WO2014001232A1 - Blood plasma biomarkers for bevacizumab combination therapies for treatment of breast cancer - Google Patents

Blood plasma biomarkers for bevacizumab combination therapies for treatment of breast cancer Download PDF

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Publication number
WO2014001232A1
WO2014001232A1 PCT/EP2013/063094 EP2013063094W WO2014001232A1 WO 2014001232 A1 WO2014001232 A1 WO 2014001232A1 EP 2013063094 W EP2013063094 W EP 2013063094W WO 2014001232 A1 WO2014001232 A1 WO 2014001232A1
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WIPO (PCT)
Prior art keywords
patient
biomarker
expression level
vegf antibody
icam
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PCT/EP2013/063094
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English (en)
French (fr)
Inventor
Ursula Klause
Nicola MOORE
Céline PALLAUD
Stefan Scherer
Norbert Wild
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F. Hoffmann-La Roche Ag
Hoffmann-La Roche Inc.
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Application filed by F. Hoffmann-La Roche Ag, Hoffmann-La Roche Inc. filed Critical F. Hoffmann-La Roche Ag
Priority to BR112014032456A priority Critical patent/BR112014032456A2/pt
Priority to RU2015102026A priority patent/RU2015102026A/ru
Priority to CA2871385A priority patent/CA2871385A1/en
Priority to KR20147035874A priority patent/KR20150024342A/ko
Priority to CN201380030336.6A priority patent/CN104364655A/zh
Priority to JP2015519012A priority patent/JP2015522815A/ja
Priority to EP13730890.4A priority patent/EP2864788A1/en
Priority to MX2014014821A priority patent/MX2014014821A/es
Publication of WO2014001232A1 publication Critical patent/WO2014001232A1/en
Priority to US14/577,138 priority patent/US20150352204A1/en
Priority to HK15102817.0A priority patent/HK1202334A1/xx

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors

Definitions

  • the present invention is directed to methods for identifying which patients diagnosed with breast cancer will most benefit from treatment with an anti-cancer therapy comprising an anti-VEGF antibody.
  • Angiogenesis contributes to benign and malignant diseases such as cancer development and, especially in cancer, is necessary for primary tumor growth, invasiveness and metastasis.
  • a tumor In order to grow, a tumor must undergo an angiogenic switch.
  • Vascular endothelial growth factor (VEGF) is required to induce this angiogenic switch.
  • VEGF and the genes in the VEGF pathway are considered important mediators of cancer progression.
  • the VEGF gene family includes the VEGF gene, also referred to as VEGFA, homologues to VEGF including, placenta growth factor (P1GF), VEGFB, VEGFC, VEGFD, the VEGF receptors, including VEGFR-1 and VEGFR-2 (also referred to as FLT1 and FLK1/KDR, respectively), the VEGF inducers, including hypoxia- inducible factors HIFl , HIF2 a, and the oxygen sensors PHD1, PHD2 and PHD3.
  • VEGFA placenta growth factor
  • VEGFB placenta growth factor
  • VEGFC VEGFC
  • VEGFD VEGFR-1 and VEGFR-2
  • the VEGF inducers including hypoxia- inducible factors HIFl , HIF2 a
  • angiogenesis inhibitors such as bevacizumab
  • pegaptanib sunitinib
  • sunitinib sunitinib
  • sorafenib vatalanib
  • vatalanib vatalanib
  • angiogenesis inhibitors such as bevacizumab
  • patients still succumb to cancer.
  • not all patients respond to angiogenesis inhibitor therapy.
  • the mechanism underlying the non-responsiveness remains unknown.
  • angiogenesis inhibitor therapy is associated with side effects, such as gastrointestinal perforation, thrombosis, bleeding, hypertension and proteinuria.
  • the present invention therefore relates to a method of determining whether a patient diagnosed with breast cancer is sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen, by determing an expression level of at least one biomarker which is selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a patient sample and comparing it with reference levels.
  • the present invention also relates to a pharmaceutical composition comprising an anti-VEGF antibody, such as bevacizumab, for the treatment of a patient diagnosed with breast cancer and having an increased expression level of at least one biomarker which is selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 relative to reference levels.
  • the present invention further relates to a method for improving the treatment effect of an anti-cancer therapy comprising a chemotherapy of a patient diagnosed with breast cancer by adding an anti-VEGF antibody, such as bevacizumab, based on an expression level of at least one biomarker which is selected from the group consisting of E- selectin, ICAM-1 and VEGFR-3 in a patient sample.
  • an anti-VEGF antibody such as bevacizumab
  • One embodiment of the invention provides in vitro methods of determining whether a patient diagnosed with breast cancer is more or less suitably treated by an anti-cancer therapy comprising an anti-VEGF antibody.
  • the methods comprise (a) determining an expression level of at least one biomarker which is selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample derived from a patient diagnosed with breast cancer, and (b) identifying the patient as more or less suitably treated by an anti-cancer therapy comprising an anti-VEGF antibody based on the expression level in accordance with (a), wherein an expression level of said biomarker at or above a reference level indicates that the patient is more suitably treated with the anti-cancer therapy, or an expression level of said biomarker below a reference level indicates that the patient is less suitably treated with the anti-cancer therapy.
  • the methods further comprise treating the patient with the anticancer therapy.
  • the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane.
  • the anti- VEGF antibody is bevacizumab.
  • the anti-HER2 antibody is trastuzumab.
  • the taxane is docetaxel or paclitaxel.
  • the expression level of said at least one bio marker is a protein expression level.
  • the sample derived from the patient is a blood plasma sample.
  • the at least one biomarker is E-selectin.
  • the at least one biomarker is ICAM-1.
  • the at least one biomarker is VEGFR-3.
  • Another embodiment of the invention provides in vitro methods of selecting a treatment for a patient diagnosed with breast cancer.
  • the methods comprise (a) assaying a biological sample from the patient, thereby determining that the patient has an expression level of at least one biomarker which is selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 at or above a reference level, and (b) based on that determination, selecting the treatment comprising an anti-cancer therapy comprising an anti-VEGF antibody.
  • whether a patient is suitably treated by an anti-cancer therapy is determined in terms of progression-free survival.
  • the methods further comprise treating the patient with the anticancer therapy.
  • the patient received no previous chemotherapeutic or radiation treatment.
  • the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane.
  • the anti-VEGF antibody is bevacizumab.
  • the anti-HER2 antibody is trastuzumab.
  • the taxane is docetaxel or paclitaxel.
  • the expression level of said at least one biomarker is a protein expression level.
  • the sample derived from the patient is a blood plasma sample.
  • the at least one biomarker is E-selectin.
  • the at least one biomarker is ICAM-1.
  • the at least one biomarker is VEGFR-3.
  • a further embodiment of the invention provides in vitro methods of determining whether a patient diagnosed with breast cancer is sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen.
  • the methods comprise (a) determining an expression level of at least one biomarker which is selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample derived 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 a chemotherapy regimen based on the expression level in accordance with (a), wherein an expression level of said biomarker at or above a reference level indicates that the patient is sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen.
  • the methods further comprise treating the patient with the anticancer therapy.
  • the patient received no previous chemotherapeutic or radiation treatment.
  • the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane.
  • the anti-VEGF antibody is bevacizumab.
  • the anti-HER2 antibody is trastuzumab.
  • the taxane is docetaxel or paclitaxel.
  • the expression level of said at least one biomarker is a protein expression level.
  • the sample derived from the patient is a blood plasma sample.
  • the at least one biomarker is E-selectin.
  • the at least one biomarker is ICAM-1.
  • the at least one biomarker is VEGFR-3.
  • Even another embodiment of the invention provides in vitro methods of selecting an anti-cancer therapy for a patient diagnosed with breast cancer.
  • the methods comprise (a) assaying a biological sample from the patient, thereby determining that the patient has an expression level of at least one biomarker which is selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 at or above a reference level, and (b) based on that determination, selecting the anticancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen.
  • whether a patient is suitably treated by an anti-cancer therapy is determined in terms of progression- free survival.
  • the methods further comprise treating the patient with the anticancer therapy.
  • the anticancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane.
  • the anti-VEGF antibody is bevacizumab.
