer Life Sciences), or using a SuperSignal West Femto Maximum sensitivity substrate kit from Pierce (Rockford, Ill.) as described (Xia et al., 2002, Oncogene 21: 6255-6263).
  • EXAMPLE 3 Procedure for Immunohistochemistry
  • [0096]
    Immunohistochemistry for detecting and measuring predictive biomarker expression, activation or both was performed as follows. HER2/neu, EGFR, HER3, IGFR, TGF-α, Heregulin (NDF), p-ERK, p-AKT, and p-S6 ribosomal protein or phosphorylation levels were quantified using alkaline phosphatase or peroxidase techniques and microscope-based image analysis of immunohistochemically stained slides (as described in Bacus et al., 1997, Analyt. Quant. Cytol. Histol. 19: 316-328). Quantification was by means of a CAS 200 image analyzer, as previously described (Bacus & Ruby, 1993, Pathol Annu, 28: 179-204). For the purpose of the analysis, tumors were classified as negative or positive for each antibody based upon the level of staining. Statistical analysis was performed using Systat to quantify frequencies and calculate Pearson Chi-squared tests of significance for interactions between variables. In all cases, the p value refers to the significance of the deviation of the distribution of samples from what would be expected based upon the overall population distribution. Comparisons were performed only on samples for which all relevant data were available. As a result, the number of patients included in most comparisons was slightly less then the total number of available samples.
  • [0097]
    Quantitative immunohistochemistry (IHC) was performed as previously described (Bacus et al., 1997, Analyt. Quant. Cytol. Histol. 19: 316-328). EGFR, and erbB-2 (HER2) immunostaining was performed using pre-diluted EGFR, and erbB-2 (HER2) antibodies from Ventana Medical Systems, Inc. (VMSI, Tucson, Ariz.) on the VMSI automated “BenchMark” staining module as described. The VMSI “I-View” detection kit was used for all three of the VMSI pre-diluted primary antibodies according to the manufacturer's instructions. Antibodies to erbB-3 (1:10), Heregulin (1:25), and TGF-a (1:20), were also used for immunostaining using the “BenchMark” with I-VIEW detection chemistry. Antibodies to Phospho-Erk (1:100) and p-AKT (1:75) were used for immunostaining using a labeled streptavidin peroxidase technique as described by the manufacturer. Phospho-Erk and p-AKT slides were antigen retrieved as described by Bacus et al. (1997, Analyt. Quant. Cytol. Histol. 19: 316-328). Slides were placed onto the Autostainer (Dako Corp.) and the “LSAB2” kit (Dako) employed as the detection chemistry. After staining, all slides were counterstained manually with 4% ethyl green (Sigma). Investigators preparing and analyzing tissue sections were blinded to both patient tumor type and response to therapy.
  • [0098]
    For IHC, antibodies to EGFR and erbB-2 were from Ventana Medical Scientific Instruments (VMSI) (Tucson, Ariz.); anti-p-AKT (Ser 437) and p-Erk1/2 were from Cell Signaling Technology Inc. (Beverly, Mass.); antibodies to TGFα, erbB3, heregulin, and IGFR-1 were from NeoMarkers.
  • EXAMPLE 4 Analysis of Breast Cancer Tissue Microarrays
  • [0099]
    Tissue microarrays derived from 250 breast cancer patients who received conventional chemotherapy together with HERCEPTIN® were obtained from Clinomics Biosciences (Pittsfield, Mass.). The histology of the tumors varied, with infiltrating ductal carcinoma being the most common. All patients had received post-surgical radiotherapy. The tissue samples in the array were taken before treatment. HER2/neu expression had been determined by using the HercepTest system (DAKO, Caprintera, Calif.) on the original biopsies for all patients. Patient response was based upon the case histories at last follow-up as decided by an independent pathologist provided by Clinomics.
  • [0100]
    Demographics of these patients are reported in Table 1. The great majority of patients had infiltrating ductal carcinomas and received anthracycline plus cyclophosphamide. Fifty-seven of the patients had metastatic diseases. All patients had received a 4 mg/kg HERCEPTIN® loading dosage and a 2 mg/kg weekly maintenance dosage.
  • [0101]
    From the original tissue arrays of 250 patients, seventy-five samples were not used in the analysis because of the lack of clinical data or because the sections did not contain useable tumor tissue. Overall, 15% of the remaining patients were disease free or had stable disease after therapy, while 85% relapsed. Of these remaining one hundred and seventy five patients, twenty-eight samples lacked HercepTest results and were therefore also excluded from further analysis. Of the samples for which HercepTest results were obtained, seventy-seven had a HercepTest score of +3, and seventy had +2 or less staining intensity (Table I).
  • [0102]
    The HercepTest staining scores were confirmed by analyzing HER2/neu expression levels using microarrays (data not shown). HER2/neu expression strongly correlated with patient response; 100% of the 0 or +1 HER2/neu patients relapsed while only 77% of the +3 patients relapsed. This response rate if similar to what has been reported previously (see Baselga, 2002, Annuals of Oncology 13: 8-9). Based on these results, further analysis of biomarkers concentrated on patients that expressed HER2 at the highest (+3) level. Of the samples that had the highest HercepTest scores (+3), seventy-four were taken from the primary tumor, two from lymph nodes, and one from an adrenal metastasis.
