JP2015522815A - Plasma biomarkers of bevacizumab combination therapy for the treatment of breast cancer - Google Patents

Abstract

The present invention relates to expression levels of E-selectin, ICAM-1, or VEGFR-3 as compared to control levels of patients diagnosed with HER2-positive breast cancer, particularly locally recurrent or metastatic HER2-positive breast cancer Suffering from HER2-positive breast cancer, particularly locally recurrent or metastatic HER2-positive breast cancer, by adding bevacizumab (Avastin®) to the chemotherapy regimen, especially by determining plasma expression levels To improve the therapeutic effect of chemotherapy regimens in patients.

Description

  The present invention is directed to a method of identifying patients who have the greatest benefit from treatment with anti-cancer therapy including anti-VEGF antibodies among patients diagnosed with breast cancer.

  Angiogenesis contributes to benign and malignant diseases such as the development of cancer, particularly in cancer, which is necessary for early tumor growth, invasiveness, and metastasis. In order to grow, the tumor must go through an angiogenic switch. Vascular endothelial growth factor (VEGF) is required to induce this angiogenic switch. VEGF and genes in the VEGF pathway are considered important mediators of cancer progression. The VEGF gene family includes VEGF genes, also called VEGFA, VEGF homologues including placental growth factor (PlGF), VEGFB, VEGFC, VEGFD, VEGFR-1 and VEGFR-2 (also called FLT1 and FLK1 / KDR, respectively). Receptors, hypoxia-inducing factors HIF1a, VEGF-inducing factors including HIF2a, and oxygen sensors PHD1, PHD2, and PHD3 are included.

  The importance of this pathway in cancer cell growth and metastasis has led to the development of anti-angiogenic agents used in cancer therapy. Such treatments include, among others, bevacizumab, begaptanib, sunitinib, sorafenib, and vatalanib. Although angiogenesis inhibitors such as bevacizumab significantly extend survival, patients still die from cancer. Furthermore, not all patients respond to treatment with angiogenesis inhibitors. The mechanism that causes the non-response remains unclear. Furthermore, treatment with angiogenesis inhibitors is associated with side effects such as gastrointestinal perforation, thrombosis, bleeding, hypertension, and proteinuria.

  Accordingly, there is a need for a method of determining patients that respond particularly well to treatment with an angiogenesis inhibitor.

  Investigation of the status of biomarkers related to angiogenesis and tumor development has shown that the expression levels of E-selectin, ICAM-1 and VEGFR-3 relative to the baseline level determined in the total biomarker patient population are the outcome of treatment in breast cancer patients It became clear that there was a correlation with the improvement. In particular, patients exhibiting elevated levels of expression of E-selectin, ICAM-1, and VEGFR-3 relative to the baseline level determined in the entire biomarker patient population responded to the addition of bevacizumab to the combination therapy of trastuzumab and docetaxel Prolonged progression-free survival.

  Thus, the present invention determines whether a patient diagnosed with breast cancer is sensitive to anti-cancer therapy including the addition of anti-VEGF antibodies to a chemotherapy regimen, in patient samples, E-selectin, ICAM-1, and Relevant to a method of determining by determining the expression level of at least one biomarker selected from the group consisting of VEGFR-3 and comparing it to a reference level. The present invention also provides treatment of a patient who has been diagnosed with breast cancer and whose expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 is increased from a reference level. For a pharmaceutical composition comprising an anti-VEGF antibody such as babacizumab. The present invention further provides that the therapeutic effect of anti-cancer therapy including chemotherapy for a patient diagnosed with breast cancer is selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a patient sample. Based on the expression level of one biomarker, it relates to a method that improves by adding an anti-VEGF antibody such as bevacizumab.

  One embodiment of the present invention provides an in vitro method for determining whether a patient diagnosed with breast cancer is adequately treated to some extent by anti-cancer therapy including anti-VEGF antibodies. This method comprises (a) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with breast cancer. And (b) based on the expression level according to (a), identifying the patient as being treated appropriately to some extent by anti-cancer therapy including anti-VEGF antibody, wherein the expression level of the biomarker is When the level is above the reference level, the patient is shown to be highly suitable for treatment with anti-cancer therapy, or when the expression level of the biomarker is below the reference level, the patient is not suitable for treatment with anti-cancer therapy. Is identified. In some embodiments, in terms of progression-free survival, it is determined whether the patient is properly treated with anti-cancer therapy. In some embodiments, the method further comprises treating the patient with anti-cancer therapy. In some embodiments, the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody, and a taxane. In some embodiments, the anti-VEGF antibody is bevacizumab. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the taxane is docetaxel or paclitaxel. In some embodiments, the expression level of the at least one biomarker is a protein expression level. In some embodiments, the patient-derived sample is a plasma sample. In some embodiments, the at least one biomarker is E-selectin. In some embodiments, the at least one biomarker is ICAM-1. In some embodiments, the at least one biomarker is VEGFR-3.

  Another embodiment of the invention provides an in vitro method of selecting treatment for a patient diagnosed with breast cancer. In this method, (a) by assaying a biological sample derived from a patient, the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 is determined based on the patient. Determining that it is at or above the level, and (b) based on the determination, selecting a treatment comprising an anti-cancer therapy comprising an anti-VEGF antibody. In some embodiments, in terms of progression-free survival, it is determined whether the patient is properly treated with anti-cancer therapy. In some embodiments, the method further comprises treating the patient with anti-cancer therapy. In some embodiments, the patient has not previously received chemotherapy or radiation therapy. In some embodiments, the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody, and a taxane. In some embodiments, the anti-VEGF antibody is bevacizumab. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the taxane is docetaxel or paclitaxel. In some embodiments, the expression level of the at least one biomarker is a protein expression level. In some embodiments, the patient-derived sample is a plasma sample. In some embodiments, the at least one biomarker is E-selectin. In some embodiments, the at least one biomarker is ICAM-1. In some embodiments, the at least one biomarker is VEGFR-3.

  A further embodiment of the present invention provides an in vitro method for determining whether a patient diagnosed with breast cancer is susceptible to anti-cancer therapy including the addition of anti-VEGF antibodies to chemotherapy. This method comprises (a) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with breast cancer. And (b) identifying the patient as sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to chemotherapy based on the expression level according to (a), wherein the expression level of the biomarker is a reference level If so, identifying the patient is shown to be susceptible to anti-cancer therapy including the addition of anti-VEGF antibodies to chemotherapy. In some embodiments, in terms of progression-free survival, it is determined whether the patient is properly treated with anti-cancer therapy. In some embodiments, the method further comprises treating the patient with anti-cancer therapy. In some embodiments, the patient has not previously received chemotherapy or radiation therapy. In some embodiments, the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody, and a taxane. In some embodiments, the anti-VEGF antibody is bevacizumab. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the taxane is docetaxel or paclitaxel. In some embodiments, the expression level of the at least one biomarker is a protein expression level. In some embodiments, the patient-derived sample is a plasma sample. In some embodiments, the at least one biomarker is E-selectin. In some embodiments, the at least one biomarker is ICAM-1. In some embodiments, the at least one biomarker is VEGFR-3.

  Yet another embodiment of the invention provides an in vitro method of selecting an anti-cancer therapy for a patient diagnosed with breast cancer. In this method, (a) by assaying a biological sample derived from a patient, the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 is determined based on the patient. Determining that it is at or above the level, and (b) selecting an anti-cancer therapy comprising the addition of an anti-VEGF antibody to the chemotherapy based on the determination. In some embodiments, in terms of progression-free survival, it is determined whether the patient is properly treated with anti-cancer therapy. In some embodiments, the method further comprises treating the patient with anti-cancer therapy. In some embodiments, the patient has not previously received chemotherapy or radiation therapy. In some embodiments, the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody, and a taxane. In some embodiments, the anti-VEGF antibody is bevacizumab. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the taxane is docetaxel or paclitaxel. In some embodiments, the expression level of the at least one biomarker is a protein expression level. In some embodiments, the patient-derived sample is a plasma sample. In some embodiments, the at least one biomarker is E-selectin. In some embodiments, the at least one biomarker is ICAM-1. In some embodiments, the at least one biomarker is VEGFR-3.

  Another embodiment of the present invention is a pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient is anti-antigen by any of the methods described herein. Provided are pharmaceutical compositions that have been identified as being highly suitable for treatment with anti-cancer therapies comprising VEGF antibodies.

  Another embodiment of the present invention is a pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient is anti-antigen by any of the methods described herein. Provided are pharmaceutical compositions identified as being highly suitable for treatment with anti-cancer therapies comprising VEGF antibodies.

  A further embodiment of the invention is a pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient is treated with a chemotherapy regimen by any of the methods described herein. A pharmaceutical composition has been identified that has been identified as being sensitive to anti-cancer therapy comprising the addition of anti-VEGF antibodies to.

  A further embodiment of the invention is a pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient is treated with a chemotherapy regimen by any of the methods described herein. Pharmaceutical compositions identified as being sensitive to anti-cancer therapies comprising the addition of anti-VEGF antibodies to are provided.

  Yet another embodiment of the present invention provides a kit for performing any of the methods described herein. The kit includes a set of compounds for detecting the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1, and VEGFR-3, the set specific to the biomarker Antibody capable of binding.

  Yet another embodiment of the present invention provides a method of improving the therapeutic effect of an anti-cancer therapy including a chemotherapy regimen in a patient diagnosed with breast cancer by adding an anti-VEGF antibody to the chemotherapy regimen. This method comprises (a) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with positive breast cancer. And (b) identifying, based on the expression level according to (a), that the patient is sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen, wherein the expression level of said biomarker is Identifying, when above a reference level, the patient is shown to be sensitive to anti-cancer therapy comprising the addition of anti-VEGF antibodies to a chemotherapy regimen; (c) an effective amount of an anti-VEGF antibody in an effective amount In combination with other chemotherapy regimens of (b) to patients identified as sensitive to anti-cancer therapy including addition of anti-VEGF antibody to chemotherapy And a Rukoto. In some embodiments, in terms of progression-free survival, it is determined whether the patient is properly treated with anti-cancer therapy. In some embodiments, the method further comprises treating the patient with anti-cancer therapy. In some embodiments, the patient has not previously received chemotherapy or radiation therapy. In some embodiments, the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody, and a taxane. In some embodiments, the anti-VEGF antibody is bevacizumab. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the taxane is docetaxel or paclitaxel. In some embodiments, the expression level of the at least one biomarker is a protein expression level. In some embodiments, the patient-derived sample is a plasma sample. In some embodiments, the at least one biomarker is E-selectin. In some embodiments, the at least one biomarker is ICAM-1. In some embodiments, the at least one biomarker is VEGFR-3.

1. Definitions As used herein, the term “administration” or “administering” refers to administration of a pharmaceutical composition, such as an angiogenesis inhibitor, to a patient in need of such treatment, or as known in the art. Means medical intervention by any appropriate means. Non-limiting routes of administration include oral, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal, or intrabronchial administration (eg, by inhalation). Particularly preferred in the context of the present invention is parenteral administration, for example intravenous administration.

  The term “anti-angiogenic agent” or “anti-angiogenic agent” refers to small molecular weight substances, polynucleotides, polypeptides, isolated proteins, recombinant proteins, antibodies, or conjugates or fusion proteins thereof, It directly or indirectly inhibits angiogenesis, vasculogenesis or unwanted vascular permeability. Anti-angiogenic agents should be understood to include agents that bind to an angiogenic factor or its receptor and block its angiogenic activity. For example, an anti-angiogenic agent is defined throughout the specification or known in the art for antibodies or other antagonists to angiogenic agents such as VEGF-A or VEGF-A receptor (eg, KDR Anti-PDGFR inhibitors such as, but not limited to, antibodies to the receptor, or Flt-1 receptor), VEGF-trap, Gleevec® (imatinib mesylate). Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin, endostatin and the like. Examples include Klagsbrun and D'Amore, Annu. Rev. Physiol., 53: 217-39 (1991); Streit and Detmar, Oncogene, 22: 3172-3179 (2003) (for example, antiangiogenic therapy for malignant melanoma) Table 3); Ferrara & Alitalo, Nature Medicine 5: 1359-1364 (1999); Tonini et al., Oncogene, 22: 6549-6556 (2003) (eg, Table 2 listing known anti-angiogenic factors); and See, Sato Int. J. Clin. Oncol., 8: 200-206 (2003) (eg, Table 1 which lists anti-angiogenic agents used in clinical trials).