  • the anti-HER2 antibody is trastuzumab.
  • the taxane is docetaxel or paclitaxel.
  • the expression level of said at least one biomarker is a protein expression level.
  • the sample derived from the patient is a blood plasma sample.
  • the at least one biomarker is E-selectin.
  • the at least one biomarker is ICAM-1.
  • the at least one biomarker is VEGFR-3.
  • compositions comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient has been identified as more suitably treated by an anti-cancer therapy comprising an anti- VEGF antibody in accordance with any of the methods described herein.
  • compositions comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient is identified as more suitably treated by an anti-cancer therapy comprising an anti-VEGF antibody in accordance with any of the methods described herein.
  • a further embodiment of the invention provides pharmaceutical compositions comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient has been identified as sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen in accordance with any of the methods described herein.
  • a further embodiment of the invention provides pharmaceutical compositions comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient is identified as sensitive to an anti-cancer therapy comprising the addition of an anti- VEGF antibody to a chemotherapy regimen in accordance with any of the methods described herein.
  • kits for carrying out any of the method described herein.
  • the kits comprise a set of compounds for detecting an expression level of at least one biomarker which is selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3, the set comprising antibodies capable of specifically binding to said biomarker.
  • Even another embodiment of the invention provides methods for improving the treatment effect of an anti-cancer therapy comprising a chemotherapy regimen in a patient diagnosed with breast cancer by adding an anti-VEGF antibody to the chemotherapy regimen.
  • the methods comprise (a) determining an expression level of at least one biomarker which is selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample derived from a patient diagnosed with positive breast cancer; (b) identifying the patient as sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen based on the expression level in accordance with (a), wherein an expression level of said biomarker at or above a reference level indicates that the patient is sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen; and (c) administering an effective amount of an anti-VEGF antibody in combination with an effective amount of a chemotherapy regimen to the patient identified as sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy
  • the methods further comprise treating the patient with the anticancer therapy.
  • the patient received no previous chemotherapeutic or radiation treatment.
  • the anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane.
  • the anti-VEGF antibody is bevacizumab.
  • the anti-HER2 antibody is trastuzumab.
  • the taxane is docetaxel or paclitaxel.
  • the expression level of said at least one biomarker is a protein expression level.
  • the sample derived from the patient is a blood plasma sample.
  • the at least one biomarker is E-selectin.
  • the at least one biomarker is ICAM-1.
  • the at least one biomarker is VEGFR-3.
  • administration means the administration of a pharmaceutical composition, such as an angiogenesis inhibitor, to a patient in need of such treatment or medical intervention by any suitable means known in the art.
  • routes of administration include by oral, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration (for example as effected by inhalation).
  • parenteral administration e.g., intravenous administration.
  • anti-angiogenesis agent or "angiogenesis inhibitor” refers to a small molecular weight substance, a polynucleotide, a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly. It should be understood that the anti-angiogenesis agent includes those agents that bind and block the angiogenic activity of the angiogenic factor or its receptor.
  • an anti-angiogenesis agent is an antibody or other antagonist to an angiogenic agent as defined throughout the specification or known in the art, e.g., but are not limited to, antibodies to VEGF-A or to the VEGF-A receptor (e.g., KDR receptor or Fit- 1 receptor), VEGF-trap, anti-PDGFR inhibitors such as GleevecTM (Imatinib Mesylate).
  • Anti-angiogensis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and DAmore, Annu. Rev.
  • antibody is herein used in the broadest sense and includes, but is not limited to, monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, CDR grafted antibodies, humanized antibodies, camelized antibodies, single chain antibodies and antibody fragments and fragment constructs, e.g., F(ab') 2 fragments, Fab- fragments, Fv-fragments, single chain Fv-fragments (scFvs), bispecific scFvs, diabodies, single domain antibodies (dAbs) and minibodies.
  • monoclonal and polyclonal antibodies multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, CDR grafted antibodies, humanized antibodies, camelized antibodies, single chain antibodies and antibody fragments and fragment constructs, e.g., F(ab') 2 fragments, Fab- fragments, Fv-fragments, single chain Fv-fragments (scFvs), bispecific scFvs, di
  • VEGF refers to, e.g. the 165-amino acid human vascular endothelial cell growth factor and related 121-, 189-, and 206- amino acid human vascular endothelial cell growth factors, as described by Leung et al. (1989) Science 246: 1306, and Houck et al. (1991) Mol. Endocrin, 5: 1806, together with the naturally occurring allelic and processed forms thereof, including the no- amino acid human vascular endothelial cell growth factor generated by plasmin cleavage of VEGFi 6 5 as described in Ferrara Mol. Biol. Cell 21 :687 (2010).
  • VEGF also refers to VEGFs from non-human species such as mouse, rat or primate. Sometimes the VEGF from a specific species are indicated by terms such as hVEGF for human VEGF, mVEGF for murine VEGF, and etc.
  • VEGF is also used to refer to truncated forms of the polypeptide comprising amino acids 8 to 109 or 1 to 109 of the 165-amino acid human vascular endothelial cell growth factor. Reference to any such forms of VEGF may be identified in the present application, e.g., by "VEGF (8-109),” “VEGF (1-109)” or
  • VEGFi65 The amino acid positions for a "truncated" native VEGF are numbered as indicated in the native VEGF sequence. For example, amino acid position 17 (methionine) in truncated native VEGF is also position 17 (methionine) in native VEGF.
  • the truncated native VEGF has binding affinity for the KDR and Fit- 1 receptors comparable to native VEGF.
  • VEGF biological activity includes binding to any VEGF receptor or any VEGF signaling activity such as regulation of both normal and abnormal angiogenesis and vasculogenesis (Ferrara and Davis-Smyth (1997) Endocrine Rev. 18:4-25; Ferrara (1999) J. Mol. Med. 77:527- 543); promoting embryonic vasculogenesis and angiogenesis (Carmeliet et al. (1996) Nature 380:435-439; Ferrara et al. (1996) Nature 380:439-442); and modulating the cyclical blood vessel proliferation in the female reproductive tract 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).
  • VEGF as a pleio tropic growth factor, exhibits multiple biological effects in other
  • VEGF antagonist or "VEGF-specific antagonist” refers to a molecule capable of binding to VEGF, reducing VEGF expression levels, or neutralizing, blocking, inhibiting, abrogating, reducing, or interfering with VEGF biological activities, including, but 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 are polypeptides that specifically bind to VEGF, anti-VEGF antibodies and antigen- binding fragments thereof, receptor molecules and derivatives which bind specifically to VEGF thereby sequestering its binding to one or more receptors, fusions proteins (e.g., VEGF-Trap (Regeneron)), and VEGFm-gelonin (Peregrine).
  • VEGF-specific antagonists also include antagonist variants of VEGF polypeptides, antisense nucleobase oligomers directed to VEGF, small RNA molecules directed to VEGF, RNA aptamers, peptibodies, and ribozymes against VEGF.
  • VEGF-specific antagonists also include nonpeptide small molecules that bind to VEGF and are capable of blocking, inhibiting, abrogating, reducing, or interfering with VEGF biological activities.
  • VEGF activities specifically includes VEGF mediated biological activities of VEGF.
  • the VEGF antagonist reduces or inhibits, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, the expression level or biological activity of VEGF.
  • an "anti-VEGF antibody” is an antibody that binds to VEGF with sufficient affinity and specificity.
  • the antibody selected will normally have a sufficiently binding affinity for VEGF, for example, the antibody may bind hVEGF with a Ka value of between 100 nM-1 pM.
  • Antibody affinities may be determined by a surface plasmon resonance based assay (such as the BIAcore assay as described in PCT Application Publication No.
  • the anti-VEGF antibody can be used as a therapeutic agent in targeting and interfering with diseases or conditions wherein the VEGF activity is involved.
  • the antibody may be subjected to other biological activity assays, e.g., in order to evaluate its effectiveness as a therapeutic.
  • Such assays are known in the art and depend on the target antigen and intended use for the antibody.
  • HUVEC inhibition assay examples include the HUVEC inhibition assay; tumor cell growth inhibition assays (as described in WO 89/06692, for example); antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (US Patent 5,500,362); and agonistic activity or hematopoiesis 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 P1GF, PDGF or bFGF.