  • [0103]
    The seventy-seven patients who overexpressed HER2/neu (+3 HercepTest staining score) were analyzed for expression levels of EGFR, HER3, IGFR, and NDF/Heregulin, and TGF-α, as well as activated downstream signals p-ERK and p-AKT (phosphorylated forms of ERK and AKT) and the downstream signal of mTOR, p-S6 (phosphorylated S6 ribosomal protein). The analysis of receptor kinases reveled that, similar to HER2/neu, EGFR expression also significantly correlated with patient response (Table 2). Among the HERCEPTIN®-treated patients that over-expressed HER-2/neu, 30% of EGFR-positive patients had stable disease or were disease free, while only 9% of EGFR-negative patients did not progress. Among the seventy-seven +3 HER2/neu patients, seventy of them expressed HER3; however, HER3 expression did not significantly correlate with patient response (although the low number of HER3-negative patients limits this comparison in the data set). The growth-factor receptor HER3 is thought to play an important role in downstream erbB signaling because it has a P13-Kinase docking site and forms active heterodimers with the other erbB receptors. The expression of other growth factor receptors may also mediate patient response, either through direct stimulation or downstream pathways or through transactivation of the erbB receptors.
  • [0104]
    Expression of erbB ligands, including NDF and TGF-α, also varied among patients (see Table 3). Approximately 70% of the patients expressed high levels of NDF, while approximately 57% expressed high levels of TGF-α. A significant correlation was observed between NDF levels and patient response. A very high proportion of HER2/neu overexpressing patients who were NDF-negative relapsed (91%), whereas only 62% of NDF-positive patients who overexpressed HER2/neu relapsed. In contrast, no predictive relationship was observed between TGF-αlevels alone and patient response (see Table 3). The combination of TGF-αor NDF expression and EGFR over-expression, however, did positively correlate with patient response in patients overexpressing HER2/neu (p=0.02 and p=0.03 respectively) (data not shown).
  • [0105]
    The activation of heterodimers of HER2 with HER3 or EGFR results in activation of the MAPK and PI3K/AKT pathways. The MAPK pathway was measured by analyzing the level of activation or phosphorylation of ERK (pERK). Analysis and comparison of the levels of activated ERK alone, among patients that overexpressed HER2/neu and who either had stable disease or who relapsed, failed to demonstrate any dramatic effect of elevated pERK levels as a factor for patient response (see Table 4). Similarly, based on this analysis, AKT activation (p-AKT) alone does not appear to be a predictive marker for response among HER2-positive patients treated with HERCEPTIN® (see Table 4). Also, analysis of S6 ribosomal protein phosphorylation, which integrates multiple signals through mTOR and p70 S6 kinase, did not significantly correlate with patient response for patients that overexpressed HER2/neu (see Table 4). If consideration of pERK and pAKT expression is limited to those patients that expressed EGFR and HER2/neu, however, low expression of either of these signaling molecules was a significant predictor of positive response to HERCEPTIN® (Table 5).
  • [0106]
    To increase the predicative power of the analysis, consideration of two or more of the biomarkers were combined in a multivariate analysis to characterize the tumor. In this analysis, the observation of the combination of HER2/neu and EGFR expression with ERK activation significantly predicated response (see Table 5). For example, patients with low EGFR expression and high ERK phosphorylation failed to respond (0% response), whereas patients with high EGFR expression and low ERK phosphorylation had a high response rate (42% response). Similarly, the combination of high EGFR and HER2/neu with high NDF expression or a combination of high EGFR and HER2/neu with high TGF-α expression predicted a better response compared to patients that had low expression of EGFR and the NDF ligand (data not shown). This comparison was often dramatic. For example, while 39% of the patients with high EGFR, HER2/neu, and NDF expression responded to therapy, none of the patients with high HER2/neu expression but low EGFR and NDF expression responded (data not shown).
  • [0107]
    The combination of high Her2/neu expression, low IGFR expression, and high S6 ribosomal protein phosphorylation gave high patient response (67%, Table 5). This is in contrast to patients with high HER2/neu and IGFR expression and high S6 ribosomal protein phosphorylation, a high percentage that did not respond to therapy. The best combination of markers for predicting whether patients that overexpressed HER2/neu would respond to HERCEPTIN® therapy were high NDF expression, low IGFR expression, and high S6 phosphorylation (Table 6). In contrast, none of the patients overexpressed HER2/neu and had low NDF expression and high IGFR expression responded to therapy, regardless of S6 status (Table 6). However, these results were obtained using a small sample population of these patients. In patients with high NDF, HER2/neu, and EGFR expression levels, phosphorylation of ERK correlated with a difference in response from 28% (high p-ERK) to 54% (low p-ERK) (Table 6). Similarly, those patients with low levels of p-AKT with any other combination of biomarkers that include the expression of HER2/neu and NDF, did better than those that over-express this protein (results not shown). Taken together, this data shows that HER2/neu together with its ligand and other erbB receptors and ligands, as well as other growth factor receptors play a role in HERCEPTIN® response. Importantly, analysis of a select combination of these proteins correlated with response rates that varied from 0 to 100%.