As used herein, the term “antibody” is used in the broadest sense and includes, but is not limited to, monoclonal and polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), chimeric antibodies, CDR-grafted antibodies, humanized Antibodies, dead antibodies, single chain antibodies, and antibody fragments and fragment constructs such as F (ab ′) ₂ fragments, Fab fragments, Fv fragments, single chain Fv fragments (scFv), bispecific scFv, bispecific Includes bispecific antibodies, single domain antibodies (dAbs), and miniantibodies.

As used herein, the term “VEGF” or “VEGF-A” refers to, for example, 165-amino acid human vascular endothelial growth factor and Leung et al. (1989) Science 246: 1306, and Houck et al. (1991) Mol. Endocrin, 5: 1806, the related 121-, 189-, and 206-amino acid human vascular endothelial growth factors, and Ferrara Mol. Biol. Cell 21: 687 (2010). Refers to naturally occurring alleles and their processed forms, including the ₁₁₀ -amino acid human vascular endothelial growth factor produced by plasmin cleavage of VEGF ₁₆₅ . The term “VEGF” also refers to VEGFs from non-human species such as mice, rats, or primates. Sometimes VEGF from a particular species is indicated by terms such as hVEGF for human VEGF and mVEGF for mouse VEGF. The term “VEGF” is also used to refer to a truncated form of a polypeptide comprising amino acids 8-109, or 1-109 of 165-amino acid human vascular endothelial growth factor. When referring to such a VEGF form, it is represented herein for example as “VEGF (8-109)”, “VEGF (1-109)” or “VEGF ₁₆₅ ”. The amino acid position of the “cut (cleaved)” native VEGF is indicated by the number shown in the native VEGF sequence. For example, amino acid position 17 (methionine) of truncated natural VEGF is also position 17 (methionine) in natural VEGF. The truncated native VEGF has a KDR and Flt-1 receptor binding affinity comparable to native VEGF.

  “VEGF bioactivity” refers to binding to either VEGF receptor or regulation of both normal and abnormal angiogenesis and angiogenesis (Ferrara and Davis-Smyth (1997) Endocrine Rev. 18: 4-25; Ferrara ( 1999) J. Mol. Med. 77: 527-543); Promotion of embryonic angiogenesis and angiogenesis (Carmeliet et al. (1996) Nature 380: 435-439; Ferrara et al. (1996) Nature 380: 439-442); and regulation of circulating vascular proliferation in female reproductive organs and for bone growth and cartilage formation (Ferrara et al. (1998) Nature Med. 4: 336-340; Gerber et al. (1999) Nature Med. 5: 623-628). In addition to being an angiogenic factor in angiogenesis and vasculogenesis, VEGF is a pleiotropic growth factor and other physiological factors such as endothelial cell survival, vascular permeability and vasodilation, monocyte chemotaxis, and calcium influx. The process exhibits multiple biological effects (Ferrara and Davis-Smyth (1997), supra and Cebe-Suarez et al. Cell. Mol. Life Sci. 63: 601-615 (2006)). Furthermore, recent studies have reported the mitogenic effect of VEGF on several non-endothelial cell types such as retinal pigment epithelial cells, pancreatic duct cells, and Schwann cells. Guerrin et al. (1995) J. Cell Physiol. 164: 385-394; Oberg-Welsh et al. (1997) Mol. Cell. Endocrinol. 126: 125-132; Sondell et al. (1999) J. Neurosci. 19: 5731-5740.

A “VEGF antagonist” or “VEGF-specific antagonist” is a molecule that has the ability to bind to VEGF, reduce VEGF expression levels, or neutralize, block, inhibit, suppress, reduce, or prevent VEGF biological activity. Such capacities include, but are not limited to, VEGF binding to one or more VEGF receptors, and VEGF-mediated angiogenesis, and endothelial cell survival or proliferation. VEGF-specific antagonists useful in the methods of the invention include VEGF, anti-VEGF antibodies, and polypeptides that specifically bind to antigen-binding fragments thereof, one or more by specifically binding to VEGF. Receptor molecules and derivatives that sequester their binding to the receptor, fusion proteins (eg, VEGF trap (Regeneron)), and VEGF ₁₂₁ -gelonine (Peregrine). VEGF-specific antagonists also include antagonist variants of VEGF polypeptides, antisense nucleobase oligomers directed against VEGF, small RNA molecules directed against VEGF, RNA aptamers, peptibodies, and ribozymes against VEGF. VEGF-specific antagonists also include non-peptide small molecules that have the ability to bind to VEGF and block, inhibit, abrogate, reduce, or prevent VEGF biological activity. Thus, the term “VEGF activity” specifically includes VEGF-mediated biological activity of VEGF. In certain embodiments, the VEGF antagonist has a VEGF expression level or biological activity of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Reduce or inhibit.

  An “anti-VEGF antibody” is an antibody that binds to VEGF with sufficient affinity and specificity. In certain embodiments, the selected antibody typically has sufficient binding affinity for VEGF, for example, the antibody may bind hVEGF with a Kd value of 100 nM to 1 pM. Antibody affinity can be determined, for example, by surface plasmon resonance based assays (such as the Biacore assay described in PCT Application Publication No. 2005/012359); enzyme-linked immunosorbent assays (ELISA); and competition experiments (eg, RIA's). Can be determined. In certain embodiments, anti-VEGF antibodies may be useful as therapeutic agents in targeting and interfering with diseases and conditions that involve VEGF activity. The antibodies can also be the subject of other biological activity assays, for example to assess their effectiveness as therapeutics. Such assays are known in the art and depend on the intended use for the target antigen and antibody. Examples include HUVEC inhibition assays; tumor cell growth inhibition assays (eg, as described in WO 89/06692); antibody-dependent cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (US Pat. No. 5,500,362) ); And agonist activity or hematopoietic assays (see WO 95/27062). An anti-VEGF antibody will usually not bind to other VEGF homologues such as VEGF-B or VEGF-C, nor other growth factors such as PlGF, PDGF or bFGF. In one embodiment, the anti-VEGF antibody is a monoclonal antibody that binds to the same epitope as monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709. In another embodiment, the anti-VEGF antibody is a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al. (1997) Cancer Res. 57: 4593-4599, including but not limited to bevacizumab (BV; AVASTIN ( Antibody known as a registered trademark.

  The anti-VEGF antibody “bevacizumab (BV)”, also known as “rhuMAb VEGF” or “AVASTIN®”, is a recombination produced by Presta et al. (1997) Cancer Res. 57: 4593-4599. Humanized anti-VEGF monoclonal antibody. This is due to the murine anti-hVEGF monoclonal antibody A.B. blocking the binding of human VEGF to its receptor. 4. Includes a mutated human IgG1 framework region from 4.6.1 and an antigen binding complementarity determining region. Approximately 93% of the amino acid sequence of bevacizumab, including most of the framework regions, is derived from human IgG1, and approximately 7% of the sequence is derived from murine antibody A4.6.1. Bevacizumab has a molecular weight of about 149,000 daltons and is glycosylated. Bevacizumab and other humanized anti-VEGF antibodies are further described in US Pat. No. 6,884,879, issued February 26, 2005, the entire disclosure of which is incorporated herein by reference.

  The term “cancer” generally refers to the physiological condition of a mammal characterized by unregulated cell growth. Examples of cancer include, but are not limited to, adenocarcinoma, lymphoma, blastoma, sarcoma and leukemia. More detailed examples of such cancers include squamous cell carcinoma, lung cancer (small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, stomach or Abdominal cancer (including gastrointestinal cancer), pancreatic cancer (including metastatic pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer (locally progressive) , Recurrent or metastatic HER-2 negative breast cancer and locally recurrent or metastatic HER-2 positive breast cancer), colorectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or kidney Cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, and B cell lymphoma (low grade / vesicular non-Hodgkin's lymphoma (NHL); small Lymphocyte type (SL) NHL; moderate grade / vesicular NHL; moderate grade diffuse NHL; high grade immunity Spherical NHL; high-grade lymphoblastic NHL; high-grade small non-cleavable cell NHL; giant lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom macroglobulinemia); Leukemia (CLL), acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disease (PTLD), and nevus, edema (such as that associated with brain tumors) , And abnormal vascular growth associated with Megs syndrome.

  Examples of “physiological or pathological angiogenesis abnormalities” include, but are not limited to, high-grade gliomas, glioblastomas, Rendu-Osler, von Hippel-Lindau disease, hemangioma, psoriasis, Kaposi's sarcoma, ocular neovascularization, rheumatoid arthritis, endometriosis, atherosclerosis, myocardial ischemia, peripheral ischemia, cerebral ischemia, And wound healing.

  The term “chemotherapeutic agent” or “chemotherapeutic regimen” includes any active agent capable of providing an anti-cancer therapeutic effect, in particular a chemical agent or biological that can interfere with cancer cells or tumor cells. It may be a drug. Certain active agents are those that act as anti-neoplastic (chemotoxic or chemostatic) agents that inhibit or prevent the development, maturation or proliferation of malignant cells. Examples of chemotherapeutic agents include alkylating agents such as nitrogen mustard (eg, mechlorethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil), nitrosourea (eg, carmustine (BCNU), lomustine (CCNU), and semustine (Methyl-CCNU)), ethyleneimine / methylmelamine (eg, triethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine)), alkyl sulfonate ( For example, busulfan), and triazines (eg, dacarbazine (DTIC)); antimetabolites such as folic acid analogs (eg, methotrexate, trimetrexate), pyrimidine analo (Eg, 5-fluorouracil, capecitabine, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2′-difluorodeoxycytidine), and purine analogs (eg, 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA)); made from natural products Mitotic inhibitors (eg, paclitaxel, vinca alkaloids (eg, vinblastine (VLB), vincristine, and vinorelbine), docetaxel, estramustine, and estramustine phosphate), Pipeodophylloxins (eg, etoposide, teniposide), antibiotics (eg, actinomycin D, daunomycin (rubidomycin), daunorubicin, doxorubicin, epirubicin, mitoxantrone, idarubicin, bleomycin, pricamycin (mitromycin), mitomycin C, actino Mycins), enzymes (eg, L-asparaginase), and biological response modifiers (eg, interferon-α, IL-2, G-CSF, GM-CSF); miscellaneous agents including platinum complex complexes ( For example, cisplatin, carboplatin, oxaliplatin), anthracenedione (eg, mitoxantrone), substituted urea (ie, hydroxyurea), methylhydrazine derivatives (eg, N Hormones and antagonists (eg, prednisone and equivalents, dexamethasone) including methylhydrazine (MIH), procarbazine), corticosteroids (eg, mitotan (o, p'-DDD), aminoglutethimide); corticosteroid antagonists , Aminoglutethimide), progestins (eg, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate), estrogens (eg, diethylstilbestrol, ethinyl estradiol and equivalents); antiestrogens (eg, Tamoxifen), androgens (eg, testosterone propionate, fluoxymesterone and equivalents), antiandrogens (eg, flutamide, gonadotropin releasing hormone analogs) , Leuprolide) and non-steroidal antiandrogens (eg, flutamide), epidermal growth factor inhibitors (eg, erlotinib, lapatinib, gefitinib) antibodies (eg, trastuzumab), irinotecan, and other drugs such as leucovorin. For the treatment of locally recurrent or metastatic HER2-positive breast cancer, chemotherapeutic agents or chemotherapy regimens for administration with bevacizumab include capecitabine, paclitaxel, docetaxel, and transizumab, and combinations thereof (See also the examples provided herein).