  • anti-VEGF antibody is a monoclonal antibody that binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709.
  • the anti-VEGF antibody is a recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al. (1997) Cancer Res. 57:4593-4599, including but not limited to the antibody known as bevacizumab (BV; AVASTIN ® ).
  • Bevacizumab also known as “rhuMAb VEGF” or
  • AVASTIN ® is a recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al. (1997) Cancer Res. 57:4593-4599. It comprises mutated human IgGl framework regions and antigen-binding complementarity-determining regions from the murine anti-hVEGF monoclonal antibody A.4.6.1 that blocks binding of human VEGF to its receptors. Approximately 93% of the amino acid sequence of Bevacizumab, including most of the framework regions, is derived from human IgGl, and about 7% of the sequence is derived from the murine antibody A4.6.1. Bevacizumab has a molecular mass of about 149,000 daltons and is glycosylated. Bevacizumab and other humanized anti-VEGF antibodies are further described in U.S. Pat. No. 6,884,879 issued Feb. 26, 2005, the entire disclosure of which is expressly incorporated herein by reference.
  • cancer refers to the physiological condition in mammals that is typically characterized by unregulated cell proliferation.
  • examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia. More particular examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer (including metastic pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including locally advanced, recurrent or metastatic HER-2 negative breast cancer and locally recurrent or metastatic HER2 positive breast cancer), colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid
  • physiological or patholigical angiogenic abnormalities include, but are not limited to, high grade glioma, glioblastoma, M. Rendu-Osler, von-Hippel-Lindau diseases, hemangiomas, psoriasis, Kaposi's sarcoma, ocular neovascularisation, rheumatoid arthritis, endometriosis, atherosclerosis, myochardial ischemia, peripheral ischemia, cerebral ischemia and wound healing.
  • chemotherapeutic agent or “chemotherapy regimen” includes any active agent that can provide an anticancer therapeutic effect and may be a chemical agent or a biological agent, in particular, that are capable of interfering with cancer or tumor cells.
  • active agents are those that act as anti-neoplastic (chemotoxic or chemostatic) agents which inhibit or prevent the development, maturation or proliferation of malignant cells.
  • alkylating agents such as nitrogen mustards (e.g., mechlorethamine,
  • cyclophosphamide ifosfamide, melphalan and chlorambucil
  • nitrosoureas e.g., carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU)
  • ethylenimines/ methylmelamines e.g., thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa)
  • HMM hexamethylmelamine
  • alkyl sulfonates e.g., busulfan
  • triazines e.g., dacarbazine (DTIC)
  • antimetabolites such as folic acid analogs (e.g., methotrexate,
  • pyrimidine analogs e.g., 5-fluorouracil, capecitabine, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine
  • purine analogs e.g., 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2- chlorodeoxyadenosine (cladribine, 2-CdA)
  • antimitotic drugs developed from natural products (e.g., paclitaxel, vinca alkaloids (e.g., vinblastine (VLB), vincristine, and vinorelbine), docetaxel, estramustine, and estramustine phosphate), epipodophylotoxins (.e
  • aminoglutethimide hormones and antagonists including adrenocorticosteroid antagonists (.e.g, prednisone and equivalents, dexamethasone, aminoglutethimide), progestins (e.g.,
  • hydroxyprogesterone caproate medroxyprogesterone acetate, megestrol acetate
  • estrogens e.g., diethylstilbestrol, ethinyl estradiol and equivalents thereof
  • antiestrogens e.g., tamoxifen
  • androgens e.g., testosterone propionate, fluoxymesterone and equivalents thereof
  • antiandrogens e.g., flutamide, gonadotropin-releasing hormone analogs, leuprolide
  • nonsteroidal antiandrogens e.g., flutamide
  • epidermal growth factor inhibitors e.g., erlotinib, lapatinib, gefitinib
  • antibodies e.g., trastuzumab
  • irinotecan e.g., trastuzumab
  • chemotherapeutic agents or chemotherapeutic regimens for administration with bevacizumab include capecitabine, paclitaxel, docetaxel and trastuzumab and combinations thereof (see also the examples herein provided).
  • docetaxel is an anti-neoplastic agent that binds to free tubulin and promotes the assembly of tubulin into stable microtubules while simultaneously inhibiting their assembly. This leads to the production of microtubule bundles without normal function and to the stabilization of microtubules, blocking cells in the M-phase of the cell cycle and leading to cell death.
  • the term "effective amount" refers to an amount of a drug alone or in combination with other drug or treatment regimen effective to treat a disease or disorder in a mammal.
  • the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.
  • the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • efficacy in vivo can, for example, be measured by assessing the duration of survival, duration of progression free survival (PFS), the response rates (RR), duration of response, and/or quality of life.
  • the term "expression level” as used herein refers may also refer to the concentration or amount of marker/indicator proteins of the present invention in a sample.
  • epitope recognized by the anti-VEGF antibody bevacizumab AVASTIN®
  • the anti-VEGF antibodies include, but are not limited to, a monoclonal antibody that binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709; a recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al. (1997) Cancer Res. 57:4593-4599.
  • the "epitope 4D5" is the region in the extracellular domain of HER2 to which the antibody 4D5 (ATCC CRL 10463) and trastuzumab bind, as described in WO2009/154651. This epitope is close to the transmembrane domain of HER2, and within Domain IV of HER2, that being amino acid residues from about 489-630 - residue numbering without signal peptide. See 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. Natl. Acad. Sci 90: 1746- 1750 (1993).
  • a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed.
  • epitope mapping can be performed to assess whether the antibody binds essentially to the 4D5 epitope of HER2 (e.g. any one or more residues in the region from about residue 529 to, about residue 625, inclusive of the HER2 extracellular domain, residue numbering including signal peptide).
  • a "HER receptor” is a receptor protein tyrosine kinase which belongs to the HER receptor family and includes EGFR, HER2, HER3 and HER4 receptors.
  • the HER receptor will generally comprise an extracellular domain, which may bind an HER ligand and/or dimerize with another HER receptor molecule; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain harboring several tyrosine residues which can be phosphorylated.
  • the HER receptor may be a "native sequence” HER receptor or an "amino acid sequence variant" thereof. In one embodiment, the HER receptor is native sequence human HER receptor.
  • ErbBl ErbBl
  • HERl epidermal growth factor receptor
  • EGFR epidermal growth factor receptor
  • HER2 protein described, for example, 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 "neu” refers to the gene encoding rat pi 85.
  • an anti-Her2 antibody is an antibody that binds to a HER2 receptor.
  • the HER antibody further interferes with HER2 activation or function.
  • an anti-HER2 antibody of the present invention is an anti-HER2 antibody that binds the 4D5 epitope on HER2 polypeptide, or in another embodiment, trastuzumab.
  • Humanized HER2 antibodies include huMAb4D5-l, huMAb4D5-2, huMAb4D5-3, huMAb4D5- 4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 or trastuzumab (i.e., HERCEPTIN®) as described in Table 3 of U.S. Patent 5,821,337.
  • trastuzumab i.e., HERCEPTIN®
  • a cancer or cancer cell or tumor that is "HER2 positive” is one which has significantly higher levels of a HER receptor protein or gene compared to a noncancerous cell of the same tissue type.
  • Such overexpression may be caused by gene amplification or by increased transcription or translation.
  • HER receptor overexpression or amplification may be determined in a diagnostic or prognostic assay by evaluating increased levels of the HER protein present on the surface of a cell (e.g. via an immunohistochemistry assay; IHC). Alternatively, or additionally, one may measure levels of HER-encoding nucleic acid in the cell, e.g.
  • FISH fluorescent in situ hybridization
  • PCR polymerase chain reaction
  • qRT-PCR quantitative real time PCR
  • various in vivo assays are available to the skilled practitioner.
  • a detectable label e.g. a radioactive isotope
  • Metastasis refers to the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant.
  • oligonucleotide and “polynucleotide” are used interchangeably and refer to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide.
  • An oligonucleotide can be derived synthetically or by cloning. Chimeras of deoxyribonucleotides and ribonucleotides may also be in the scope of the present invention.
  • overall survival refers to the length of time during and after treatment the patient survives.