  • EXAMPLE 5 Analysis of Breast Cancer Samples
  • [0108]
    Samples from seven breast cancer patients were obtained from Yale Univeristy. The clinical history of these seven patients varied, with some given HERCEPTIN® in combination with chemotherapy as a first line therapy while others were given HERCEPTIN® as an adjuvant therapy. These seven samples were analyzed for receptor, ligand, and signaling protein expression or phosphorylation, and the results compared to the results with the tissue microarray analysis.
  • [0109]
    All seven patients over-expressed HER2/neu, as determined at the time of analysis with the other antibodies immunologically specific for non-HER2/neu polypeptides. The case histories of the patients varied. For example, patient #1 was given HERCEPTIN® plus docetaxel after relapsing with metastatic disease four years after initial presentation. This patient had stable disease for more than a year after commencing combination therapy. Patient #7 was given HERCEPTIN® plus vinorelbine following the discovery of a solitary metastasis seven months after initial radiotherapy. After eight weeks of combination therapy there was progression of disease. Of the seven patients, three showed response to HERCEPTIN® while the other four failed to respond (Table 7). One of the responders did not express IGFR but did express EGFR and showed positive downstream signaling. The other one of these responders expressed IGFR and EGFR but did not show active downstream signaling in S6 or ERK. All of the non-responders expressed IGFR and had positive S6 phosphorylation. Two of the non-responders also expressed EGFR. These results are consistent with the results obtained from the microarray analysis. Patients with active IGFR receptors as demonstrated by IGFR expression plus S6 phosphorylation are unlikely to respond to HERCEPTIN®, while patients that lack IGFR or have IGFR but no downstream signaling are more likely to respond.
  • [0110]
    It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.
    TABLE 1
    Demographics
    number
    of disease-free or
    patients stable disease relapse
    all patients included in study 175 15% 85%
    Histology
    infiltrating ductal carcinoma 109 17% 83%
    lobular carcinoma 7 43% 57%
    medullary carcinoma 3 33% 67%
    metastatic breast carcinoma 19 5% 95%
    papillary carcinoma 3 0% 100%
    scirrhous carcinoma 3 100% 0%
    tubular carcinoma 3 0% 100%
    treatment following surgery
    (followed by Herceptin ®)
    Doxorubicin 44 2% 98%
    anthracycline plus 100 23% 77%
    cyclophosphamide
    Paclitaxel 3 100% 0%
    HER2/neu expression tumor
    0 or 1 17 0% 100%
    2 53 8% 92%
    3 77 30% 70%
  • [0111]
    [0111]
    TABLE 2
    patient group n % responders % relapse P value
    EGFR positive 43 30% 70% 0.002
    EGFR negative 23 9% 91%
    HER3 positive 70 29% 71% 0.43
    HER3 negative 7 43% 57%
    IGFR positive 33 24% 76% 0.16
    IGFR negative 35 40% 60%
  • [0112]
    Receptor tyrosine kinase expression versus patient response. Analysis on tissue array samples for which clinical and Herceptest data was available and who over-expressed HER2/neu.
    TABLE 3
    patient group n % responders % relapse P value
    NDF positive 55 39% 62% 0.01
    NDF negative 22 9% 91%
    TGF-α positive 38 34% 66% 0.56
    TGF-α negative 29 28% 72%
  • [0113]
    Receptor tyrosine kinase ligand expression versus patient response following therapy. Analysis on tissue array samples for which clinical and Herceptest data was available and who over-expressed HER2/neu.
    TABLE 4
    patient group n % responders % relapse P value
    p-ERK positive 36 25% 75% 0.43
    p-ERK negative 39 33% 67%
    p-AKT positive 24 25% 75% 0.53
    p-AKT negative 53 32% 68%
    p-S6 positive 27 33% 67% 0.74
    p-S6 negative 44 30% 70%
  • [0114]
    Downstream protein activation versus patient response following therapy. Analysis on tissue array samples for which clinical and Herceptest data was available and who over-expressed HER2/neu.
    TABLE 5
    patient group n % responders % relapse P value
    EGFR pos/p-ERK pos 21 14% 86% 0.04
    EGFR pos/p-ERK neg 19 42% 58%
    EGFR neg/p-ERK pos 9 0% 100%
    EGFR neg/p-ERK neg 14 14% 86%
    EGFR pos/p-AKT pos 17 18% 82% 0.07
    EGFR pos/p-AKT neg 26 38% 62%
    EGFR neg/p-AKT pos 5 20% 80%
    EGFR neg/p-AKT neg 18 6% 94%
    IGFR pos/p-S6 pos 13 8% 92% 0.01
    IGFR pos/p-S6 neg 20 35% 65%
    IGFR neg/p-S6 pos 12 67% 33%
    IGFR neg/p-S6 neg 23 26% 74%
  • [0115]
    Analysis of receptor and downstream protein activation versus response in patients following therapy. Analysis on tissue array samples for which clinical and Herceptest data was available and who over-expressed HER2/neu.