  The term “docetaxel” is an antineoplastic agent that binds to free tubulin and promotes the association of tubulin with stable microtubules while at the same time inhibiting the association thereof. This leads to the generation of microtubule bundles that do not have normal functions and the stabilization of microtubules, blocking cells in the M phase of the cell cycle and causing cell death.

  The term “effective amount” refers to an amount of an agent effective in treating a mammalian disease or disorder, alone or in combination with other agents, or in a treatment regimen. In the case of cancer, a therapeutically effective amount of the drug reduces the number of cancer cells and reduces the size of the tumor; inhibits the invasion of cancer cells into surrounding organs (ie slows, preferably stops) to some extent. Inhibit tumor metastasis (ie slow, preferably stop) to some extent; inhibit tumor growth to some extent; and / or moderate one or more symptoms associated with the disease. To the extent that an agent can prevent and / or kill the growth of existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer treatment, for example, efficacy in vivo can be measured by assessing duration of survival, duration of progression-free survival (PFS), response rate (RR), duration of response, and / or quality of life. .

  The term “expression level” as used herein also refers to the concentration or amount of a marker / indicator protein of the invention in a sample.

  The term “epitope A4.6.1” refers to the anti-VEGF antibody bevacizumab (AVASTIN®) (see Muller Y et al., Structure 15 September 1998, 6: 1153-1167). In a particular embodiment of the invention, the anti-VEGF antibody is a monoclonal antibody that binds to the same epitope as monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709; Presta et al. (1997) Cancer Res. 57: 4593-4599, including but not limited to recombinant humanized anti-VEGF monoclonal antibody.

  “Epitope 4D5” is a region in the extracellular domain of HER2 to which antibody 4D5 (ATCC CRL 10463) and trastuzumab are bound, as described in WO2009 / 154651. This epitope is an amino acid residue from about 489-630 residue numbering near the HER2 transmembrane domain and within HER2 domain IV and without a signal peptide. Garrett et al Mol Cell. 1 1: 495-505 (2003), Cho et al Nature 421: 756-760 (2003), Franklin et al Cancer Cell 5: 317-328 (2004), and Plowman et al. Proc. See Natl. Acad. Sci 90: 1746-1750 (1993). To screen for antibodies that essentially bind to the 4D5 epitope, a routine cross-blocking assay as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) can be performed. . Alternatively, the antibody consists essentially of any one or more residues (residue numbering within the region from about residue 529 to about residue 625, including the HER2 extracellular domain, including the HER2 extracellular domain). In order to assess whether it contains a signal peptide)), epitope mapping can be performed.

  “HER receptor” is a receptor protein tyrosine kinase that belongs to the HER receptor family and includes EGFR, HER2, HER3 and HER4 receptors. A HER receptor usually comprises an extracellular domain, which can bind to a HER ligand and / or another HER receptor molecule; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; It dimerizes with the carboxyl-terminal signaling domain with a tyrosine residue inside. The HER receptor may be a “native sequence” HER receptor or an “amino acid sequence variant” thereof. In one embodiment, the HER receptor is a native sequence human HER receptor. The terms “ErbB1,” “HER1,” “epidermal growth factor receptor,” and “EGFR” are used interchangeably herein, eg, Carpenter et al, Ann. Rev. Biochem. 56: 881-914 (1987). EGFR, as disclosed in U.S.), which includes naturally occurring mutant forms (eg, deletions as described in Humphrey et al. PNAS (USA) 87: 4207-421 1 (1990)). Mutant). erbB refers to the gene encoding the EGFR protein product.

  The expressions “ErbB2” and “HER2” are used interchangeably herein to refer to a human antigen. For example, the HER2 protein described in Semba et al., PNAS (USA) 82: 6497-6501 (1985) and Yamamoto et al. Nature 319: 230-234 (1986) (Genebank accession number X03363). The term “erbB2” refers to the gene encoding human ErbB2, and the term “neu” refers to the gene encoding mouse pi85.

  An “anti-Her2” antibody is an antibody that binds to the HER2 receptor. Optionally, the HER antibody further interferes with HER2 activity or function. In one embodiment, the anti-HER2 antibody of the invention is an anti-HER2 antibody that binds to a 4D5 epitope on a HER2 polypeptide, or in another embodiment, trastuzumab.

  Humanized HER2 antibodies are huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 or trastuzumab as described in Table 3 of US Pat. No. 5,821,337. (Ie, HERCEPTIN®).

  A cancer, cancer cell, or tumor that is “HER2-positive” has a significantly higher level of HER receptor protein or gene compared to a non-cancerous cell of the same tissue type. Such overexpression may be due to gene growth or increased transcription or translation. HER receptor overexpression or proliferation can be determined by assessing elevated levels of HER protein present on the surface of cells in a diagnostic or prognostic assay (eg, immunohistologic assay; by IHC). Alternatively or additionally, for example, fluorescence in situ hybridization (FISH; see, eg, WO 98/45479 published October 1998), Southern blotting, or quantitative real-time PCR (qRT-PCR) The level of HER-encoding nucleic acid in cells can be measured by the polymerase chain reaction (PCR) technique. It is also possible to study HER receptor overexpression or proliferation by measuring antigens (eg, HER extracellular domain) that flow in biological fluids such as serum (eg, US published on June 12, 1990). Patent No. 4933294; WO 91/05264 issued April 18, 1991; US Pat. No. 5,401,638 issued March 28, 1995; and Sias et al. J. Immunol. Methods 132: 73-80. (1990)). Apart from the above assays, various in vivo assays are available to practitioners with advanced skills. For example, cells inside the patient's body can be exposed to an antibody that may be labeled with a detectable label, such as a radioisotope, and binding of the antibody to the patient's cells can be determined by, for example, external X-ray examination of radiation. Alternatively, it can be assessed by analyzing a biopsy taken from the patient prior to exposure to the antibody.

  “Metastasis” or “metastatic” means the spread of cancer from the primary site of cancer in the body to other locations. Cancer cells can detach from the primary tumor, penetrate lymphatic vessels and blood vessels, circulate through the bloodstream, and grow (metastasize) at distant lesions in normal tissues in other parts of the body. The metastasis can be local or remote. Metastasis is a sequential process that is conditional on tumor cells detaching from the primary tumor, moving through the bloodstream, and stopping at the distant site. At the new site, the cells can establish a blood supply and proliferate to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within tumor cells control this behavior, and interactions between tumor cells and host cells at remote sites are also important.

  The terms “oligonucleotide” and “polynucleotide” are used interchangeably and refer to a molecule composed of two or more, preferably more than three deoxypolynucleotides, or ribonucleotides. Its exact size depends on many factors, which depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be obtained synthetically or by cloning. Deoxyribonucleotides and ribonucleotide chimeras are also within the scope of the present invention.

  The term “overall survival (OS)” refers to the length of time a patient survives during and after treatment. As will be appreciated by those skilled in the art, if a patient belongs to a subgroup of patients that has a significantly longer average survival time than another subgroup of patients, the overall survival of the patient is improved or improved.

  The term “patient” refers to any single animal, particularly a mammal (such as a non-human animal such as a dog, cat, horse, rabbit, zoo animal, cow, pig, sheep, and Including non-human primates). More specifically, the patient herein is a human.

  The expression “patient suffering from” refers to clinical indications for diseases including physiological and pathological angiogenesis and / or neoplastic diseases such as breast cancer, particularly locally recurrent or metastatic HER2-positive breast cancer. Refers to patients showing

  The term “pharmaceutical composition” is sterile and is in a form that allows the biological activity of the drug to be effective and does not contain other ingredients that are unacceptably toxic to the subject to whom the formulation is administered. Refers to the preparation of

  “Progression-free survival (PFS)” refers to the length of time during which a patient's disease does not worsen, ie does not progress, according to the evaluation of the clinician or investigator during and after treatment. As will be appreciated by those skilled in the art, if a patient belongs to a subgroup of patients who do not progress disease for longer than the mean or mean progression-free survival time of patients in a similar situation, the patient's absence. The exacerbation survival rate has improved or improved.

  The term “polypeptide” refers to a peptide, protein, oligopeptide, or polypeptide that includes a chain of amino acids of a given length, wherein the amino acid residues are linked by covalent peptide bonds. However, such protein / polypeptide peptidomimetics are also encompassed by the present invention, in which the amino acid and / or peptide bond is a functional analog, eg, of 20 gene-encoded amino acids such as selenocysteine. Substituted by other than one amino acid residue. Peptides, oligopeptides, and proteins are referred to as polypeptides. The terms polypeptide and protein are used interchangeably herein. The term polypeptide also refers to, but does not exclude, modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation and the like. Such modifications are described in detail in basic textbooks, as well as in more detailed research books, and a vast research literature. The term polypeptide also refers to and includes the term “antibody” as used herein.

  The expression “responsive to” in the context of the present invention refers to a subject suffering from, suspected of suffering from or susceptible to suffering from breast cancer, particularly locally recurrent or metastatic HER2-positive breast cancer. / Show patient responds to chemotherapy regimens including bevacizumab. One skilled in the art will be able to determine whether a person treated with bevacizumab according to the methods of the present invention will respond. For example, the response may be reduced pain due to suffering from breast cancer, particularly locally recurrent or metastatic HER2-positive breast cancer, such as reduced and / or stopped tumor growth, reduced tumor size, and / or cancer Reflected by improvement of one or more symptoms. Preferably, the response is reflected by a reduction or reduction in the index of metastatic transformation of breast cancer, such as prevention of metastasis formation, or a reduction in the number or size of metastases (eg, Eisenhauser et al., New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1) Eur. J. Cancer 2009 45: 228-247).

  As used herein, the term “reference sample” refers to any sample, standard, or level used for comparison purposes. In one embodiment, the reference sample is obtained from a healthy and / or unaffected part of the same subject or patient's body (eg, tissue or cell). In another embodiment, the reference sample is obtained from untreated tissue and / or cells of the same subject or patient body. In yet another embodiment, the reference sample is obtained from a healthy and / or unaffected part of the body (eg, tissue or cell) of an individual that is not the subject or patient. In yet another embodiment, the reference sample is obtained from an untreated tissue and / or cellular portion of the body of an individual who is not the subject or patient. In certain embodiments, the reference sample is a single sample from the same subject or patient obtained at one or more different time points other than when the test sample is obtained, or a combination of multiple samples It is. For example, the reference sample is obtained from the same subject or patient at a time earlier than when the test sample is obtained. Such a reference sample can be useful when a reference sample is acquired during the initial diagnosis of cancer and then a test sample is acquired when the cancer becomes metastatic. In certain embodiments, reference samples include all types of biological samples defined above as the term “sample” obtained from one or more individuals who are not the subject or patient. In certain embodiments, the reference sample is obtained from one or more individuals having an angiogenic disorder (eg, cancer) that is not the subject or patient. In certain embodiments, the reference sample is a combination of multiple samples from one or more healthy individuals that are not subjects or patients. In certain embodiments, the reference sample is a combination of multiple samples from one or more individuals with a disease or disorder (eg, an angiogenic disorder such as cancer) that is not the subject or patient. In certain embodiments, the reference sample is a normal tissue or pooled plasma from one or more individuals who are not subjects or patients, or a pooled RNA sample from a serum sample. In certain embodiments, the reference sample is from a tumor tissue or pooled plasma or serum sample from one or more individuals with a disease or disorder (eg, an angiogenic disorder such as cancer) that is not the subject or patient. , A pooled RNA sample.

  The term “reference level” refers herein to a predetermined value. One skilled in the art will understand that the reference level is predetermined and set to meet the requirements in terms of specificity and / or sensitivity. These requirements can vary, for example, depending on the regulatory body. For example, assay sensitivity or specificity must each be set to a specific limit, such as 80%, 90%, or 95%. These requirements can also be defined in terms of positive or negative predictive values. Nevertheless, based on the teachings given in the present invention, there is always the possibility of reaching a reference level that meets those requirements. In one embodiment, the reference level is determined in healthy individuals. The reference value of one embodiment is predetermined in the disease to which the patient belongs. In certain embodiments, the reference level can be set, for example, to any percentage between 25% and 75% of the overall distribution of disease values investigated. In other embodiments, the reference level can be set to, for example, a median value, a quartile value, or a quartile value that is determined based on the overall distribution of values of the disease being investigated. In one embodiment, the reference level can be set to a median value that is determined based on the overall distribution of disease values being investigated.