  • OS all survival
  • patient refers to any single animal, more specifically a mammal (including such non- human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates) for which treatment is desired. Even more specifically, the patient herein is a human.
  • a patient suffering from refers to a patient showing clinical signs in respect to a disease involving physiological and pathological angiogenesis and/or tumorous disease, such as breast cancer, in particular locally recurrent or metastatic HER2 positive breast cancer.
  • pharmaceutical composition refers to a sterile preparation that is in such form as to permit the biological activity of the medicament to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.
  • progression- free survival refers to the length of time during and after treatment during which, according to the assessment of the treating physician or investigator, the patient's disease does not become worse, i.e., does not progress.
  • progression- free survival is improved or enhanced if the patient belongs to a subgroup of patients that has a longer length of time during which the disease does not progress as compared to the average or mean progression free survival time of a control group of similarly situated patients.
  • polypeptide relates to a peptide, a protein, an oligopeptide or a polypeptide which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds.
  • peptidomimetics of such proteins/polypeptides are also encompassed by the invention wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs, e.g., an amino acid residue other than one of the 20 gene-encoded amino acids, e.g., selenocysteine.
  • Peptides, oligopeptides and proteins may be termed polypeptides.
  • the terms polypeptide and protein are used interchangeably herein.
  • polypeptide also refers to, and does not exclude, modifications of the polypeptide, e.g., glycosylation, acetylation, phosphorylation and the like. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
  • polypeptide also refers to and encompasses the term "antibody" as used herein.
  • a subject/patient suffering, suspected to suffer or prone to suffer breast cancer shows a response to a chemotherapy regimen comprising bevacizumab.
  • a chemotherapy regimen comprising bevacizumab.
  • a skilled person will readily be in a position to determine whether a person treated with bevacizumab according to the methods of the invention shows a response.
  • a response may be reflected by decreased suffering from the breast cancer, in particular locally recurrent or metastatic HER2 positive breast cancer, such as a diminished and/or halted tumor growth, reduction of the size of a tumor, and/or amelioration of one or more symptoms of the cancer.
  • the response may be reflected by decreased or diminished indices of the metastatic conversion of the breast cancer such as the prevention of the formation of metastases or a reduction of number or size of metastases (see, e.g., Eisenhauser et ah, New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1) Eur. J. Cancer 2009 45: 228-247).
  • a reference sample refers to any sample, standard, or level that is used for comparison purposes.
  • a reference sample is obtained from a healthy and/or non-diseased part of the body (e.g., tissue or cells) of the same subject or patient.
  • a reference sample is obtained from an untreated tissue and/or cell of the body of the same subject or patient.
  • a reference sample is obtained from a healthy and/or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the subject or patient.
  • a reference sample is obtained from an untreated tissue and/or cell part of the body of an individual who is not the subject or patient.
  • a reference sample is a single sample or combined multiple samples from the same subject or patient that are obtained at one or more different time points than when the test sample is obtained. For example, a reference sample is obtained at an earlier time point from the same subject or patient than when the test sample is obtained. Such reference sample may be useful if the reference sample is obtained during initial diagnosis of cancer and the test sample is later obtained when the cancer becomes metastatic.
  • a reference sample includes all types of biological samples as defined above under the term
  • sample that is obtained from one or more individuals who is not the subject or patient.
  • a reference sample is obtained from one or more individuals with an angiogenic disorder (e.g., cancer) who is not the subject or patient.
  • a reference sample is a combined multiple samples from one or more healthy individuals who are not the subject or patient.
  • a reference sample is a combined multiple samples from one or more individuals with a disease or disorder (e.g., an angiogenic disorder such as, for example, cancer) who are not the subject or patient.
  • a reference sample is pooled RNA samples from normal tissues or pooled plasma or serum samples from one or more individuals who are not the subject or patient.
  • a reference sample is pooled RNA samples from tumor tissues or pooled plasma or serum samples from one or more individuals with a disease or disorder (e.g., an angiogenic disorder such as, for example, cancer) who are not the subject or patient.
  • a disease or disorder e.g., an angiogenic disorder such as, for example, cancer
  • reference level refers to a predetermined value.
  • the reference level is predetermined and set to meet the requirements in terms of e.g. specificity and/or sensitivity. These requirements can vary, e.g. from regulatory body to regulatory body. It may for example be that assay sensitivity or specificity, respectively, has to be set to certain limits, e.g. 80%, 90% or 95%. These requirements may also be defined in terms of positive or negative predictive values. Nonetheless, based on the teaching given in the present invention it will always be possible to arrive at the reference level meeting those requirements.
  • the reference level is determined in healthy individuals.
  • the reference value in one embodiment has been predetermined in the disease entity to which the patient belongs.
  • the reference level can e.g. be set to any percentage between 25% and 75% of the overall distribution of the values in a disease entity investigated. In other embodiments the reference level can e.g. be set to the median, tertiles or quartiles as determined from the overall distribution of the values in a disease entity investigated. In one embodiment the reference level is set to the median value as determined from the overall distribution of the values in a disease entity investigated.
  • the term "increase” or “above” refers to a level above the reference level or to an overall increase of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or greater, in E-selectin, ICAM-1 or VEGFR-3 level detected by the methods described herein, as compared to the E-selectin, ICAM-1 and VEGFR-3 level from a reference sample.
  • the term increase refers to the increase in E-selectin, ICAM-1 and VEGFR-3 wherein, the increase is at least about 1.5-, 1.75-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 40-, 50-, 60-, 70-, 75-, 80-, 90-, or 100- fold higher as compared to the E- selectin, ICAM-1 and VEGFR-3 level e.g. predetermined from a reference sample.
  • the term increased level relates to a value at or above a reference level.
  • the term “decrease” or “below” herein refers to a level below the reference level or to an overall reduction of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in E-selectin, ICAM-1 and VEGFR-3 level detected by the methods described herein, as compared to the E-selectin, ICAM-1 and VEGFR-3 level from a reference sample.
  • the term decrease refers to the decrease in E-selectin, ICAM-1 and VEGFR-3 level, wherein the decreased level is at most about 0.9-, 0.8-, 0.7-, 0.6-, 0.5-, 0.4-, 0.3-, 0.2-, 0.1-, 0.05-, or 0.01- fold the E-selectin, ICAM-1 and VEGFR-3 level from the reference sample or lower.
  • the term "at a reference level” refers to a level that is the same as E- selectin, ICAM-1 and VEGFR-3 level detected by the methods described herein, from a reference sample.
  • a "recurrent" cancer is one which has regrown, either at the initial site or at a distant site, after a response to initial therapy.
  • the term "sensitive to” in the context of the present invention indicates that a subject/patient suffering, suspected to suffer or prone to suffer from breast cancer, in particular locally recurrent or metastatic HER2 positive breast cancer, shows in some way a positive reaction to treatment with bevacizumab in combination with a chemotherapy regimen.
  • the reaction of the patient may be less pronounced when compared to a patient "responsive to” as described hereinabove.
  • the patient may experience less suffering associated with the disease, though no reduction in tumor growth or metastatic indicator may be measured, and/or the reaction of the patient to the bevacizumab in combination with the chemotherapy regimen may be only of a transient nature, i.e., the growth of (a) tumor and/or (a) metastasis(es) may only be temporarily reduced or halted.
  • survival refers to the subject remaining alive, and includes progression free survival (PFS) and overall survival (OS). Survival can be estimated by the Kaplan-Meier method, and any differences in survival are computed using the stratified log-rank test.
  • extending survival or “increasing the likelihood of survival” is meant increasing PFS and/or OS in a treated subject relative to an untreated subject (i.e. relative to a subject not treated with a VEGF antibody), or relative to a control treatment protocol, such as treatment only with the chemotherapeutic agent, such as those use in the standard of care for locally recurrent or metastatic breast cancer, e.g., capecitabine, taxane, anthracycline, paclitaxel, docetaxel, paclitaxel protein-bound particles (e.g., Abraxane®), doxorubicin, epirubicin, 5-fluorouracil, cyclophosphamide, or trastuzumab (e.g., Herceptin®), or combinations thereof.