    TABLE 6
    %
    patient group n responders % relapse P value
    NDF neg/p-S6 pos/IGFR neg 2 50% 50% 0.003
    NDF neg/p-S6 neg/IGFR neg 9 11% 89%
    NDF neg/p-S6 neg/IGFR pos 4 0% 100%
    NDF neg/p-S6 pos/IGFR pos 4 0% 100%
    NDF pos/p-S6 pos/IGFR neg 7 100% 0%
    NDF pos/p-S6 neg/IGFR pos 16 44% 56%
    NDF pos/p-S6 neg/IGFR neg 14 36% 64%
    NDF neg/p-ERK pos/EGFR neg 3 0% 100% 0.08
    NDF neg/p-ERK neg/EGFR neg 4 0% 100%
    NDF neg/p-ERK neg/EGFR pos 10 20% 80%
    NDF neg/p-ERK pos/EGFR pos 6 0% 100%
    NDF pos/p-ERK pos/EGFR neg 5 0% 100%
    NDF pos/p-ERK neg/EGFR pos 13 54% 46%
    NDF pos/p-ERK neg/EGFR neg 6 17% 83%
    NDF pos/p-ERK pos/EGFR pos 18 28% 72%
  • [0116]
    Analysis of ligand and receptor expression and downstream protein activation versus patient response in patients following therapy. Analysis on tissue array samples for which clinical and Herceptest data was available and who over-expressed HER2/neu.
    TABLE 7
    Patient IGFR EGFR p-S6 p-AKT p-ERK Response
    #1 + + yes
    #2 + + + + yes
    #3 + + + yes
    #4 + + + + no
    #5 + + + + no
    #6 + + + no
    #7 + + + + + no
  • [0117]
    Receptor tyrosine kinase expression, downstream protein activation and patient response to therapy in seven breast cancer patients. Analysis was of whole tissue sections.
    TABLE 8
    AKT (NP 005154 GI:4885061) 480 AMINO ACIDS (SEQ ID NO:1)
    See, e.g., Staal, S.P., Proc. Natl. Acad. Sci. U.S.A. 84 (14),
    5034-5037 (1987).
    MSDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQREAPLNNFSVAQCQL (SEQ ID NO:1)
    MKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGLKKQEEEEMDFRSGS
    PSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVI
    VAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSED
    RARFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFC
    GTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTL
    GPEAKSLLSGLLKKDPKQRLGGGSEDAKEIMQHRFFAGIVWQHVYEKKLSPPFKPQVTSETD
    TRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASSTA
    S6 (NP 001001, GI:17158044) 249 AMINO ACIDS (SEQ ID NO:2)
    See, e.g., Pata et al., (1992) Gene 121 (2), 387-392.
    MKLNISFPATGCQKLIEVDDERKLRTFYEKRMATEVAADALGEEWKGYVVRISGGNDKQGFP (SEQ ID NO:2)
    MKQGVLTHGRVRLLLSKGHSCYRPRRTGERKRKSVRGCIVDANLSVLNLVIVKKGEKDIPGL
    TDTTVPRRLGPKRASRIRKLFNLSKEDDVRQYVVRKPLNKEGKKPRTKAPKIQRLVTPRVLQ
    HKRRRIALKKQRTKKNKEEAAEYAKLLAKRMKEAKEKRQEQIAKRRRLSSLRASTSKSESSQ
    K
    ERK (XP 055766, GI:20562757) 379 AMINO ACIDS (SEQ ID NO:3)
    See, e.g., Butch et al., J Biol Chem., 1996., 271(8): 4230-5.
    MAAAAAQGGGGGEPRRTEGVGPGVPGEVEMVKGQPFDVGPRYTQLQYIGEGAYGMVSSAYDH (SEQ ID NO:3)
    VRKTRVAIKKISPFEHQTYCQRTLREIQILLRFRHENVIGIRDILRASTLEAMRDVYIVQDL
    METDLYKLLKSQQLSNDHICYFLYQILRGLKYIHSANVLHRDLKPSNLLINTTCDLKICDFG
    LARIADPEHDHTGFLTEYVATRWYRAPEIMLNSKGYTKSIDIWSVGCILAEMLSNRPIFPGK
    HYLDQLNHILGILGSPSQEDLNCIINMKARNYLQSLPSKTKVAWAKLFPKSDSKALDLLDRM
    LTFNPNKRITVEEALAHPYLEQYYDPTDEPVAEEPFTFAMELDDLPKERLKELIFQETARFQ
    PGVLEAP
  • [0118]
    [0118]
  • 1 3 1 480 PRT Homo sapiens 1 Met Ser Asp Val Ala Ile Val Lys Glu Gly Trp Leu His Lys Arg Gly 1 5 10 15 Glu Tyr Ile Lys Thr Trp Arg Pro Arg Tyr Phe Leu Leu Lys Asn Asp 20 25 30 Gly Thr Phe Ile Gly Tyr Lys Glu Arg Pro Gln Asp Val Asp Gln Arg 35 40 45 Glu Ala Pro Leu Asn Asn Phe Ser Val Ala Gln Cys Gln Leu Met Lys 50 55 60 Thr Glu Arg Pro Arg Pro Asn Thr Phe Ile Ile Arg Cys Leu Gln Trp 65 70 75 80 Thr Thr Val Ile Glu Arg Thr Phe His Val Glu Thr Pro Glu Glu Arg 85 90 95 Glu Glu Trp Thr Thr Ala Ile Gln Thr Val Ala Asp Gly Leu Lys Lys 100 105 110 Gln Glu Glu Glu Glu Met Asp Phe Arg Ser Gly Ser Pro Ser Asp Asn 115 120 125 Ser Gly Ala Glu Glu Met Glu Val Ser Leu Ala Lys Pro Lys His Arg 130 135 140 Val Thr Met Asn Glu Phe Glu Tyr Leu Lys Leu Leu Gly Lys Gly Thr 145 150 155 160 Phe Gly Lys Val Ile Leu Val Lys Glu Lys Ala Thr Gly Arg Tyr Tyr 165 170 175 Ala Met Lys Ile Leu Lys Lys Glu Val Ile Val Ala Lys Asp Glu Val 180 185 190 Ala His Thr Leu Thr Glu Asn Arg Val Leu Gln Asn Ser Arg His Pro 195 200 205 Phe Leu Thr Ala Leu Lys Tyr Ser