  In certain embodiments, expressions such as “increased (increased, increased)” or “above (above)” are levels above reference levels, or E-selectin, ICAM-1 and VEGFR from a reference sample. 5%, 10%, 20%, 25%, 30 at the level of E-selectin, ICAM-1, or VEGFR-3 detected by the methods described herein as compared to the level of -3 %, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or more. In certain embodiments, the expression “elevated” has an increase of at least about 1.5 when compared to levels of E-selectin, ICAM-1 and VEGFR-3 determined in advance based on a reference sample. 1.75, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 100 times , E-selectin, ICAM-1, or VEGFR-3. In a preferred embodiment, the term “elevated level” relates to a reference level value or a value above the reference level.

  In certain embodiments, expressions such as “decrease (decrease, decrease)” or “below (below)” are levels below reference levels, or E-selectin, ICAM-1 and VEGFR from a reference sample. 5%, 10%, 20%, 25%, 30 in the levels of E-selectin, ICAM-1, and VEGFR-3 detected by the methods described herein as compared to the levels of -3 %, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more. In certain embodiments, the expression “decreased” means that the level after reduction is up to about 0.9, 0.8, 0 of the level of E-selectin, ICAM-1, and VEGFR-3 in the reference sample. 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 times or less, E-selectin, ICAM -1, and refers to a decrease in the level of VEGFR-3.

  In certain embodiments, the term “reference level” refers to the same level as the level of E-selectin, ICAM-1, and VEGFR-3 detected from a reference sample by the methods described herein.

  A “recurrent” cancer is a cancer that has regrown at the primary or distal site after responding to initial therapy.

  The expression “sensitive to” in the context of the present invention refers to a subject who is suffering from, suspected of suffering or susceptible to breast cancer, particularly locally recurrent or metastatic HER2-positive breast cancer / This suggests that the patient has some positive response to treatment with bevacizumab in combination with a chemotherapy regimen. The patient response may not be significant when compared to the “reactive” patients described above. For example, patient suffering associated with disease may be reduced, but no reduction in tumor growth or metastasis index is measured, and / or patient response to a combination of bevacizumab and chemotherapy regimen is transient It may only have properties, i.e. tumor growth and / or metastasis may only be temporarily reduced or stopped.

  The term “survival” refers to the subject being alive and includes progression free survival (PFS) and overall survival (OS). Survival can be estimated by the Kaplan-Meier method and any difference in survival is calculated using a stratified log rank test.

  “Prolonging life” or “increasing likelihood of survival” is compared to an untreated subject (ie, compared to a subject not treated with a VEGF antibody) or locally recurrent or Chemotherapeutic agents such as those used for standard care of metastatic breast cancer, such as capecitabine, taxane, anthracycline, paclitaxel, docetaxel, paclitaxel protein binding particles (eg Abraxane®), doxorubicin, epirubicin, 5 -PFS and / or OS found in the treated subject compared to a control treatment protocol, such as treatment with fluorouracil, cyclophosphamide, or trastuzumab (eg, Herceptin®), or combinations thereof only Means the rise. In one embodiment, such standard care for locally recurrent or metastatic breast cancer is a combination of treatments including trastuzumab and docetaxel. Survival is at least about 1 month, about 2 months, about 4 months, about 6 months, about 9 months, or at least about 1 year, or at least about 2 years, or at least about 3 years after initiation of treatment or after initial diagnosis Or at least about 4 years, or at least about 5 years, or at least about 10 years.

  The term “hazard ratio (HR)” is a statistical definition of the rate of events. For the purposes of the present invention, hazard ratio is defined as representing the probability of an event in the experimental group divided by the probability of the event in the control group at any particular time. “Hazard ratio” in progression-free survival analysis is a summary of the differences between two progression-free survival curves and represents a reduced risk of treatment death compared to controls over the period of follow-up .

  As used herein, “therapy” or “treatment” refers to clinical intervention that attempts to alter the natural course of the individual or cell being treated, for prevention or in the course of clinical pathology. Can be executed. The desired effect of treatment is to prevent the onset or recurrence of the disease, alleviate symptoms, reduce the direct or indirect pathological consequences of the disease, prevent metastases, slow the rate of progression of the disease, Including amelioration or alleviation of disease state and improvement of remission or prognosis.

  The term “therapeutic effect” encompasses the terms “overall survival” and “progression free survival”.

  The term “E-selectin” refers to endothelial adhesion molecules induced by various inflammatory stimuli (Bevilacqua, PP et al., Proc. Natl. Acad. Sci. USA 84, 9238-9242 (1987); Luscinskas , FW et al., J. Immunol. 142 2257-2263 (1989); Kuijpers, TW et al., J. Immunol. 147 1369-1376 (1991)). A cloned gene (ELAM-1) encoding E-selectin is disclosed in US Pat. No. 5,081,034. E-selectin is illustrated by UniProtKB accession number P16581 and gene ID (NCBI): 6401. The term “E-selectin” encompasses homologs and isoforms thereof. In the context of the present invention, the term “E-selectin” refers to antibody clones such as BBIG-E5 or 5D11, for which mutant proteins (including isoforms), homologous proteins, and / or fragments are available, for example, from R & D Systems. Also included are variants thereof, as well as fragments of sequences, if recognized by one or more E-selectin specific antibodies.

  The term “ICAM-1” refers to the intercellular adhesion molecule 1 illustrated by UniProtKB accession number P05362 and gene ID (NCBI): 3383. The term “ICAM-1” encompasses homologs and isoforms thereof. In the context of the present invention, the term “ICAM-1” refers to mutant proteins (including isoforms), homologous proteins, and / or fragments such as antibody clones such as 11C81 or 14C11 available from R & D Systems, If recognized by one or more ICAM-1-specific antibodies, variants thereof as well as fragments of the sequence are also included.

  The term “VEGFR-3” refers to vascular endothelial growth factor receptor 3 exemplified by UniProtKB accession number P35916 and gene ID (NCBI) 2324. The term “VEGFR-3” encompasses homologs and isoforms thereof. In the context of the present invention, the term “VEGFR-3” refers to antibody clones 54716 or 5B6, in which mutant proteins (including isoforms), homologous proteins, and / or fragments are available, for example from R & D Systems and Abnova, respectively. If recognized by one or more VEGFR-3 isomeric antibodies, variants thereof as well as fragments of the sequence are also included.

2. Detailed Embodiments In the present invention, E-selectin, ICAM-1, and VEGFR-3 have been identified as markers of survival or predictive biomarkers with anti-angiogenic therapy. The terms “marker”, “biomarker”, and “predictive biomarker” can be used interchangeably and refer to the expression levels of E-selectin, ICAM-1, and VEGFR-3. In one embodiment, E-selectin, ICAM-1, and VEGFR-3 can also be used as diagnostic biomarkers for breast cancer, specifically HER2-positive breast cancer.

Thus, the present invention provides an in vitro method for determining whether patients diagnosed with breast cancer are adequately treated to some extent by anti-cancer therapy including anti-VEGF antibodies,
(A) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with breast cancer; and (b) Identifying the patient's suitability for treatment with an anti-cancer therapy comprising an anti-VEGF antibody based on the expression level according to (a), wherein the patient's anti-cancer if the expression level of the biomarker is above a reference level Identifying, indicating that the suitability for treatment with therapy is high, or that the expression level of the biomarker is below a reference level, indicates that the patient is less suitable for treatment with anticancer therapy. In one embodiment, in terms of progression-free survival, it is determined whether the patient is properly treated with anti-cancer therapy. In one embodiment, the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody, and a taxane.

The present invention further comprises a pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer,
(A) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with breast cancer; and (b) Identifying the patient's suitability for treatment with an anti-cancer therapy comprising an anti-VEGF antibody based on the expression level according to (a), wherein the patient's anti-cancer if the expression level of the biomarker is above a reference level An in vitro method comprising identifying a high suitability for treatment with a therapy or an indication that the patient's suitability for treatment with an anti-cancer therapy is low if the expression level of the biomarker is below a reference level Provides a pharmaceutical composition identified as having high suitability for treatment with an anti-cancer treatment comprising an anti-VEGF antibody . In one embodiment, in terms of progression-free survival, it is determined whether the patient is properly treated with anti-cancer therapy. In one embodiment, the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody, and a taxane.

The present invention further comprises a pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer,
(A) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with breast cancer; and (b) Based on the expression level according to (a), identifying a patient that is appropriately treated to some extent by anti-cancer therapy including anti-VEGF antibody, wherein the expression level of the biomarker is above the reference level The patient is shown to be more suitable for treatment with anti-cancer therapy, or if the expression level of the biomarker is below a reference level, the patient is shown to be less suitable for treatment with anti-cancer therapy. A pharmaceutical that has been identified as having high suitability for treatment by anti-cancer therapy including anti-VEGF antibodies by an in vitro method comprising identifying To provide a Narubutsu. In one embodiment, in terms of progression-free survival, it is determined whether the patient is properly treated with anti-cancer therapy. In one embodiment, the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody, and a taxane.

The present invention further provides an in vitro method for determining whether a patient diagnosed with breast cancer is susceptible to anti-cancer therapy including the addition of anti-VEGF antibodies to chemotherapy, said method comprising:
(A) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with breast cancer; and (b) Based on the expression level according to (a), identifying a patient as sensitive to anti-cancer therapy including addition of anti-VEGF antibody to chemotherapy, wherein the expression level of the biomarker is above a reference level Identifying, indicating that the patient is susceptible to anti-cancer therapy, including the addition of anti-VEGF antibodies to chemotherapy. In one embodiment, whether a patient is sensitive to an anti-cancer therapy that includes the addition of anti-VEGF antibodies to chemotherapy is determined in terms of progression-free survival. In one embodiment, the chemotherapeutic regimen comprises an anti-HER2 antibody and a taxane.

The present invention further comprises a pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer,
(A) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with breast cancer; and (b) Based on the expression level according to (a), identifying a patient as sensitive to anti-cancer therapy including the addition of anti-VEGF antibodies to a chemotherapy regimen, wherein the expression level of the biomarker is above a reference level In vitro methods, including identifying, indicating that the patient is susceptible to anti-cancer therapy including addition of anti-VEGF antibodies to the chemotherapy regimen, including adding anti-VEGF antibodies to the chemotherapy regimen. Pharmaceutical compositions that have been identified as susceptible to cancer therapy are provided. In one embodiment, whether a patient is sensitive to an anti-cancer therapy that includes the addition of anti-VEGF antibodies to chemotherapy is determined in terms of progression-free survival. In one embodiment, the chemotherapeutic regimen comprises an anti-HER2 antibody and a taxane.

The present invention further comprises a pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer,
(A) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with breast cancer; and (b) Based on the expression level according to (a), identifying a patient as sensitive to anti-cancer therapy including the addition of anti-VEGF antibodies to a chemotherapy regimen, wherein the expression level of the biomarker is above a reference level An anti-cancer therapy comprising adding an anti-VEGF antibody to a chemotherapy regimen by an in vitro method comprising identifying, wherein the patient is shown to be sensitive to an anti-cancer therapy comprising adding an anti-VEGF antibody to the chemotherapy Pharmaceutical compositions identified as being sensitive to are provided. In one embodiment, whether a patient is sensitive to an anti-cancer therapy that includes the addition of anti-VEGF antibodies to chemotherapy is determined in terms of progression-free survival. In one embodiment, the chemotherapeutic regimen comprises an anti-HER2 antibody and a taxane.