  • chemotherapeutic agent such as those use in the standard of care for locally recurrent or metastatic breast cancer, e.g., capecitabine, taxane, anthracycline, paclit
  • such standard of care for treating locally recurrent or metastatic breast cancer is a treatment combination comprising trastuzumab and docetaxel. Survival is monitored for at least about one month, about two months, about four months, about six months, about nine months, or at least about 1 year, or at least about 2 years, or at least about 3 years, or at least about 4 years, or at least about 5 years, or at least about 10 years, etc., following the initiation of treatment or following the initial diagnosis.
  • hazard ratio is a statistical definition for rates of events.
  • hazard ratio is defined as representing the probability of an event in the experimental arm divided by the probability of an event in the control arm at any specific point in time.
  • Hazard ratio in progression free survival analysis is a summary of the difference between two progression free survival curves, representing the reduction in the risk of death on treatment compared to control, over a period of follow-up.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • treatment effect encompasses the terms “overall survival” and “progression- free survival”.
  • E-selectin refers to an endothelial adhesion molecule that is induced by various inflammatory stimuli (Bevilacqua, P. P. et al, Proc. Natl. Acad. Sci. USA 84, 9238-9242 (1987) ; Luscinskas, F.W. et al, J. Immunol. 142 2257- 2263 (1989) ; Kuijpers, T.W. et al, J. Immunol. 147 1369-1376 (1991)).
  • ELAM-1 A cloned gene encoding E-Selectin (ELAM-1) is disclosed in U. S. Patent No. 5,081,034.
  • E-selectin is exemplified by UniProtKB Accession Number P16581 and Gene ID (NCBI): 6401.
  • the term "E-selectin” encompasses its homologues and isoforms.
  • the term “E-selectin” also encompasses variants thereof, as well as fragments of the sequences, provided that the variant proteins (including isoforms), homologous proteins and/or fragments are recognized by one or more E-selectin specific antibodies, such as antibody clone BBIG-E5 or 5D11, which are available from R&D Systems.
  • ICAM-1 refers to intercellular adhesion molecule 1 exemplified by UniProtKB Accession Number P05362 and Gene ID (NCBI): 3383.
  • the term “ICAM-1” encompasses its homologues and isoforms.
  • the term “ICAM-1” also encompasses variants thereof, as well as fragments of the sequences, provided that the variant proteins (including isoforms), homologous proteins and/or fragments are recognized by one or more ICAM-1 specific antibodies, such as antibody clone 11C81 or 14C11, which are available from R&D Systems.
  • 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 its homologues and isoforms.
  • the term “VEGFR-3” also encompasses variants thereof, as well as fragments of the sequences, provided that the variant proteins (including isoforms), homologous proteins and/or fragments are recognized by one or more VEGFR-3 specific antibodies, such as antibody clone 54716 or 5B6, which are available from R&D Systems and Abnova, respectively.
  • E-selectin, ICAM-1 and VEGFR-3 were identified as markers or predictive biomarkers for survival with an anti-angiogenesis therapy.
  • the terms “marker”, “biomarker” and “predictive biomarker” can be used interchangeably and refer to expression levels of E-selectin, ICAM-1 and VEGFR-3.
  • E-selectin, ICAM-1 and VEGFR-3 can also be used as prognostic biomarkers for breast cancer, more specifically HER2 positive breast cancer.
  • the present invention provides an in vitro method of determining whether a patient diagnosed with breast cancer is more or less suitably treated by an anti-cancer therapy comprising an anti-VEGF antibody, the method comprising:
  • an anti-cancer therapy comprising an anti-VEGF antibody based on the expression level in accordance with (a), wherein an expression level of said bio marker at or above a reference level indicates that the patient is more suitably treated with the anti-cancer therapy, or an expression level of said biomarker below a reference level indicates that the patient is less suitably treated with the anticancer therapy.
  • whether a patient is suitably treated by an anti-cancer therapy is determined in terms of progression- free survival.
  • said anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane.
  • the present invention further provides a pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient has been identified as more suitably treated by an anti-cancer therapy comprising an anti-VEGF antibody by an in vitro method comprising:
  • an anti-cancer therapy comprising an anti-VEGF antibody based on the expression level in accordance with (a), wherein an expression level of said biomarker at or above a reference level indicates that the patient is more suitably treated with the anti-cancer therapy, or an expression level of said biomarker below a reference level indicates that the patient is less suitably treated with the anticancer therapy.
  • whether a patient is suitably treated by an anti-cancer therapy is determined in terms of progression- free survival.
  • said anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane.
  • the present invention further provides a pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient is identified as more suitably treated by an anti-cancer therapy comprising an anti-VEGF antibody by an in vitro method comprising:
  • an anti-cancer therapy comprising an anti-VEGF antibody based on the expression level in accordance with (a), wherein an expression level of said bio marker at or above a reference level indicates that the patient is more suitably treated with the anti-cancer therapy, or an expression level of said biomarker below a reference level indicates that the patient is less suitably treated with the anticancer therapy.
  • whether a patient is suitably treated by an anti-cancer therapy is determined in terms of progression- free survival.
  • said anti-cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane.
  • the present invention also provides an in vitro method of determining whether a patient diagnosed with breast cancer is sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen, said method comprising:
  • identifying the patient as sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen based on the expression level in accordance with (a), wherein an expression level of said biomarker at or above a reference level indicates that the patient is sensitive to an anti-cancer therapy comprising the addition of an anti- VEGF antibody to a chemotherapy regimen.
  • whether a patient is sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen is determined in terms of progression-free survival.
  • said chemotherapy regimen comprises an anti-HER2 antibody and a taxane.
  • the present invention further provides a pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient has been identified as sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen by an in vitro method comprising:
  • identifying the patient as sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen based on the expression level in accordance with (a), wherein an expression level of said biomarker at or above a reference level indicates that the patient is sensitive to an anti-cancer therapy comprising the addition of an anti- VEGF antibody to a chemotherapy regimen.
  • whether a patient is sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen is determined in terms of progression-free survival.
  • said chemotherapy regiment comprises an anti-HER2 antibody and a taxane.
  • the present invention further provides a pharmaceutical composition comprising an anti-VEGF antibody for the treatment of a patient diagnosed with breast cancer, wherein the patient is identified as sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen by an in vitro method comprising:
  • identifying the patient as sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen based on the expression level in accordance with (a), wherein an expression level of said biomarker at or above a reference level indicates that the patient is sensitive to an anti-cancer therapy comprising the addition of an anti- VEGF antibody to a chemotherapy regimen.
  • whether a patient is sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen is determined in terms of progression-free survival.
  • said chemotherapy regiment comprises an anti-HER2 antibody and a taxane.
  • the present invention also provides a method for improving the treatment effect of an anticancer therapy comprising a chemotherapy regimen in a patient diagnosed with breast cancer by adding an anti-VEGF antibody to the chemotherapy regimen, the method comprising:
  • whether a patient is sensitive to an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a chemotherapy regimen is determined in terms of progression- free survival.
  • said chemotherapy regimen comprises an anti-HER2 antibody and a taxane.
  • said determination of the biomarker is carried out either a) by contacting the sample with an agent that specifically binds to the biomarker, thereby forming a complex between the agent and the biomarker, detecting the amount of complex formed, thereby measuring the level of the biomarker; or b) by amplifying the biomarker present in the sample and detecting the amplified biomarker with an agent that specifically binds to the amplified biomarker, thereby measuring the level of the biomarker.
  • said patient is diagnosed with HER2 positive breast cancer, more specifically, locally recurrent or metastatic HER2 positive breast cancer. In one embodiment, said patient received no previous chemotherapeutic or radiation treatment.
  • said anti-VEGF antibody binds the A4.6.1 epitope. More specifically, said anti-VEGF antibody is bevacizumab.
  • said taxane is docetaxel or paclitaxel, more specifically, docetaxel.
  • said anti-HER2 antibody binds the 4D5 epitope. More specifically, said anti-HER2 antibody is trastuzumab.
  • said expression level is a protein expression level.
  • said sample is a blood plasma sample.
  • said expression level is an expression level of E-selectin.
  • said expression level is an expression level of ICAM-1.
  • said expression level is an expression level of VEGFR-3.
  • the expression levels, in particular protein expression levels, of E-selectin, ICAM-1 and VEGFR-3 may be considered separately, as individual markers, or in groups of two or more, as an expression profile or marker panel.