Phe Gln Thr His Asp Arg Leu Cys 210 215 220 Phe Val Met Glu Tyr Ala Asn Gly Gly Glu Leu Phe Phe His Leu Ser 225 230 235 240 Arg Glu Arg Val Phe Ser Glu Asp Arg Ala Arg Phe Tyr Gly Ala Glu 245 250 255 Ile Val Ser Ala Leu Asp Tyr Leu His Ser Glu Lys Asn Val Val Tyr 260 265 270 Arg Asp Leu Lys Leu Glu Asn Leu Met Leu Asp Lys Asp Gly His Ile 275 280 285 Lys Ile Thr Asp Phe Gly Leu Cys Lys Glu Gly Ile Lys Asp Gly Ala 290 295 300 Thr Met Lys Thr Phe Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val 305 310 315 320 Leu Glu Asp Asn Asp Tyr Gly Arg Ala Val Asp Trp Trp Gly Leu Gly 325 330 335 Val Val Met Tyr Glu Met Met Cys Gly Arg Leu Pro Phe Tyr Asn Gln 340 345 350 Asp His Glu Lys Leu Phe Glu Leu Ile Leu Met Glu Glu Ile Arg Phe 355 360 365 Pro Arg Thr Leu Gly Pro Glu Ala Lys Ser Leu Leu Ser Gly Leu Leu 370 375 380 Lys Lys Asp Pro Lys Gln Arg Leu Gly Gly Gly Ser Glu Asp Ala Lys 385 390 395 400 Glu Ile Met Gln His Arg Phe Phe Ala Gly Ile Val Trp Gln His Val 405 410 415 Tyr Glu Lys Lys Leu Ser Pro Pro Phe Lys Pro Gln Val Thr Ser Glu 420 425 430 Thr Asp Thr Arg Tyr Phe Asp Glu Glu Phe Thr Ala Gln Met Ile Thr 435 440 445 Ile Thr Pro Pro Asp Gln Asp Asp Ser Met Glu Cys Val Asp Ser Glu 450 455 460 Arg Arg Pro His Phe Pro Gln Phe Ser Tyr Ser Ala Ser Ser Thr Ala 465 470 475 480 2 249 PRT Homo sapiens 2 Met Lys Leu Asn Ile Ser Phe Pro Ala Thr Gly Cys Gln Lys Leu Ile 1 5 10 15 Glu Val Asp Asp Glu Arg Lys Leu Arg Thr Phe Tyr Glu Lys Arg Met 20 25 30 Ala Thr Glu Val Ala Ala Asp Ala Leu Gly Glu Glu Trp Lys Gly Tyr 35 40 45 Val Val Arg Ile Ser Gly Gly Asn Asp Lys Gln Gly Phe Pro Met Lys 50 55 60 Gln Gly Val Leu Thr His Gly Arg Val Arg Leu Leu Leu Ser Lys Gly 65 70 75 80 His Ser Cys Tyr Arg Pro Arg Arg Thr Gly Glu Arg Lys Arg Lys Ser 85 90 95 Val Arg Gly Cys Ile Val Asp Ala Asn Leu Ser Val Leu Asn Leu Val 100 105 110 Ile Val Lys Lys Gly Glu Lys Asp Ile Pro Gly Leu Thr Asp Thr Thr 115 120 125 Val Pro Arg Arg Leu Gly Pro Lys Arg Ala Ser Arg Ile Arg Lys Leu 130 135 140 Phe Asn Leu Ser Lys Glu Asp Asp Val Arg Gln Tyr Val Val Arg Lys 145 150 155 160 Pro Leu Asn Lys Glu Gly Lys Lys Pro Arg Thr Lys Ala Pro Lys Ile 165 170 175 Gln Arg Leu Val Thr Pro Arg Val Leu Gln His Lys Arg Arg Arg Ile 180 185 190 Ala Leu Lys Lys Gln Arg Thr Lys Lys Asn Lys Glu Glu Ala Ala Glu 195 200 205 Tyr Ala Lys Leu Leu Ala Lys Arg Met Lys Glu Ala Lys Glu Lys Arg 210 215 220 Gln Glu Gln Ile Ala Lys Arg Arg Arg Leu Ser Ser Leu Arg Ala Ser 225 230 235 240 Thr Ser Lys Ser Glu Ser Ser Gln Lys 245 3 379 PRT Homo sapiens 3 Met Ala Ala Ala Ala Ala Gln Gly Gly Gly Gly Gly Glu Pro Arg Arg 1 5 10 15 Thr Glu Gly Val Gly Pro Gly Val Pro Gly Glu Val Glu Met Val Lys 20 25 30 Gly Gln Pro Phe Asp Val Gly Pro Arg Tyr Thr Gln Leu Gln Tyr Ile 35 40 45 Gly Glu Gly Ala Tyr Gly Met Val Ser Ser Ala Tyr Asp His Val Arg 50 55 60 Lys Thr Arg Val Ala Ile Lys Lys Ile Ser Pro Phe Glu His Gln Thr 65 70 75 80 Tyr Cys Gln Arg Thr Leu Arg Glu Ile Gln Ile Leu Leu Arg Phe Arg 85 90 95 His Glu Asn Val Ile Gly Ile Arg Asp Ile Leu Arg Ala Ser Thr Leu 100 105 110 Glu Ala Met Arg Asp Val Tyr Ile Val Gln Asp Leu Met Glu Thr Asp 115 120 125 Leu Tyr Lys Leu Leu Lys Ser Gln Gln Leu Ser Asn Asp His Ile Cys 130 135 140 Tyr Phe Leu Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala 145 150 155 160 Asn Val Leu His Arg Asp Leu Lys Pro Ser Asn Leu Leu Ile Asn Thr 165 170 175 Thr Cys Asp Leu Lys Ile Cys Asp Phe Gly Leu Ala Arg Ile Ala Asp 180 185 190 Pro Glu His Asp His Thr Gly Phe Leu Thr Glu Tyr Val Ala Thr Arg 195 200 205 Trp Tyr Arg Ala Pro Glu Ile Met Leu Asn Ser Lys Gly Tyr Thr Lys 210 215 220 Ser Ile Asp Ile Trp Ser Val Gly Cys Ile Leu Ala Glu Met Leu Ser 225 230 235 240 Asn Arg Pro Ile Phe Pro Gly Lys His Tyr Leu Asp Gln Leu Asn His 245 250 255 Ile Leu Gly Ile Leu Gly Ser Pro Ser Gln Glu Asp Leu Asn Cys Ile 260 265 270 Ile Asn Met Lys Ala Arg Asn Tyr Leu Gln Ser Leu Pro Ser Lys Thr 275 280 285 Lys Val Ala Trp Ala Lys Leu Phe Pro Lys Ser Asp Ser Lys Ala Leu 290 295 300 Asp Leu Leu Asp Arg Met Leu Thr Phe Asn Pro Asn Lys Arg Ile Thr 305 310 315 320 Val Glu Glu Ala Leu Ala His Pro Tyr Leu Glu Gln Tyr Tyr Asp Pro 325 330 335 Thr Asp Glu Pro Val Ala Glu Glu Pro Phe Thr Phe Ala Met Glu Leu 340 345 350 Asp Asp Leu Pro Lys Glu Arg Leu Lys Glu Leu Ile Phe Gln Glu Thr 355 360 365 Ala Arg Phe Gln Pro Gly Val Leu Glu Ala Pro 370 375