The present invention also provides a method for improving the therapeutic effect of anti-cancer therapy, including chemotherapy, in patients diagnosed with breast cancer by adding an anti-VEGF antibody to the chemotherapy regimen, the method comprising:
(A) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with positive breast cancer;
(B) identifying a patient as being sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen based on the expression level according to (a), wherein the expression level of the biomarker is above a reference level Wherein the patient is shown to be susceptible to anti-cancer therapy including the addition of anti-VEGF antibodies to chemotherapy, and identifying
(C) administering an effective amount of an anti-VEGF antibody in combination with an effective amount of a chemotherapeutic regimen to a patient identified by (b) as being sensitive to anti-cancer therapy including the addition of anti-VEGF antibody to chemotherapy Including that. In one embodiment, whether a patient is sensitive to an anti-cancer therapy that includes the addition of anti-VEGF antibodies to chemotherapy is determined in terms of progression-free survival. In one embodiment, the chemotherapeutic regimen comprises an anti-HER2 antibody and a taxane.

  In one embodiment, said determination of the biomarker comprises: a) contacting the sample with an agent that specifically binds to the biomarker to form a complex between the agent and the biomarker, and the formed complex Measuring the level of the biomarker by detecting the amount of the body, or b) growing the biomarker present in the sample and growing with a drug that specifically binds to the grown biomarker This is done by measuring the level of the biomarker by detecting the marker.

  In one embodiment, the patient is diagnosed with HER2-positive breast cancer, specifically locally recurrent or metastatic HER2-positive breast cancer. In one embodiment, the patient has not previously received chemotherapy or radiation therapy.

  In one embodiment, the anti-VEGF antibody binds to the A4.6.1 epitope. Specifically, the anti-VEGF antibody is bevacizumab.

  In one embodiment, the taxane is docetaxel or paclitaxel, specifically docetaxel.

  In one embodiment, the anti-HER2 antibody binds to a 4D5 epitope. Specifically, the anti-HER2 antibody is trastuzumab.

  In one embodiment, the expression level is a protein expression level.

  In one embodiment, the sample is a plasma sample.

  In one embodiment, the expression level is the expression level of E-selectin. In one embodiment, the expression level is the expression level of ICAM-1. In one embodiment, the expression level is the expression level of VEGFR-3.

  In the context of the invention described herein, the expression levels of E-selectin, ICAM-1, and VEGFR-3, in particular protein expression levels, are considered separately as individual markers or expression profiles or markers As a panel, it is considered in two or more groups. In the context of the invention described herein, an expression profile or marker panel when the expression profiles of two or more markers are considered together is also referred to as a combination of expression levels. For example, the expression levels of two or more markers are summed and compared to a combination of control expression levels determined in the same manner. Accordingly, the methods of the present invention include the determination of an expression profile comprising a combination of expression levels based on the expression level of one or more markers.

  In order to consider E-selectin, ICAM-1 and VEGFR-3 separately in the context of the invention described herein and according to the accompanying examples, the following values correspond to the corresponding high expression values of the markers: Or as low expression values: high E-selectin (≧ 36.9 ng / mL), low E-selectin (<36.9 ng / mL), high ICAM-1 (≧ 210 ng / mL), low ICAM-1 (<210 ng / mL), high VEGFR-3 (≧ 10.6 ng / mL), low VEGFR-3 (<10.6 ng / mL). Such a level was determined as the sample median for each predictive analysis plan. In addition, the optimization level resulting in a cut-off value between high and low expression of a particular marker can be cut off until a subset of patients above and below the cut-off meets the relevant statistical optimality criteria. It is determined by changing. For example, the optimal cut point can be chosen to maximize the difference in treatment hazard ratio between the upper and lower subsets, or the therapeutic effect of one subgroup or any other relevant statistic You can choose to maximize the treatment effect of the baseline. However, one of ordinary skill in the art will appreciate that the expression level of a particular marker, and therefore the high or low expression level, can vary from patient to patient and patient population. Accordingly, those of skill in the art will identify when using detection methods other than those described in the following examples, and when studying patients and patient populations other than those described in the following examples. It will be understood that what is considered a high and / or low expression level of a biomarker can vary from the values described herein. Given the methods described herein, one of ordinary skill in the art can determine what results in high and / or low levels of expression of a particular biomarker.

  In one embodiment, the reference level of E-selectin, ICAM-1, and VEGFR-3 is set to the median value in the sample concentration obtained from the patient group. In one example, the reference level of E-selectin is about 36.9 ng / mL in plasma. In one example, the reference level of ICAM-1 is about 210 ng / mL in plasma. In one example, the reference level of VEGFR-3 is about 10.6 ng / mL in plasma. In one embodiment, the reference level of E-selectin, ICAM-1, and VEGFR-3 is determined by quartile analysis of samples obtained from a group of patients. In one embodiment, the reference level of E-selectin, ICAM-1, and VEGFR-3 is set to a first quartile value for increasing the concentration in a sample obtained from a patient group. In one example, the reference level of E-selectin is about 26.7 ng / mL in plasma. In one example, the reference level of ICAM-1 is about 170.4 ng / mL in plasma. In one example, the reference level of VEGFR-3 is about 7.8 ng / mL in plasma. In one embodiment, the reference level of E-selectin, ICAM-1, and VEGFR-3 is set to a third quartile value to increase the concentration in the sample obtained from the patient group. In one example, the reference level of E-selectin is about 49.1 ng / mL in plasma. In one example, the reference level of ICAM-1 is about 272.2 ng / mL in plasma. In one example, the reference level of VEGFR-3 is about 13.0 ng / mL in plasma.

  As will be appreciated by those skilled in the art, there are many ways to use measurements of two or more markers to improve the interview under study. A very simple but often effective approach assumes a positive result if the sample is positive for at least one of the markers under investigation.

  However, marker combinations may be evaluated. The values measured for markers in the marker panel, eg, E-selectin, ICAM-1, and VEGFR-3, are mathematically combined and the combined value (or combination of expression levels) is used in the underlying interrogation. Can be correlated. Marker values can be combined by any suitable conventional mathematical method. Well-known mathematical methods for correlating marker combinations with disease or treatment effects are discriminant analysis (DA) (ie linear, quadratic, normalized -DA), kernel method (ie SVM), non-parametric method (ie , K-neighbor classifier), PLS (partial least squares), tree type method (ie logistic regression, CART, random forest method, boosting / bagging method), generalized linear model (ie logistic regression), principal component Such methods as the method based on (ie SIMCA), general additive model, method based on fuzzy theory, neural network, and genetic algorithm method are adopted. Those skilled in the art will be able to select an appropriate method for evaluating the marker combinations of the present invention without difficulty. Marker combinations according to the invention disclosed herein can be used for example for overall survival, progression-free survival, reactivity or sensitivity to addition of bevacizumab to a chemotherapeutic agent / chemotherapeutic regimen, and / or (one or more chemistry Methods used to correlate with improved prediction of response or sensitivity to bevacizumab (added to the therapeutic / chemotherapeutic regimen) are DA (ie, linear, quadratic, normalized discriminant analysis), kernel method ( Ie, SVM), non-parametric method (ie, k-nearest classifier), PLS (partial least squares), tree-type method (ie, logistic regression, CART, random forest method, boosting method), or generalized linear model (Ie, logistic regression). Details relating to these statistical methods are found in the following references. Ruczinski, I., et al, J. of Computational and Graphical Statistics, 12 (2003) 475-511; Friedman, JH, J. of the American Statistical Association 84 (1989) 165-175; Hastie, Trevor, Tibshirani, Robert , Friedman, Jerome, The Elements of Statistical Learning, Springer Series in Statistics, 2001; Breiman, L., Friedman, JH, Olshen, RA, Stone, CJ (1984) Classification and regression trees, California: Wadsworth; Breiman, L. , Random Forests, Machine Learning, 45 (2001) 5-32; Pepe, MS, The Statistical Evaluation of Medical Tests for Classification and Prediction, Oxford Statistical Science Series, 28 (2003); and Duda, RO, Hart, PE, Stork , DG, Pattern Classification, Wiley Interscience, 2nd Edition (2001).

  Accordingly, the invention disclosed herein is anti-cancer for the combination of the underlying biological markers and for distinguishing between state A and state B, for example including the addition of bevacizumab to a chemotherapy regimen It is associated with the use of an optimized multivariable cut-off to distinguish patients responsive or sensitive to therapy from patients who are inadequately responding to the addition of bevacizumab therapy to a chemotherapy regimen. In this type of analysis, the markers are not independent but form a marker panel or a combination of expression levels.

3. Detection of the expression level of E-selectin, ICAM-1, or VEGFR-3 The expression level of one or more of the markers E-selectin, ICAM-1, or VEGFR-3 is known in the art, An antibody that can be assessed by any method suitable for determining the level of a particular protein in a patient sample, preferably specific to one or more of E-selectin, ICAM-1, or VEGFR-3 Determined by immunoassay using, for example, ELISA. Such methods are well known and routinely performed in the art, and corresponding commercially available antibodies and / or kits are readily available. For example, commercially available antibodies / test kits for E-selectin, ICAM-1, or VEGFR-3 are from R & D Systems as clones BBIG-E5 and 5D11, from R & D Systems as clones 11C81 and 14C11, and as clones 54716 and 5B6 Each can be acquired from Abnova. Preferably, the expression level of the marker / indicator protein of the present invention is assessed using the antibody and kit manufacturer's reagents and / or protocol recommendations. Those skilled in the art are also aware of additional means for determining the level of expression of one or more of E-selectin, ICAM-1, and VEGFR-3 by immunoassay. Thus, the expression level of one or more of the markers / indicators of the present invention can be determined in a routine and reproducible manner by those skilled in the art without undue burden. However, to ensure accurate and reproducible results, the present invention also includes testing patient samples in specialized laboratories that can reliably verify test procedures.

  E-selectin, ICAM-1, and VEGFR-3 protein or nucleic acid can be detected using any method known in the art. For example, assays of mammalian tissue or cell samples can be performed using, for example, Western blots and ELISAs for proteins and Northern blots, dot plots, or polymerase chain reactions for mRNAs or DNAs from the gene biomarker of interest. Conveniently using commercially available DNA SNP chip microarrays including (PCR) analysis, array hybridization, RNase protection assay, or DNA microarray snapshots. For example, real-time PCR (RT-PCR) assays such as quantitative PCR assays are well known in the art. In an exemplary embodiment of the invention, a method for detecting mRNA from a gene biomarker of interest in a biological sample generates cDNA from the sample by reverse transcription using at least one primer; Amplifying the amplified cDNA; and detecting the presence of the amplified cDNA. In addition, such methods allow determination of the level of mRNA in a biological sample (eg, by simultaneously examining the level of a comparative control mRNA sequence of a “housekeeping” gene such as an actin family member) One or more steps can be included, optionally the sequence of the amplified cDNA can be determined.

  A number of criteria are available that provide guidance in applying the above techniques (Kohler et al., Hybridoma Techniques (Cold Spring Harbor Laboratory, New York, 1980); Tijssen, Practice and Theory of Enzyme Immunoassays ( Elsevier, Amsterdam, 1985); Campbell, Monoclonal Antibody Technology (Elsevier, Amsterdam, 1984); Hurrell, Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Boca Raton, FL, 1982); and Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-1 58 (CRC Press, Inc., 1987).

  The expression level of one or more of E-selectin, ICAM-1, and VEGFR-3 is assessed in a patient sample that is a biological sample. The patient sample can be a blood sample, a serum sample, or a plasma sample. Methods for obtaining blood samples, serum samples, and plasma samples are known in the art. Patient samples can be obtained from patients before or after neoadjuvant therapy, or before or after adjuvant therapy.

4). Methods of Treatment In the context of the present invention, bevacizumab is in addition to or with one or more chemotherapeutic agents administered as part of a standard chemotherapeutic regimen, as is known in the art. It is administered as a combination therapy or a combination therapy. Examples of drugs included in such standard chemotherapy regimens include 5-fluorouracil, leucovorin, irinotecan, gemcitabine, eroticinib, capecitabine, taxanes such as docetaxel and paclitaxel, interferon alpha, vinorelbine, and platinum Agents such as carboplatin, cisplatin, and oxaliplatin are included. As demonstrated in the examples below, the addition of bevacizumab is in patients and / or patient populations defined and selected by the expression level of one or more of E-selectin, ICAM-1, and VEGFR-3. Increased progression-free survival. Thus, bevacizumab can be combined with a chemotherapy regimen such as docetaxel therapy, as demonstrated in the examples below.