  • an expression profile or marker panel wherein the expression profiles of two or more markers may be considered together may also be referred to as a combined expression level.
  • the expression levels of two or more markers may be added together and compared to a similarly determined control combined expression level. Therefore, the methods of the invention encompass determination of an expression profile, including a combined expression level, based on the expression level of one or more of the markers.
  • optimized levels constituting the cut-off value between high and low expression of a particular marker may be determined by varying the cut-off until the subset of patients above and below the cut-off satisfy a relevant statistical optimality criterion.
  • an optimal cut-point may be chosen to maximize the differences in treatment Hazard Ratio between the subset above and below, or to maximize treatment effect in one sub-group, or any other relevant statistical criterion.
  • the skilled person will, however, understand that the expression level of the particular marker and, therefore, what constitutes a high or low expression level may vary by patient and by patient population.
  • said reference level of E-selectin, ICAM-1 and VEGFR-3 is set to the median value of concentrations in samples derived from a patient group. In one example, said reference level of E-selectin is about 36.9 ng/mL in plasma. In one example, said reference level of ICAM-1 is about 210 ng/mL in plasma. In one example, said reference level of VEGFR-3 is about 10.6 ng/mL in plasma. In one embodiment, said reference level of E-selectin, ICAM-1 and VEGFR-3 is determined by a quartile analysis of samples derived from a patient group.
  • said reference level of E-selectin, ICAM-1 and VEGFR-3 is set to the 1st quartile value in order of increasing concentrations in samples derived from a patient group.
  • said reference level of E-selectin is about 26.7 ng/mL in plasma.
  • said reference level of ICAM-1 is about 170.4 ng/mL in plasma.
  • said reference level of VEGFR-3 is about 7.8 ng/mL in plasma.
  • said reference level of E- selectin, ICAM-1 and VEGFR-3 is set to the 3rd quartile value in order of increasing concentrations in samples derived from a patient group.
  • said reference level of E-selectin is about 49.1 ng/mL in plasma.
  • said reference level of ICAM-1 is about 272.2 ng/mL in plasma.
  • said reference level of VEGFR-3 is about 13.0 ng/mL in plasma.
  • markers of a marker panel may be mathematically combined and the combined value may be correlated to the underlying diagnostic question.
  • Marker values may be combined by any appropriate state of the art mathematical method.
  • Well-known mathematical methods for correlating a marker combination to a disease or to a treatment effect employ methods like, discriminant analysis (DA) (i.e. linear-, quadratic-, regularized-DA), Kernel Methods (i.e. SVM), Nonparametric Methods (i.e.
  • k- Nearest-Neighbor Classifiers PLS (Partial Least Squares), Tree-Based Methods (i.e. Logic Regression, CART, Random Forest Methods, Boosting/Bagging Methods), Generalized Linear Models (i.e. Logistic Regression), Principal Components based Methods (i.e. SIMCA), Generalized Additive Models, Fuzzy Logic based Methods, Neural Networks and Genetic Algorithms based Methods.
  • PLS Partial Least Squares
  • Tree-Based Methods i.e. Logic Regression, CART, Random Forest Methods, Boosting/Bagging Methods
  • Generalized Linear Models i.e. Logistic Regression
  • Principal Components based Methods i.e. SIMCA
  • Generalized Additive Models Fuzzy Logic based Methods, Neural Networks and Genetic Algorithms based Methods.
  • the skilled artisan will have no problem selecting an appropriate method to evaluate a marker combination of the present
  • the method used in correlating marker combinations in accordance with the invention herein disclosed with, for example improved overall survival, progression free survival, responsiveness or sensitivity to addition of bevacizumab to chemotherapeutic agents/chemo therapy regimen and/or the prediction of a response to or sensitivity to bevacizumab (in addition to one or more chemotherapeutic agents/chemo therapy regimen) is selected from DA (i.e. Linear-, Quadratic-, Regularized Discriminant Analysis), Kernel Methods (i.e. SVM), Nonparametric Methods (i.e. k-Nearest-Neighbor Classifiers), PLS (Partial Least Squares), Tree-Based Methods (i.e.
  • DA i.e. Linear-, Quadratic-, Regularized Discriminant Analysis
  • Kernel Methods i.e. SVM
  • Nonparametric Methods i.e. k-Nearest-Neighbor Classifiers
  • PLS Partial Least Squares
  • the invention herein disclosed relates to the use of an optimized multivariate cutoff for the underlying combination of biological markers and to discriminate state A from state B, e.g. patients responsive to or sensitive to an anti-cancer therapy comprising the addition of bevacizumab to a chemotherapy regimen from patients that are poor responders to the addition of bevacizumab therapy to a chemotherapy regimen.
  • state A e.g. patients responsive to or sensitive to an anti-cancer therapy comprising the addition of bevacizumab to a chemotherapy regimen from patients that are poor responders to the addition of bevacizumab therapy to a chemotherapy regimen.
  • the markers are no longer independent but form a marker panel or a combined expression level.
  • the expression level of one or more of the markers E-selectin, ICAM-1 and VEGFR-3 may be assessed by any method known in the art suitable for determination of specific protein levels in a patient sample and is preferably determined by an immunoassay method, such as ELISA, employing antibodies specific for one or more of E-selectin, ICAM-1 and VEGFR-3.
  • an immunoassay method such as ELISA
  • Such methods are well known and routinely implemented in the art and corresponding commercial antibodies and/or kits are readily available.
  • E-selectin, ICAM-1 and VEGFR-3 can be obtained from R&D Systems as clone BBIG- E5 and 5D11, from R&D systems as clone 11C81 and 14C11 and from R&D Systems and Abnova as clone 54716 and 5B6, respectively.
  • the expression levels of the marker/indicator proteins of the invention are assessed using the reagents and/or protocol recommendations of the antibody or kit manufacturer.
  • the skilled person will also be aware of further means for determining the expression level of one or more of E-selectin, ICAM-1 and VEGFR-3 by immunoassay methods.
  • the expression level of one or more of the markers/indicators of the invention can be routinely and reproducibly determined by a person skilled in the art without undue burden.
  • the invention also encompasses the testing of patient samples in a specialized laboratory that can ensure the validation of testing procedures.
  • E-selectin, ICAM-1 and VEGFR-3 protein or nucleic acids can be detected using any method known in the art.
  • tissue or cell samples from mammals can be conveniently assayed for, e.g., proteins using Westerns and ELISAs, mRNAs or DNAs from a genetic biomarker of interest using Northern, dot-blot, or polymerase chain reaction (PCR) analysis, array hybridization, RNase protection assay, or using DNA SNP chip microarrays, which are commercially available, including DNA microarray snapshots.
  • RT- PCR real-time PCR assays such as quantitative PCR assays are well known in the art.
  • a method for detecting mRNA from a genetic biomarker of interest in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced; and detecting the presence of the amplified cDNA.
  • such methods can include one or more steps that allow one to determine the levels of mRNA in a biological sample ⁇ e.g., by simultaneously examining the levels a comparative control mRNA sequence of a "housekeeping" gene such as an actin family member).
  • the sequence of the amplified cDNA can be determined.
  • the expression level of one or more of E-selectin, ICAM-1 and VEGFR-3 may be assessed in a patient sample that is a biological sample.
  • the patient sample may be a blood sample, blood serum sample or a blood plasma sample. Methods of obtaining blood samples, blood serum samples and blood plasma samples are well known in the art.
  • the patient sample may be obtained from the patient prior to or after neoadjuvant therapy or prior to or after adjuvant therapy.
  • bevacizumab is to be administered in addition to or as a co-therapy or co-treatment with one or more chemotherapeutic agents administered as part of standard chemotherapy regimen as known in the art.
  • agents included in such standard chemotherapy regimens include 5-fluorouracil, leucovorin, irinotecan, gemcitabine, erlotinib, capecitabine, taxanes, such as docetaxel and paclitaxel, interferon alpha, vinorelbine, and platinum-based chemotherapeutic agents, such as, carboplatin, cisplatin and oxaliplatin.
  • bevacizumab effected an increase in the progression free survival in the patients and/or patient population defined and selected according to the expression level of one or more of E-selectin, ICAM-1 and VEGFR-3.