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US7381535Jan 16, 2003Jun 3, 2008The Board Of Trustees Of The Leland Stanford JuniorMethods and compositions for detecting receptor-ligand interactions in single cells
US7393656Jul 21, 2004Jul 1, 2008The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for risk stratification
US7563584Jul 21, 2009The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for detecting the activation state of multiple proteins in single cells
US7695924Apr 13, 2010The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for detecting receptor-ligand interactions in single cells
US7695926Jan 18, 2007Apr 13, 2010The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for detecting receptor-ligand interactions in single cells
US7695929Apr 13, 2010Ventana Medical Systems, Inc.Haptens, hapten conjugates, compositions thereof and method for their preparation and use
US7700299May 19, 2006Apr 20, 2010Hoffmann-La Roche Inc.Method for predicting the response to a treatment
US7846440Feb 15, 2008Dec 7, 2010Merrimack Pharmaceuticals, Inc.Antibodies against ErbB3 and uses thereof
US7939278May 10, 2011The Board Of Trustees Of Leland Stanford Junior UniversityMethods and compositions for risk stratification
US7981418 *Jul 19, 2011Genentech, Inc.Predicting response to a HER inhibitor
US7985557May 22, 2008Jul 26, 2011Ventana Medical Systems, Inc.Polymeric carriers for immunohistochemistry and in situ hybridization
US8017321Jan 24, 2005Sep 13, 2011The Regents Of The University Of Colorado, A Body CorporateGefitinib sensitivity-related gene expression and products and methods related thereto
US8133692Jul 20, 2006Mar 13, 2012University Of South FloridaMethods of predicting responsiveness to chemotherapeutic agents and selecting treatments
US8148094Feb 17, 2009Apr 3, 2012The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for detecting the activation state of multiple proteins in single cells
US8198037Apr 26, 2011Jun 12, 2012The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for detecting receptor-ligand interactions in single cells
US8206939May 2, 2011Jun 26, 2012The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for risk stratification
US8227202Jul 10, 2009Jul 24, 2012Nodality, Inc.Methods for diagnosis, prognosis and methods of treatment
US8273544Apr 8, 2011Sep 25, 2012Nodality, Inc.Methods for diagnosis, prognosis and methods of treatment
US8309316Nov 13, 2012The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for risk stratification
US8349574Jan 15, 2010Jan 8, 2013Laboratory Corporation Of America HoldingsMethods of determining patient response by measurement of Her-3
US8394599Mar 12, 2013The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for risk stratification
US8399206Oct 22, 2010Mar 19, 2013Nodality, Inc.Methods for diagnosis, prognosis and methods of treatment
US8445191May 21, 2013Ventana Medical Systems, Inc.Polymeric carriers for immunohistochemistry and in situ hybridization
US8470542Dec 1, 2009Jun 25, 2013Laboratory Corporation Of America HoldingsMethods and assays for measuring p95 and/or p95 complexes in a sample and antibodies specific for p95
US8486620Apr 29, 2011Jul 16, 2013Ventana Medical Systems, Inc.Polymeric carriers for immunohistochemistry and in situ hybridization
US8618265Mar 2, 2010Dec 31, 2013Ventana Medical Systems, Inc.Haptens, hapten conjugates, compositions thereof and method for their preparation and use
US8623592Aug 17, 2009Jan 7, 2014Merrimack Pharmaceuticals, Inc.Methods and systems for predicting response of cells to a therapeutic agent
US8691225Aug 21, 2009Apr 8, 2014Merrimack Pharmaceuticals, Inc.Antibodies against the ectodomain of ErbB3 and uses thereof
US8703490Jun 1, 2009Apr 22, 2014Ventana Medical Systems, Inc.Compositions comprising nanomaterials and method for using such compositions for histochemical processes