  Common modes of administration include bolus administration or infusion over a set time, such as 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 3 minutes Parenteral administration is included as a total daily dosage over a period of 4 hours, 5 hours, or 6 hours. For example, 2.5 mg to 15 mg of bevacizumab per kg body weight (Avastin®) can be administered weekly, every two weeks, or every three weeks, depending on the type of cancer being treated. Examples of doses include 2.5 mg, 5 mg, 7.5 mg, 10 mg, and 15 mg per kg body weight weekly, every 2 weeks, or every 3 weeks. Further examples of doses include 5 mg per kg body weight every 2 weeks, 10 mg per kg body weight every 2 weeks, 7.5 mg per kg body weight every 3 weeks, and 15 mg per kg body weight every 3 weeks. In the context of the invention described herein, low doses of bevacizumab include, for example, 2.5 mg / kg body weight every week, 5 mg / kg body weight every 2 weeks, and 7.5 mg / kg body weight every 3 weeks. included. In the context of the invention described herein, a high dose of bevacizumab includes, for example, 5 mg / kg body weight weekly, 10 mg / kg body weight every 2 weeks, and 15 mg / kg body weight every 3 weeks.

  One skilled in the art will recognize that additional dosage models of bevacizumab are covered by the present invention as determined by the particular patient and chemotherapeutic regimen, and that the particular dosage model and therapeutic dose are in accordance with methods known in the art. It will be recognized that it is best determined by the clinician.

  Patients selected according to the methods of the invention can be treated with bevacizumab in combination with a chemotherapy regimen and treated with one or more additional anti-cancer therapies. In certain embodiments, the one or more additional anticancer therapies is radiation.

5. Kits The present invention also relates to diagnostic compositions or kits comprising oligonucleotides or polypeptides suitable for determining the expression level of one or more of E-selectin, ICAM-1 and VEGFR-3. As detailed in the present invention, DNA, RNA, or a mixture of DNA and RNA probes can be used to detect the mRNA level of a marker / indicator protein, while the polypeptide can undergo specific protein-protein interactions. Used to directly detect the protein level of the marker / indicator protein. In a preferred embodiment of the invention, a polypeptide that is included as a probe for the expression level of one or more of E-selectin, ICAM-1 and VEGFR-3, and is included in a kit or diagnostic composition described herein Are antibodies specific for these proteins or specific for their homologues and / or truncated forms.

  Accordingly, further embodiments of the invention described herein include oligonucleotides or polypeptides that can determine the expression level of one or more of E-selectin, ICAM-1 and VEGFR-3. A kit useful for performing the method is provided. Oligonucleotides include primers and / or probes specific for mRNA encoding one or more of the markers / indicators described herein, and the polypeptide is a marker / indicator protein, eg, marker / indicator specific Includes proteins capable of specifically interacting with an antibody or antibody fragment.

  In addition to the methods described above, the present invention further provides for assessing or determining the expression level of one or more of E-selectin, ICAM-1 and VEGFR-3, eg, by Western blotting and ELISA based detection. It also includes immunoassays. Also, as understood in the art, the expression level of the marker / indicator protein of the present invention can be determined by mRNA by any suitable method known in the art, such as Northern blotting, real-time PCR, and RT PCR. Rated by level. Immunoassays and systems, as well as mRNA-based detection methods and systems are well known in the art, and standard textbooks such as Lottspeich (Bioanalytik, Spektrum Akademisher Verlag, 1998) or Sambrook and Russell (Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA, 2001). The methods described are for E-selectin, ICAM-1 and VEGFR in patients or patient groups compared to established control levels in populations diagnosed with breast cancer, particularly locally recurrent or metastatic HER2-positive breast cancer. Especially used to determine the expression level of -3.

  In addition, the expression level of one or more of E-selectin, ICAM-1 and VEGFR-3 can be expressed by immunoagglutination, immunoprecipitation (eg, immunodiffusion, immunoelectrophoresis, immunofixation), Western blot technique (eg, ( In situ) immunocytochemistry, affinity chromatography, enzyme immunoassay, etc. can be used to determine at the protein level. Also, the amount of purified polypeptide in solution is determined by physical methods such as photometry. The method of quantifying a particular polypeptide in a mixture usually depends on the specific binding of, for example, an antibody. Specific detection and quantification methods that take advantage of antibody specificity include, for example, immunoassays. For example, the concentration / amount of the marker / indicator protein of the present invention in a patient sample is determined by enzyme linked immunosorbent assay (ELISA). Alternatively, Western blot analysis or immunostaining can be performed. Western blotting combines the separation of a protein mixture by electrophoresis with specific detection using antibodies. Electrophoresis can be multidimensional like 2D electrophoresis. Polypeptides are usually separated in 2D electrophoresis by their apparent molecular weight along one dimension and their isoelectric point along the other dimension.

  As mentioned above, the expression level of the marker / indicator protein according to the present invention is also reflected in increased expression of the corresponding genes encoding E-selectin, ICAM-1 and VEGFR-3. Thus, a quantitative assessment of the gene product (spliced mRNA, unspliced mRNA, or partially spliced mRNA) prior to translation can be performed to assess the expression of the corresponding gene. One skilled in the art will know the standard methods used in this context or will be able to infer these methods from standard textbooks (eg, Sambrook, 2001, loc. Cit.). For example, quantitative data regarding the relative concentration / amount of mRNA encoding one or more of E-selectin, ICAM-1, and vEGFR-3 can be obtained by Northern blot, real-time PCR, and the like.

  In a further aspect of the invention, the kit of the invention is advantageously used to carry out the method of the invention and can be used in various applications, especially in the diagnostic field or as a research tool. The parts of the kit of the present invention can be individually packaged in vials or can be packaged in a container or combination of multiple container units. The manufacture of the kit preferably follows standard procedures known to those skilled in the art. Kits or diagnostic compositions can be used for E-selectin, ICAM-1, and VEGFR-3 in accordance with the methods of the invention described herein, eg, using the immunohistochemical techniques described herein. Used for detection of one or more expression levels.

  When used in the detection methods described herein, one of skill in the art has the ability to label a polypeptide, eg, antibody, or oligonucleotide encompassed by the present invention. As routinely practiced in the art, hybridization probes used for the detection of mRNA levels and / or antibodies or antibody fragments used in immunoassays are standard methods known in the art. Non-limiting examples of widely used systems that can be labeled and visualized according to, include radiolabels, enzyme labels, fluorescent tags, biotin-avidin complexes, chemiluminescence, and the like.

  The invention is further illustrated by the following non-limiting examples.

Example 1-Comparison of bevacizumab and trastuzumab / docetaxel combination with trastuzumab / docetaxel alone as first line therapy for patients with HER2 positive locally recurrent or metastatic breast cancer-AVEREL study The primary purpose of the clinical trials disclosed herein is to improve the survival rate of patients randomized to bevacizumab and trastuzumab / docetaxel combination versus patients randomized to trastuzumab / docetaxel alone (PFS) was to be compared. Secondary objectives are overall survival (OS); best overall response rate (OR); duration of response (DR); successful treatment period (TTF); safety and tolerability of combining bevacizumab with trastuzumab and docetaxel; It was to evaluate the quality of life.

  Specifically, the study described herein uses (1) a loading dose of 15 mg / kg bevacizumab + 8 mg / kg trastuzumab every 3 weeks, then 6 mg / kg every 3 weeks until disease progression + Using docetaxel 100 mg / 2 every 3 weeks for a minimum of 6 cycles, loading dose of trastuzumab 8 mg / kg, then 6 mg / kg every 3 weeks until disease progression + Docetaxel 100 mg / m2 every 3 weeks for a minimum of 6 Providing favorable treatment outcomes for the main variables of PFS when compared to cycle use; and (2) a loading dose of 15 mg / kg bevacizumab + 8 mg / kg trastuzumab every 3 weeks, then 6 mg / kg every 3 weeks Use at least 6 cycles of docetaxel 100 mg / m2 every 3 weeks It was used to determined to have an acceptable safety profile.

Study Design The trial was a randomized, open-label, 2 treatment group, multicenter, phase III trial. Patients were assigned to treatment groups on a 1: 1 basis by a central randomization process. A block design randomization procedure was used. In order to avoid an imbalance of important prognostic factors in the patient population between the two treatment groups, patients were stratified according to the following criteria:
Elapsed time since last administration of prior adjuvant / neoadjuvant / taxane / adjuvant / neoadjuvant chemotherapy. Patients were first stratified for prior treatment with taxanes (yes versus no). If “no taxane history”, the second stratification, ie, who has never received adjuvant / neoadjuvant chemotherapy, or the time elapsed since the last dose of chemotherapy <12 months, Elapsed time since last dose ≧ 12 months was implemented.
• Trastuzumab as part of adjuvant treatment for no trastuzumab • Hormone receptor (ER / PgR) status (positive vs. negative), and • Measurable disease (yes vs. no)

Patients were randomly assigned to one of the following two treatment groups.
Treatment group A trastuzumab loading dose of 8 mg / kg, then 6 mg / kg every 3 weeks until disease progression + docetaxel 100 mg / m2 every 3 weeks for a minimum of 6 cycles (or disease progression or unacceptable toxicity) Used up to the earlier of occurrence). After 6 cycles without progression or toxicity, docetaxel was continued for additional cycles at the investigator's discretion.
Treatment group B 8 mg / kg loading dose of trastuzumab, then 6 mg / kg every 3 weeks until disease progression + docetaxel 100 mg / m2 every 3 weeks for a minimum of 6 cycles (or disease progression or occurrence of unacceptable toxicity) (Whichever comes first). After 6 cycles without progression or toxicity, docetaxel was continued for additional cycles at the investigator's discretion, plus 15 mg / kg bevacizumab every 3 weeks until disease progression.

Table 1: Study summary and dosing regimen

Study duration 424 patients from 60 centers were recruited over approximately 29 months and followed for approximately 26 weeks for primary endpoint (PFS).

END OF STUDY This was an event driven test. The primary endpoint analysis was performed when 310 events were identified in 424 randomly assigned patients. An additional overall survival analysis was performed approximately 36 months after the last patient randomization, at which point the trial was terminated. The study ended on the last visit date of the last patient participating in the trial, which was coincident with the last overall survival analysis performed approximately 36 months after the final patient randomization.

  After this clinical cut-off for the final overall survival analysis, patients who benefited from study treatment were able to continue bevacizumab treatment until disease progression.

Number of patients / assignment to treatment groups where 424 patients were randomized 1: 1 into two study groups:
205 HER2 positive patients in the trastuzumab / docetaxel treatment group (treatment group A); and 205 HER2 positive patients in the bevacizumab + trastuzumab / docetaxel treatment group (treatment group B).

  Patients were randomly assigned to treatment groups. Patients received the initial dose of study treatment on the day of randomization, at the latest within 5 business days after randomization. Whatever the circumstances, patients enrolled in the study were re-randomized into the study and allowed to enroll in a second course of treatment.

Eligible Patient Registration Criteria Premenopausal and postmenopausal female and male patients with locally recurrent or metastatic HER2-positive breast cancer (excluding primary tumor-T4d inflammatory breast cancer). Patients were completed 3 weeks prior to randomization and could have previously received radiation therapy for metastatic breast cancer (MBC) if irradiation did not exceed 30% of bones including bone marrow . Prior adjuvant radiation therapy was allowed if completed at least 6 months prior to randomization.

  Patients who received trastuzumab as adjuvant therapy were allowed to enroll if ≧ 6 months had passed since the last adjuvant administration of trastuzumab. Patients had to have a baseline proper left ventricular ejection fraction defined as LVEF as measured by echocardiography or MUGA. Patients treated with anthracyclines for adjuvant disease could be included in the study if the maximum cumulative dose was 360 mg / m 2 or less doxorubicin or 720 mg / m 2 epirubicin.