  • bevacizumab may be combined with a chemotherapy regimen, such as docetaxel therapy as demonstrated in the appended illustrative example.
  • Common modes of administration include parenteral administration as a bolus dose or as an infusion over a set period of time, e.g., administration of the total daily dose over 10 min., 20 min., 30 min., 40 min., 50 min., 60 min., 75 min., 90 min., 105 min., 120 min., 3 hr., 4 hr., 5 hr. or 6 hr.
  • administration of the total daily dose over 10 min., 20 min., 30 min., 40 min., 50 min., 60 min., 75 min., 90 min., 105 min., 120 min., 3 hr., 4 hr., 5 hr. or 6 hr.
  • 2.5 mg/kg of body weight to 15 mg/kg of body weight bevacizumab can be administered every week, every 2 weeks or every 3 weeks, depending on the type of cancer being treated.
  • Examples of dosages include 2.5 mg/kg of body weight, 5 mg/kg of body weight, 7.5 mg/kg of body weight, 10 mg/kg of body weight and 15 mg/kg of body weight given every week, every 2 weeks or every 3 weeks. Further examples of dosages are 5 mg/kg of body weight every 2 weeks, 10 mg/kg every 2 weeks, 7.5 mg/kg of body weight every 3 weeks and 15 mg/kg of body weight every 3 weeks.
  • low dose bevacizumab includes, for example, dosages of 2.5 mg/kg of body weight every week, 5 mg/kg of body weight every 2 weeks and 7.5 mg/kg of body weight every 3 weeks.
  • high dose bevacizumab includes, for example, dosages of 5 mg/kg of body weight every week, 10 mg/kg of body weight every 2 weeks and 15 mg/kg of body weight every 3 weeks.
  • the patients selected according to the methods of the present invention are treated with bevacizumab in combination with a chemotherapy regimen, and may be further treated with one or more additional anti-cancer therapies.
  • the one or more additional anticancer therapy is radiation.
  • the present invention also relates to a diagnostic composition or kit comprising oligonucleotides or polypeptides suitable for the determination of expression levels of one or more of E-selectin, ICAM-1 and VEGFR-3.
  • oligonucleotides such as DNA, RNA or mixtures of DNA and RNA probes may be of use in detecting mRNA levels of the marker/indicator proteins, while polypeptides may be of use in directly detecting protein levels of the marker/indicator proteins via specific protein-protein interaction.
  • the polypeptides encompassed as probes for the expression levels of one or more of E-selectin, ICAM-1 and VEGFR-3, and included in the kits or diagnostic compositions described herein are antibodies specific for these proteins, or specific for homologues and/or truncations thereof. Accordingly, in a further embodiment of the present invention provides a kit useful for carrying out the methods herein described, comprising oligonucleotides or polypeptides capable of determining the expression level of one or more of E-selectin, ICAM-1 and VEGFR-3.
  • the oligonucleotides may comprise primers and/or probes specific for the mRNA encoding one or more of the markers/indicators described herein, and the polypeptides comprise proteins capable of specific interaction with the marker/indicator proteins, e.g., marker/indicator specific antibodies or antibody fragments.
  • the invention also encompasses further immunoassay methods for assessing or determining the expression level of one or more of E- selectin, ICAM-1 and VEGFR-3, such as by Western blotting and ELISA-based detection.
  • the expression level of the marker/indicator proteins of the invention may also be assessed at the mRNA level by any suitable method known in the art, such as Northern blotting, real time PCR, and RT PCR.
  • Immunoassay- and mRNA-based detection methods and systems are well known in the art and can be deduced from 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, U.S.A., 2001).
  • the described methods are of particular use for determining the expression levels of E-selectin, ICAM-1 and VEGFR-3 in a patient or group of patients relative to control levels established in a population diagnosed with breast cancer, in particular locally recurrent or metastatic HER2 positive breast cancer.
  • the expression level of one or more of E-selectin, ICAM-1 and VEGFR-3 can also be determined on the protein level by taking advantage of immunoagglutination, immunoprecipitation ⁇ e.g., immunodiffusion, immunelectrophoresis, immune fixation), western blotting techniques ⁇ e.g., ⁇ in situ) immuno cytochemistry, affinitychromatography, enzyme immunoassays), and the like.
  • Amounts of purified polypeptide in solution may also be determined by physical methods, e.g. photometry. Methods of quantifying a particular polypeptide in a mixture usually rely on specific binding, e.g., of antibodies. Specific detection and quantitation methods exploiting the specificity of antibodies comprise for example immunoassay methods.
  • concentration/amount of marker/indicator proteins of the present invention in a patient sample may be determined by enzyme linked-immunosorbent assay (ELISA).
  • ELISA enzyme linked-immunosorbent assay
  • Western Blot analysis or immuno staining can be performed. Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies. Electrophoresis may be multi-dimensional such as 2D electrophoresis. Usually, polypeptides are separated in 2D electrophoresis by their apparent molecular weight along one dimension and by their isoelectric point along the other direction.
  • the expression level of the marker/indicator proteins according to the present invention may also be reflected in an increased expression of the corresponding gene(s) encoding E-selectin, ICAM-1 and VEGFR-3. Therefore, a quantitative assessment of the gene product prior to translation (e.g. spliced, unspliced or partially spliced mRNA) can be performed in order to evaluate the expression of the corresponding gene(s).
  • a quantitative assessment of the gene product prior to translation e.g. spliced, unspliced or partially spliced mRNA
  • the person skilled in the art is aware of standard methods to be used in this context or may deduce these methods from standard textbooks (e.g. Sambrook, 2001 , loc. cit.).
  • quantitative data on the respective concentration/amounts 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.
  • the kit of the invention may advantageously be used for carrying out a method of the invention and could be, inter alia, employed in a variety of applications, e.g., in the diagnostic field or as a research tool.
  • the parts of the kit of the invention can be packaged individually in vials or in combination in containers or multicontainer units.
  • Manufacture of the kit follows preferably standard procedures which are known to the person skilled in the art.
  • the kit or diagnostic compositions may be used for detection of the expression level of one or more of E-selectin, ICAM-1 and VEGFR-3 in accordance with the herein-described methods of the invention, employing, for example, immunohistochemical techniques described herein.
  • the skilled person has the ability to label the polypeptides, for example antibodies, or oligonucleotides encompassed by the present invention.
  • hybridization probes for use in detecting mRNA levels and/or antibodies or antibody fragments for use in immunoassay methods can be labelled and visualized according to standard methods known in the art, nonlimiting examples of commonly used systems include the use of radiolabels, enzyme labels, fluorescent tags, biotin-avidin complexes, chemiluminescence, and the like.
  • the present invention is further illustrated by the following non- limiting illustrative example.
  • Example 1 Bevacizumab in combination with trastuzumab/docetaxel compared with trastuzumab/docetaxel alone as first line treatment for patients with HER2 positive locally recurrent or metastatic breast cancer - AVEREL study
  • the primary objective of the clinical trial disclosed herein was to compare Progression Free Survival (PFS) in patients randomized to bevacizumab in combination with trastuzumab / docetaxel versus patients randomized to trastuzumab / docetaxel alone.
  • the secondary objectives were to evaluate Overall Survival (OS); Best Overall Response (OR); Duration of Response (DR); Time to Treatment Failure (TTF); Safety and tolerability of combining bevacizumab with trastuzumab and docetaxel; and finally Quality of Life.
  • the study described herein were to determine (1) that bevacizumab at 15 mg/kg every 3 weeks + trastuzumab at 8 mg/kg loading dose followed by 6 mg/kg every 3 weeks until disease progression + docetaxel 100 mg/m2 every 3 weeks for a minimum of 6 Cycles confers a positive treatment effect on the primary variable of PFS when compared to trastuzumab 8 mg/kg loading dose followed by 6 mg/kg every 3 weeks until disease progression + docetaxel 100 mg/m2 every 3 weeks for a minimum of 6 Cycles; and (2) that bevacizumab at 15 mg/kg every 3 weeks + trastuzumab 8 mg/kg loading dose followed by 6 mg/kg every 3 weeks until disease progression + docetaxel 100 mg/m2 every 3 weeks for a minimum of 6 Cycles has an acceptable safety profile.