US8778620May 17, 2012Jul 15, 2014Nodality, Inc.Methods for diagnosis, prognosis and methods of treatment
US8815527Apr 26, 2011Aug 26, 2014The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for detecting the activation state of multiple proteins in single cells
US8846320Feb 17, 2010Sep 30, 2014Ventana Medical Systems, Inc.Haptens, hapten conjugates, compositions thereof and method for their preparation and use
US8865420May 22, 2008Oct 21, 2014The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for risk stratification
US8895001Mar 11, 2011Nov 25, 2014Merrimack Pharmaceuticals, Inc.Use of ErbB3 inhibitors in the treatment of triple negative and basal-like breast cancers
US8940302Oct 9, 2012Jan 27, 2015Genentech, Inc.Predicting response to a HER inhibitor
US8961966Oct 14, 2010Feb 24, 2015Merrimack Pharmaceuticals, Inc.Antibodies against ERBB3 and uses thereof
US8962263Apr 26, 2011Feb 24, 2015The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for detecting the activation state of multiple proteins in single cells
US9017954Apr 23, 2014Apr 28, 2015Ventana Medical Systems, Inc.Polymeric carriers for immunohistochemistry and in situ hybridization
US9081019Jun 6, 2013Jul 14, 2015Laboratory Corporation Of America HoldingsMethods and assays for measuring p95 and/or p95 complexes in a sample and antibodies specific for p95
US9103822May 30, 2013Aug 11, 2015Ventana Medical Systems, Inc.Polymeric carriers for immunohistochemistry and in situ hybridization
US9110066Nov 7, 2012Aug 18, 2015Laboratory Corporation Of America HoldingsHER-3 antibodies and methods of use
US9115384May 2, 2011Aug 25, 2015The Board Of Trustees Of The Leland Stanford Junior UniversityMethods and compositions for detecting receptor-ligand interactions in single cells
US9434994Jul 5, 2012Sep 6, 2016The Regents Of The University Of Colorado, A Body CorporateMethods for prediction of clinical outcome to epidermal growth factor receptor inhibitors by non-small cell lung cancer patients
US20050112700 *Jul 21, 2004May 26, 2005Perez Omar D.Methods and compositions for risk stratification
US20060073474 *Jul 10, 2002Apr 6, 2006Perez Omar DMethods and compositions for detecting the activation state of multiple proteins in single cells
US20060121044 *Dec 6, 2005Jun 8, 2006Genentech, Inc.Selecting patients for therapy with a her inhibitor
US20060281977 *May 26, 2006Dec 14, 2006Michael SoppetDiagnostic and treatment planning calculator
US20070031871 *Jul 20, 2006Feb 8, 2007University Of South FloridaMethods of predicting responsiveness to chemotherapeutic agents and selecting treatments
US20070037228 *May 19, 2006Feb 15, 2007Joachim MoecksMethod for predicting the response to a treatment
US20070196869 *Jan 18, 2007Aug 23, 2007Perez Omar DMethods and compositions for detecting receptor-ligand interactions in single cells
US20070270505 *Jan 24, 2005Nov 22, 2007The Regents Of The University Of ColoradoGefitinib Sensitivity-Related Gene Expression and Products and Methods Related Thereto
US20080090233 *May 26, 2005Apr 17, 2008The Regents Of The University Of ColoradoMethods for Prediction of Clinical Outcome to Epidermal Growth Factor Receptor Inhibitors by Cancer Patients
US20080113874 *Jul 23, 2007May 15, 2008The Regents Of The University Of ColoradoGefitinib sensitivity-related gene expression and products and methods related thereto
US20080182262 *Jan 17, 2008Jul 31, 2008Perez Omar DMethods and compositions for risk stratification
US20080234265 *Mar 13, 2006Sep 25, 2008The Regents Of The University Of ColoradoHistone Deacetylase Inhibitors Sensitize Cancer Cells to Epidermal Growth Factor Inhibitors
US20080254489 *Apr 2, 2008Oct 16, 2008Perez Omar DMethods and compositions for risk stratification
US20080305497 *May 22, 2008Dec 11, 2008Ventana Medical Systems, Inc.Polymeric carriers for immunohistochemistry and in situ hybridization
US20080317753 *May 1, 2008Dec 25, 2008Genentech, Inc.Selecting patients for therapy with a her inhibitor
US20090068681 *May 22, 2008Mar 12, 2009Perez Omar DMethods and compositions for risk stratification