  The patient has pre-menopausal or post-menopausal breast adenocarcinoma with histologically or cytologically confirmed HER2 positive, measurable or non-measurable locally recurrent or metastatic disease I had to. Patients must have good general condition (ECOG 0-1), normal liver, kidney, and bone marrow function and have other severe illnesses that can affect protocol compliance or interpretation of results. I didn't. The patient's risk of gastrointestinal perforation, hypertension, proteinuria, and wound healing complications, thromboembolism, or bleeding should not be high. Patients with metastatic CNS disease caused by metastasis or spinal cord compression were not eligible. The patient must have had no other primary tumor within the past 5 years (except for well-treated cervical CIS, squamous cell carcinoma of the skin, or basal cell skin cancer). Pregnant or breastfeeding women were excluded.

  As long as patients were at a stable level of anticoagulation for at least two weeks at the time of randomization, full anticoagulation at the time of study entry was allowed.

Eligibility criteria:
1. Patient age ≧ 18 years;
2. Follow the protocol;
3. ECOG PS ≦ 1;
4). Life expectancy ≧ 12 weeks 5. Premenopausal or postmenopausal patients with histologically or cytologically confirmed breast cancer (mammary adenocarcinoma) who are candidates for chemotherapy and are locally relapsed or metastatic that cannot be measured Patients with lesions (except primary tumor-T4d-inflammatory breast cancer). Locally recurrent disease had no possibility of resection for curative purposes. ER / PgR and HER2 status had to be documented.
6). Patients may have HER2 protein overexpression (3+) as determined by immunohistochemistry (IHC) of primary tumors or metastases confirmed by the central laboratory prior to randomization; or fluorescence in situ hybridization (FISH) or chromogenic. Had to have HER2 / c-erbB2 amplification determined by in situ hybridization (CISH). Confirmation of HER2 positivity of the primary tumor by the central laboratory is not required in this trial for patients who previously participated in an adjuvant trastuzumab funded by Roche or Genentech where HE2 status was confirmed centrally It was. (Eg, HERA, BCIRG006, NSABP B31, or Intergroup / NCCTG / H2061 trial);
7). Patients who received trastuzumab in adjuvant therapy were eligible unless they had relapsed within 6 months after the last dose of trastuzumab;
8). Patients treated with anthracyclines in adjuvant or non-adjuvant therapy are eligible only if the final dose is> 6 months prior to randomization. The maximum cumulative dose should not exceed 360 mg / m ² for doxorubicin and 720 mg / m ² for epirubicin.
9. Patients treated with taxanes were eligible only if the last adjuvant or non-adjuvant chemotherapy was> 12 months prior to randomization.
10. Baseline left ventricular ejection fraction (LVEF) measured by echocardiography or MUGA does not fall below 50%.
11. Use of a sufficient amount of oral or parenteral anticoagulant was permitted only as long as the patient's anticoagulation level was stable for at least two weeks at the time of randomization.
・ Patients with heparin treatment whose baseline PTT was 1.5 to 2.5 times that of patients before UNL or heparin treatment ・ Receiving heparin (LMWH) at a low molecular weight of 1.5 daily Patients: 2 mg / kg (enoxaparin) or appropriate dose of the corresponding anticoagulant, depending on the package insert. INR assessed at baseline of 2 consecutive measurements 1 to 4 days apart. Patients treated with coumarin derivatives that were 3.0. Patients who did not receive anticoagulant were treated with 1.5 times INR ≤ ULN and 1 with aPTT ≤ ULN within 7 days prior to randomization. Had to have .5 times.

Exclusion criteria:
1. Prior chemotherapy for metastatic or locally recurrent breast cancer. Prior hormonal treatment was allowed but had to be completed at least 2 weeks prior to randomization.
2. Prior chemotherapy for the treatment of metastatic breast cancer was not allowed in the following cases:
If more than 30% of bone, including bone marrow, has been irradiated. If the last part of chemotherapy is given within 3 weeks prior to randomization, it will end at least 6 months before randomization. If so, prior adjuvant radiation therapy for breast cancer was allowed.
3. Other primary tumors (including primary brain tumors) within the past 5 years prior to randomization. Exceptions were properly treated cervical carcinoma in situ, squamous cell carcinoma of the skin, or some appropriately controlled basal cell skin cancers.
4). Evidence of spinal cord compression or latest evidence of CNS metastasis. If there was clinical suspicion of brain metastasis, a CT or MRI scan of the brain was required (within 4 weeks prior to randomization).
5. 5. History or evidence from physical / neurological examination of CNS disease (unrelated to cancer) (unless properly treated with standard medical therapy), eg uncontrolled seizures. Prediction that major surgical procedures, direct biopsies, or major trauma, or major surgery will be required during the course of research treatment within 28 days prior to the start of study treatment. Preexisting peripheral neuropathy at the time of randomization> CTC grade 2
8). Insufficient bone marrow function: ANC <1.5 × 109 / L, platelet count <100 × 109 / L and Hb <9 g / dL.
9. Insufficient liver function:
Serum (total) bilirubin> ULN
・ AST and ALT> 2.5 × ULN
AST or ALT> 1.5 × ULN, at the same time serum alkaline phosphatase level> 2.5 × baseline ULN
10. Insufficient renal function:
i. Serum creatinine> 2.0 mg / dL or 177 μmol / L
ii. Urinary dipstick for proteinuria ≧ 2 +. Patients with baseline proteinuria ≧ 2 + on dipstick urinalysis should collect 24-hour urine and show ≦ 1 g protein / 24 hours.
11. Long-term daily treatment with corticosteroids (> 10 mg / day dose of methylprednisolone equivalent) (excluding inhaled steroids)
12 Long-term daily treatment with aspirin (> 325 mg / day) or clopidogrel (> 75 mg / day) 13. Uncontrolled hypertension (systolic> 150 mmHg and / or diastolic 100 mmHg) or clinically significant (ie active) cardiovascular disease: CVA / beat (≤6 months prior to randomization), myocardial infarction (no 13. Pre-randomization ≤ 6 months), unstable angina, New York Heart Association (NYHA, package 6) Congestive heart failure of class 2 or higher, or serious arrhythmia requiring medication. 15. History or evidence of an inherited hemorrhagic predisposition or coagulation disorder at risk of bleeding History of abdominal fistula, gastrointestinal perforation, or intraperitoneal tumor within 6 months prior to randomization 16. Active infections that require intravenous antibiotics at the time of randomization 17. 18. Serious non-healing wound, peptic ulcer, or fracture Any other disease, evidence of metabolic or mental dysfunction, a disease or condition contraindicated for the use of study drug, or a disease that may affect patient routine compliance or increase the risk of patient complications Or a clinical laboratory or physics laboratory finding that gives a reasonable suspicion of the condition 19. A pregnant or entrusted woman. Serum pregnancy test will be evaluated within 7 days prior to study treatment or within 14 days of urine pregnancy test for confirmation within 7 days prior to study treatment.
20. Non-hormonal means of effective birth control (intrauterine device combined with sperm jelly or birth control, contraceptive barrier method), possibly pregnant (women after last menstruation <2 years) )
21. 21. Current or recent (within 30 days prior to start of study treatment), treatment with another study drug, or participation in another study Known hypersensitivity to either study drug or excipients 23. Hypersensitivity to Chinese hamster ovary cell products or other recombinant human or humanized antibodies

result:
Improvements in investigator-assessed PFS (not stratified for treatments other than those specified in the protocol (NPT) and not censored) are, as described above, PFS in the study control treatment group. It was calculated to be 2.8 months longer than Specifically, as summarized in Table 2, the median PFS was 13.7 months in the control group (treatment group A) versus 16.5 in the “bevacizumab” group (treatment group B). The hazard ratio was 0.82 at 95% CI 0.65, 1.02), p-value = 0.0775.

Table 2: PFS by investigator evaluation

  In addition, the PFS results from the independent review committee (IRC) assessment, stratified and censored for NPT, were statistically significant. Specifically, as summarized in Table 3, the median PFS was 13.9 months for the control group (treatment group A) versus 16.8 for the “bevacizumab” group (treatment group B). The hazard ratio was 0.72 at 95% CI (0.54, 0.94), p-value = 0.0162. In general, the median PFS in the “Treatment Group B” or “Bevacizumab” group was 2.9 months longer than the PFS in the study control treatment group.

Table 3: Independent Review Committee (IRC) PFS

  Table 4 shows both the investigator evaluation results (INV) and the independent review committee (IRC) evaluation results as the objective response rate (ORR) results of the study. In particular, for the INV-rated ORR, the study showed 69.9% in the control treatment group versus 74.3% in “bevacizumab” or treatment group B. The difference between the two was 95% CI (-5.2%, 14.0%), 4.43%, and the p-value was 0.3492. For IRC-evaluated ORRs, the study showed 65.9% in the control treatment group versus 76.5% in “bevacizumab” or treatment group B. The difference between the two was 95% CI (1.0%, 20.2%), 10.59%, and the p value was 0.0265.

Table 4: Objective response rate (ORR)

  The provisional overall survival (OS) results of the study are summarized in Table 5, which shows a median survival rate of 38.3 months in the control treatment group compared to 38.3% in the “bevacizumab” treatment group B. 5 months, 95% CI (0.74, 1.38), hazard ratio (HR) of 1.01, and p-value of 0.9543.

Table 5: Provisional overall survival (OS)

  In addition, a preliminary safety assessment demonstrated that there were no new safety signals presented by patients in this study.

Example 2 Exploratory Biomarker Analysis Patients and Immunochemical Methods in the AVEREL Study In this study, plasma baseline samples for analysis were available from 162 patients.

Plasma analysis Plasma for biomarker discovery and confirmation was collected from patients who agreed to study BO20231. Plasma (total of about 20 mL) was collected at baseline (after randomization and before the first dose of study drug) and at the time of disease progression.

  A total of 4.9 mL of blood was drawn into S-monovette® (EDTA) tubes. Immediately thereafter, it was mixed by gently inverting the tube and centrifuged at 1500 g in a centrifuge within 30 minutes (10 minutes at room temperature). Immediately thereafter, the supernatant plasma was dispensed into a clear polypropylene 5 mL transfer tube. The plasma was then dispensed into 2 plastic storage tubes (approximately 1.25 ml each). Samples were stored at -70 ° C in an upright position. In some cases, samples were stored at -20 ° C for up to 1 month and then transferred to -70 ° C.

  Samples were prepared using interleukin-8 (Il-8), intercellular adhesion molecule 1 (ICAM-1), VEGFA, VEGF-C, VEGF receptor-using Roche Diagnostics GmbH's immunological MultiParameter Chip Technique (IMPACT). Of 1 (VEGFR1), VEGF receptor 2 (VEGFR2), VEGF receptor-3 (VEGFR3), basic fibroblast growth factor (bFGF), platelet derived growth factor-C (PDGF-C), and E-selectin levels. Used for measurement.

IMPACT Multiplex Assay Technology Roche Professional Diagnostics (Roche Diagnostics GmbH) has developed a multi-marker platform under the use name IMPACT (immunology multi-parameter chip technology). This technique was used to measure protein markers as described above in the “Plasma Analysis” section. This technique is based on small polystyrene chips made by the procedure disclosed in EP0939319 and EP161129. For all assays, the chip surface was coated with a streptavidin layer, and then a biotinylated antibody was placed in a patch on it. For each marker, antibody plaques were added vertically on the chip. During the assay, the array was probed with a specimen sample containing a specific analyte.