  • the trial was a randomized, open label, 2-arm, multicentre, phase III study. Patients were randomly assigned to treatment groups on a 1 : 1 basis through 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 arms, patients were stratified, according to the following criteria:
  • adjuvant/neoadjuvant chemotherapy Patients were initially stratified for prior treatment with taxanes (Yes versus No). If 'no prior taxanes', a second stratification was performed i.e. never received adjuvant/neo-adjuvant chemotherapy or relapse > 12 months since last dose of chemotherapy versus ⁇ 12 months since last dose of chemotherapy.
  • trastuzumab as part of adjuvant treatment versus no trastuzumab
  • Hormone receptor (ER/PgR) status (positive versus negative);
  • Arm B Trastuzumab 8 mg/kg loading dose followed by 6 mg/kg every 3 weeks until disease progression + Docetaxel 100 mg/m2 every 3 weeks for a minimum of 6 Cycles (or up to disease progression or unacceptable toxicity, whichever occurs first). After 6 Cycles with no progression or toxicity docetaxel may be continued for additional Cycles at the discretion of the investigator + Bevacizumab 15 mg/kg every 3 weeks until disease progression.
  • End of Study This was an event driven trial. The analysis of the primary endpoint was performed when 310 events were confirmed in the 424 patients randomized. An additional analysis of overall survival took place approximately 36 months after randomization of the last patient and the trial ended at this point. End of study occurred at the date of the last visit of the last patient participating in this trial which coincided with the final Overall Survival analysis which took place approximately 36 months after randomization of the last patient.
  • MBC metastatic breast cancer
  • trastuzumab in the adjuvant setting were allowed to be enrolled, provided that > 6 months had elapsed since last adjuvant administration of trastuzumab. Patients had to have an adequate Left Ventricular Ejection Function at baseline defined as LVEF not below 50% as measured by either echocardiography or MUGA. Patients who were treated with
  • anthracyclines for adjuvant disease could have been included into the study if the maximum cumulative dose was less/equal to 360 mg/m2 of doxorubicin or 720 mg/m2 of epirubicin.
  • IHC immunohistochemistry
  • FISH fluorescent in situ hybridization
  • CISH chromogenic in situ hybridization
  • trastuzumab in the adjuvant setting 7. Patients who received trastuzumab in the adjuvant setting were eligible as long as they did not relapsed within 6 months after the last dose of trastuzumab;
  • LVEF Baseline Left Ventricular Ejection Fraction
  • urinalysis at baseline should undergo 24 hours urine collection and must demonstrate ⁇ 1 g of protein/24 hr.
  • Chronic daily treatment with corticosteroids dose of > 10 mg/day methylprednisolone equivalent) (excluding inhaled steroids).
  • Chronic daily treatment with aspirin > 325 mg / day) or clopidogrel (> 75 mg / day).
  • Serum pregnancy test to be assessed within 7 days prior to study treatment start, or within 14 days with a confirmatory urine pregnancy test within 7 days prior to study treatment start.
  • the independent review committee assessed PFS results, which were stratified and censored for NPT, was statistically significant. Specifically, as summarized on Table 3, the median PFS was 13.9 months for the control group (Arm A) versus 16.8 months for the
  • Blood samples for biomarker discovery and validation were collected from consenting patients in study BO20231. Blood samples (approx 20 mL in total) were collected at baseline (after randomization but before the first administration of study medication) and at time of disease progression.
  • a total of 4.9 mLs of blood were drawn into a S-monovette® (EDTA) tube. They were mixed immediately thereafter by gentle invertion of the tube and were centrifuged within 30 minutes at approximately 1500g in a centrifuge (room temperature for 10 minutes). Immediately hereafter, supernatant plasma was aliquoted in a clear polypropylene 5mL transfer tube. Thereafter, plasma was aliquoted into 2 plastic storage tubes (approximately 1.25 ml each). Samples were stored in an upright position at -70°C. In some cases, samples were stored at -20°C for up to one month and then transferred to -70°C.
  • S-monovette® EDTA
  • Interleukin-8 11-8
  • Inter-Cellular Adhesion Molecule 1 IMM-1
  • VEGFA VEGFA
  • VEGF-C VEGF receptor- 1
  • VEGFR2 VEGF Receptor 2
  • VEGFR3 VEGF receptor-3
  • bFGF basic Fibroblast Growth Factor
  • PDGF-C Platelet Derived Growth Factor-C
  • IMPACT Immunological Multiparameter Chip Technique
  • IMPACT Immunological Multiparameter Chip Technique
  • the plasma volume required per specimen for measuring all markers on one chip was 20 ⁇ for chip 1 and 8 for chip 2 and chip 3 (see below).
  • the sample volume was applied together with incubation buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 0.1% Thesit, 0.5% bovine serum albumin and 0.1% Oxypyrion as a preservative agent) to give a total reaction volume of 40 ⁇ per chip.
  • the digoxigenylated detection antibody mix was added (40 ⁇ ⁇ of incubation buffer including a mix of the analyte-specific antibodies labeled with Digoxigenin) and was incubated for an additional 6 minutes to bind onto the captured analytes.
  • the second antibody was finally detected after washing with 40 ⁇ ⁇ of a 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%) Oxypyrion) including an anti-digoxigenin antibody conjugate coupled to fluorescent latex.
  • Chips were transported into the detection unit, and a charge coupled device (CCD) camera generated an image that was transformed into signal intensities using dedicated software. Individual spots were automatically located at predefined positions and quantified by image analysis. For each marker, lines of 10-12 spots were loaded on the chips, and the concentration of the markers was calculated as mean of at least 5 spots from the respective line on the chip.
  • CCD charge coupled device
  • the advantages of the technology are the ability of multiplexing up to 10 parameters in a sandwich or competitive format.
  • the calibrators and patient samples were measured in duplicate. One run was designed to contain a total of 100 determinations, including calibrators and 2 multi-controls as a run control. Since some of the selected analytes react with each other (i.e VEGFA with VEGFR1 or VEGRF2 ), the analytes were divided on three different chips as follows:
  • Chip 1 VEGFA, VEGF-C, PDGF-C
  • Chip 2 VEGFR1, VEGFR2, VEGFR3, 11-8, bFGF,
  • Chip 3 E-selectin, ICAM-1
  • Sample median was used to dichotomize biomarker values as low (below median) or high (at or above median).
  • a biomarker evaluable population was defined in this study, consisting of all patients who received any component of study medication and had marker levels at baseline for any of the following biomarkers assessed as described above and with commercially available antibodies: VEGF-A, VEGF-C, VEGF-R1, VEGF-R2, E-selectin, VEGFR-3, IL-8, bFGF, PDGF-C, ICAM- 1.
  • PFS and OS have been defined as specified in the Data Reporting Analysis Manual (DRAM) for Study BO20231.
  • DRAM Data Reporting Analysis Manual
  • the analyses of biomarker data has been based on the PFS (Investigator assessed) data at the time of final PFS analysis.
  • the sample median biomarker concentration was used as the cut point to group patients (high vs. low levels of concentration).
  • An interaction test for biomarker evaluable patients has been performed to assess whether the biomarker is predictive of treatment benefit on PFS with bevacizumab for patients with HER2 positive locally recurrent or metastatic breast cancer, using a Cox model that includes baseline biomarker levels (as a binary variable dichotomized as high and low at the sample median), treatment, baseline prognostic factors and the interaction term (baseline biomarker level by treatment).
  • Table 8 presents the results of the analysis of the association of E-selectin, ICAM-1 and VEGFR-3 with treatment effect on Investigator assessed progression free survival.
  • aModel includes prognostic factors
  • T docetaxel
  • H trastuzumab
  • BV bevacizumab
  • L low
  • H high
  • E-selectin High E-selectin (> 36.9 ng/mL)
  • Low E-selectin ⁇ 36.9 ng/mL
  • ICAM-1 High ICAM-1 (> 210 ng/mL)
  • VEGFR-3 High VEGFR-3 (> 10.6 ng/mL)
  • Low VEGFR-3 ⁇ 10.6 ng/mL
  • VEGFR-3 and E-selectin showed potential predictive value. Therefore, ICAM-1, E-selectin and VEGFR-3 can be independent predictive biomarkers for bevacizumab treatment effect on Progression Free Survival.

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