US20090155804 *Dec 9, 2008Jun 18, 2009Peter Blume-JensenDisease pathway-based method to generate biomarker panels tailored to specific therapeutics for individualized treatments
US20090291085 *Feb 15, 2008Nov 26, 2009Merrimack Pharmaceuticals, Inc.Antibodies against erbb3 and uses thereof
US20090291458 *Nov 26, 2009Nodality, Inc.Method for Determining the Status of an Individual
US20100021424 *May 31, 2007Jan 28, 2010Vincent BrichardMethod For Identifying Whether A Patient Will Be Responder or Not to Immunotherapy
US20100112603 *Nov 24, 2009May 6, 2010Joachim MoecksMethod for predicting the response to a treatment
US20100216718 *Oct 20, 2008Aug 26, 2010Cell Signaling Technology, Inc.Cancer Classification and Methods of Use
US20100221750 *May 12, 2010Sep 2, 2010Perez Omar DMethods and Compositions for Risk Stratification
US20100233732 *Jan 15, 2010Sep 16, 2010Laboratory Corporation Of America HoldingsMethods of Determining Patient Response By Measurement of HER-2 Expression
US20100266584 *Aug 21, 2009Oct 21, 2010Merrimack Pharmaceuticals, Inc.Antibodies against the ectodomain of erbb3 and uses thereof
US20110027291 *Feb 17, 2010Feb 3, 2011Merrimack Pharmaceuticals, Inc.Methods, systems and products for predicting response of tumor cells to a therapeutic agent and treating a patient according to the predicted response
US20110070268 *Mar 24, 2011Glaxosmithkline Biologicals SaMethod
US20110123523 *Oct 14, 2010May 26, 2011Merrimack Pharmaceuticals, Inc.Antibodies against erbb3 and uses thereof
US20110159513 *Aug 17, 2009Jun 30, 2011Merrimack Pharmaceuticals, Inc.Methods and systems for predicting response of cells to a therapeutic agent
US20110201018 *Aug 18, 2011Perez Omar DMethods and compositions for risk stratification
US20110201019 *Aug 18, 2011Perez Omar DMethods and Compositions for Detecting Receptor-Ligand Interactions in Single Cells
US20110207146 *Aug 25, 2011Perez Omar DMethods and compositions for detecting receptor-ligand interactions in single cells
US20110207149 *Aug 25, 2011Perez Omar DMethods and compositions for risk stratification
EP2196547A1 *May 24, 2006Jun 16, 2010F.Hoffmann-La Roche AgMethod for predicting the response to a treatment with a HER dimerization inhibitor
WO2005117553A3 *May 26, 2005Aug 28, 2008Univ ColoradoMethods for prediction of clinical outcome to epidermal growth factor receptor inhibitors by cancer patients
WO2007013946A2 *Jul 20, 2006Feb 1, 2007University Of South FloridaMethod of predicting responsiveness to chemotherapy and selecting treatments
WO2007019899A2 *May 24, 2006Feb 22, 2007F. Hoffmann-La Roche AgMethod for predicting the response to a treatment with a her dimerization inhibitor
WO2007019899A3 *May 24, 2006Apr 5, 2007Hoffmann La RocheMethod for predicting the response to a treatment with a her dimerization inhibitor
WO2009086197A1 *Dec 19, 2008Jul 9, 2009Monogram Biosciences, Inc.Her-2 diagnostic methods
Classifications
U.S. Classification435/6.14, 435/7.23
International ClassificationG01N33/574
Cooperative ClassificationG01N2333/912, G01N2333/9121, G01N2333/4756, G01N33/574, G01N33/5041, G01N2800/52, C07K2317/24, G01N2333/71, C07K16/2863, G01N2440/14, G01N33/6893, G01N2333/4703, C07K16/40, G01N33/57415
European ClassificationG01N33/50D2E14, G01N33/574, G01N33/574C4
Legal Events
DateCodeEventDescription
Jul 19, 2004ASAssignment
Owner name: VENTANA MEDICAL SYSTEMS, INC., ARIZONA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BACUS, SARAH S.;REEL/FRAME:015587/0447
Effective date: 20040629
Owner name: CELL SIGNALING TECHNOLOGY INC., MASSACHUSETTS
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SMITH, BRADLEY L.;REEL/FRAME:015584/0759
Effective date: 20040603
Owner name: VENTANA MEDICAL SYSTEMS, INC., ARIZONA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BACUS, SARAH S.;REEL/FRAME:015590/0063
Effective date: 20040629
Owner name: CELL SIGNALING TECHNOLOGY, INC., MASSACHUSETTS
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SMITH, BRADLEY L.;REEL/FRAME:015591/0797
Effective date: 20040603