The plasma volume required per specimen to measure all markers on the chip was 20 μl for chip 1 and 8 μL for chip 2 and chip 3 (see below). To bring the total reaction dose per chip to 40 μl, the sample volume was incubation buffer (50 mM HEPES, pH 7.2, 150 mM NaCl, 0.1% Thesit, 0.5% bovine serum albumin, and preservatives). As 0.1% Oxypyrion). After a 12 minute incubation and a chip wash with wash buffer (5 mM Tris, pH 7.9, 0.01% Thesit and 0.001% oxypyrion), a conjugated detection antibody is added ( 40 μL of incubation buffer containing a mixture of analyte-specific antibodies labeled with Digixigenin) and incubated for an additional 6 minutes to bind onto the captured analyte. 40 μL reagent buffer (62.5 mM TAPS, pH 8.7, 1.25 M NaCl, 0.5% bovine serum albumin, 0.063% TWEEN 20 and 0.1% containing anti-digoxigenin antibody conjugate conjugated to fluorescent latex After washing with oxypyrion), the second antibody was finally detected. Using this label, ten individual binding events could be detected in a single spot, resulting in very high sensitivity up to a concentration of fmol / L. The chip was transported to a detection unit and an image converted to signal intensity using dedicated software was generated by a charge coupled device (CCD) camera. Individual spots were automatically located at predetermined positions and quantified by image analysis. For each marker, a line of 10-12 spots was placed on the chip and the marker concentration was calculated as the average of at least 5 spots from each line on the chip. The advantage of this technique is that it can multiplex up to 10 parameters in a sandwich or competitive format. Calibrator and patient samples were measured in duplicate. A single run was designed to include a total of 100 decisions, including calibrators and 2 multiple controls as run controls. Since some of the selected analytes react with each other (ie, VEGFA and VEGFR1 or VEGFR2), the analyte was divided into three different chips as follows:
Chip 1: VEGFA, VEGF-C, PDGF-C
Chip 2: VEGFR1, VEGFR2, VEGFR3, Il-8, bFGF,
Chip 3: E-selectin, ICAM-1

Table 6. Antibodies used in this assay

Statistical analysis Sample median was used to bisect biomarker values to low (less than median) or high (above median).

  The hazard ratio of treatment effect in a subgroup of patients using high or low biomarker values was estimated using proportional hazard cox regression analysis.

  In addition, proportional hazard Cox regression was used to assess the association between myomarker values and treatment effects. The model consists of the following covariates: study treatment, biomarker levels, binary stratification factors (ER / PgR status, disease measurable at baseline, preadjuvant / neoadjuvant taxane / adjuvant / neoadjuvant chemotherapy The time course after the last dose, prior trastuzumab neoadjuvant therapy), and treatment interaction terms with biomarker levels. An interaction term Wald test was used to determine the association between mio marker levels and therapeutic effects. P values below 0.05 were considered significant.

Statistical Method Analysis Population In this study, the biomarker-evaluable population is the following biomarkers administered with any component of the study drug and evaluated as described above and commercially available antibodies: VEGF-A, VEGF Consists of all patients who had marker levels at baseline for any of C, VEGF-R1, VEGF-R2, E-selectin VEGFR-3, IL-8, bFGF, PDGF-C, ICAM-1 Defined to be.

Tuning for diversity Hierarchies were applied to the biomarkers shown below to avoid the expansion of type I errors. Each biomarker was tested at the two-sided 5% alpha level, and the next biomarker in the hierarchy was tested only if there was statistical significance.
1. VEGF-A
2. VEGF-R2
3. ICAM-1
4). bFGF
5. IL-8
6). VEGF-R1
7). PDGF-C
8). VEGF-C
9. VEGFR-3
10. E-selectin

Analysis of Effectiveness PFS and OS are defined as specified in the Data Reporting Analysis Manual (DRAM) of Research BO20231. Biomarker data analysis was based on PFS (as assessed by investigator assessment) data at the time of the last PFS analysis. The sample median biomarker concentration was used as a cut point for patients in the group (high level versus low level).

Analysis of progression-free analysis The following analysis was performed on the PFS of the biomarker-evaluable population.
Median PFS (95% CI) estimated based on Kaplan-Meier curves for patients with low and high biomarker levels
Unstratified based on Cox model of patients with low and high biomarker levels (& HR stratified for PFS (95% CI)
-Unstratified HR (95% CI) based on the Cox model with quartiles at the biomarker level
To assess whether biomarkers can predict the therapeutic effects of bevacizumab on PFS in patients with HER2-positive locally recurrent or metastatic breast cancer, baseline biomarker levels (high and low in the sample median) Perform a biomarker-evaluable patient correlation test using a Cox model that includes treatment, baseline prognostic factors, and interaction terms (baseline biomarker level with treatment) as a binary variable dichotomized as low It was done.

result
Baseline descriptive statistics for plasma marker biomarkers are presented in Table 7.

Table 7: Descriptive statistics of biomarker values (baseline)

  Table 8 presents the results of an analysis of the association of E-selectin, ICAM-1 and VEGFR-3 with treatment effects on progression-free survival by investigator assessment.

Table 8

  In this analysis, E-selectin is high (≧ 36.9 ng / mL), E-selectin is low (<36.9 ng / mL), and ICAM-1 is high (≧ 3). 210 ng / mL), ICAM-1 is low (<210 ng / mL), VEGFR-3 is high (≧ 10.6 ng / mL), and VEGFR-3 is low (<10.6 ng / mL) Used.

  High baseline ICAM-1 was statistically significantly correlated with increased PFS benefit from bevacizumab (α = 0.05). VEGFR-3 and E-selectin showed potential as predictive values. Thus, ICAM-1, E-selectin, and VEGFR-3 could be independent predictive biomarkers for the therapeutic effect of bevacizumab on progression-free survival.

Moreover, in the exploratory quartile analysis of ICAM-1, the effect of ICAM-1 on PFS was clear (Table 9).

Claims (43)

An in vitro method for determining whether a patient diagnosed with breast cancer is adequately treated to a greater or lesser extent by anti-cancer therapy including anti-VEGF antibodies,
(A) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with breast cancer; and (b) Based on the expression level according to (a), the patient is identified as being appropriately treated to some extent by anti-cancer therapy including an anti-VEGF antibody, and the expression level of the biomarker is above the reference level In some cases, the patient is shown to be more suitable for treatment with anti-cancer therapy, or when the expression level of the biomarker is below a reference level, the patient is shown to be less suitable for treatment with anti-cancer therapy. A method comprising identifying.   The method of claim 1, wherein in terms of progression free survival, it is determined whether the patient is properly treated with anti-cancer therapy.   3. The method of claim 1 or 2, further comprising treating the patient with anticancer therapy.   The method according to any one of claims 1 to 3, wherein the anticancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody, and a taxane. An in vitro method of selecting treatment for a patient diagnosed with breast cancer, comprising:
(A) By assaying a biological sample derived from a patient, the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in the patient is equal to or higher than a reference level. And (b) selecting a treatment comprising an anti-cancer therapy comprising an anti-VEGF antibody based on the determination.   6. The method of claim 5, further comprising treating the patient with anti-cancer therapy.   The method according to claim 5 or 6, wherein the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody, and a taxane. An in vitro method for determining whether a patient diagnosed with breast cancer is susceptible to anti-cancer therapy including the addition of anti-VEGF antibodies to chemotherapy,
(A) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with breast cancer; and (b) Based on the expression level according to (a), identifying a patient as sensitive to anti-cancer therapy including addition of anti-VEGF antibody to chemotherapy, wherein the expression level of the biomarker is above a reference level Identifying, wherein the patient is shown to be susceptible to anti-cancer therapy including the addition of anti-VEGF antibodies to chemotherapy.   9. The method of claim 8, further comprising treating the patient with anticancer therapy.   10. The method of claim 8 or 9, wherein whether a patient is sensitive to an anti-cancer therapy that includes the addition of anti-VEGF antibodies to chemotherapy is determined in terms of progression-free survival.   11. The method according to any one of claims 8 to 10, wherein the chemotherapy comprises an anti-HER2 antibody and a taxane. An in vitro method of selecting an anti-cancer therapy for a patient diagnosed with breast cancer, comprising:
(A) When the patient-derived biological sample is assayed, the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 is equal to or higher than a reference level. And (b) selecting an anti-cancer therapy comprising the addition of an anti-VEGF antibody to the chemotherapy based on the determination.   13. The method of claim 12, further comprising treating the patient with anticancer therapy.   14. The method of claim 12 or 13, wherein the chemotherapy comprises an anti-HER2 antibody and a taxane.   15. The method of any one of claims 1 to 14, wherein the patient has been diagnosed with locally recurrent or metastatic HER2-positive breast cancer.   16. The method according to any one of claims 1 to 15, wherein the patient has never received chemotherapy or radiation therapy.   The method according to any one of claims 1 to 16, wherein the anti-VEGF antibody is bevacizumab.   18. A method according to any one of claims 4, 7, 11, and 14 to 17, wherein the taxane is docetaxel or paclitaxel.   The method of claim 18, wherein the taxane is doxtaxel.   20. The method according to any one of claims 4, 7, 11, and 14 to 19, wherein the anti-HER2 antibody is trastuzumab.   21. The method according to any one of claims 1 to 20, wherein the expression level is a protein expression level.   The method according to any one of claims 1 to 21, wherein the sample is a plasma sample.   23. The method of claim 22, wherein the expression levels of E-selectin, ICAM-1, and VEGFR-3 are about 36.9 ng / mL, about 210 ng / mL, and about 10.6 ng / mL, respectively.   24. The method of any one of claims 1 to 23, wherein the at least one biomarker is E-selectin.   24. The method of any one of claims 1 to 23, wherein the at least one biomarker is ICAM-1.   24. The method of any one of claims 1 to 23, wherein the at least one biomarker is VEGFR-3.   27. A pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient receives an anti-VEGF antibody according to the method of any one of claims 1-4, 15-26. A pharmaceutical composition that has been identified as being highly suitable for treatment with an anti-cancer therapy.   27. A pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient is treated with an anti-VEGF antibody against a chemotherapy regimen according to the method of any one of claims 8 to 26. A pharmaceutical composition that has been identified as being sensitive to anti-cancer therapy, including additional.   A set of compounds for detecting the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3, which can specifically bind to the biomarker 27. A kit for performing the method according to any one of claims 1 to 26, comprising a set of compounds comprising: A method for improving the therapeutic effect of anticancer therapy, including chemotherapy, on a patient diagnosed with breast cancer by adding an anti-VEGF antibody to the chemotherapy regimen, comprising:
(A) determining the expression level of at least one biomarker selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample from a patient diagnosed with positive breast cancer;
(B) identifying a patient as being sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen based on the expression level according to (a), wherein the expression level of the biomarker is above a reference level Wherein the patient is shown to be susceptible to anti-cancer therapy including the addition of anti-VEGF antibodies to a chemotherapy regimen; and
(C) administering an effective amount of an anti-VEGF antibody in combination with an effective amount of a chemotherapeutic regimen to a patient identified by (b) as being sensitive to anti-cancer therapy including the addition of anti-VEGF antibody to chemotherapy A method involving that.   32. The method of claim 30, wherein the chemotherapy regimen comprises an anti-HER2 antibody and a taxane.   32. The method of claim 30 or 31, wherein the patient has been diagnosed with locally recurrent or metastatic HER2-positive breast cancer.   33. A method according to any one of claims 30 to 32, wherein the patient has never received chemotherapy or radiation therapy.   34. The method of any one of claims 30 to 33, wherein the anti-VEGF antibody is bevacizumab.   32. The method of claim 31, wherein the taxane is doxtaxel or paclitaxel.   36. The method of claim 35, wherein the taxane is doxtaxel.   32. The method of claim 31, wherein the anti-HER2 antibody is trastuzumab.   38. The method of any one of claims 30 to 37, wherein the expression level is a protein expression level.   39. A method according to any one of claims 30 to 38, wherein the sample is a plasma sample.   40. The method of claim 39, wherein the expression levels of E-selectin, ICAM-1, and VEGFR-3 are about 36.9 ng / mL, about 210 ng / mL, and about 10.6 ng / mL, respectively.   41. The method according to any one of claims 30 to 40, wherein the at least one biomarker is E-selectin.   41. The method of any one of claims 30 to 40, wherein the at least one biomarker is ICAM-1.   41. The method of any one of claims 30 to 40, wherein the at least one biomarker is VEGFR-3.