[go: up one dir, main page]

WO2012175711A1 - Procédé pour prédire la réponse à une chimiothérapie d'un patient atteint d'un ostéosarcome - Google Patents

Procédé pour prédire la réponse à une chimiothérapie d'un patient atteint d'un ostéosarcome Download PDF

Info

Publication number
WO2012175711A1
WO2012175711A1 PCT/EP2012/062147 EP2012062147W WO2012175711A1 WO 2012175711 A1 WO2012175711 A1 WO 2012175711A1 EP 2012062147 W EP2012062147 W EP 2012062147W WO 2012175711 A1 WO2012175711 A1 WO 2012175711A1
Authority
WO
WIPO (PCT)
Prior art keywords
integrin
fak
gsk
expression
chemotherapy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2012/062147
Other languages
English (en)
Inventor
Anne Gomez-Brouchet
Elizabeth MOYAL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut National de la Sante et de la Recherche Medicale INSERM
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut National de la Sante et de la Recherche Medicale INSERM filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Publication of WO2012175711A1 publication Critical patent/WO2012175711A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1777—Integrin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to an ex vivo method for predicting the responsiveness of a patient affected with an osteosarcoma to chemotherapy, comprising the step of determining the level expression of at least one marker selected in the group consisting of ⁇ 5 integrin, FAK or GSK-3P in a tumor sample from said patient.
  • the invention relate to an antagonist of ⁇ 5 integrin, FAK or GSK-3P or an inhibitor of the expression of ⁇ 5 integrin, FAK or GSK-3P for use in a method for enhancing clinical efficiency of chemotherapy.
  • Primary osteosarcoma is the most common primary bone tumour that predominantly develops in adolescents and young adults. Survival has been improved, during the last two decades by the introduction of neoadjuvant chemotherapy, to induce necrosis of the tumour before surgery, combined with wide-margin, limb sparing surgery. Nevertheless, recurrent disease still occurs in about 30-40 % of patients and more than 70 % of them die of their tumour.
  • the main prognostic factors are the presence of metastases and the response to chemotherapy evaluated by the percentage of tumoral necrosis of the surgical resection [Meyers et al, 1992 and Bacci et al, 1997]. A more intensified therapy didn't give a survival remedie in poor responders, suggesting that there may be an intrinsic biological difference between good and poor responsive tumours to chemotherapy.
  • Ezrin member of the ERM (Ezrin-Radixin-Moesin) family which is a membrane cytoskeletal linker protein which is produced by the expression of the Vil2 gene
  • Ezrin-Radixin-Moesin Ezrin-Radixin-Moesin family which is a membrane cytoskeletal linker protein which is produced by the expression of the Vil2 gene
  • HSD 17B 10 gene expression a mitochondrial enzyme implicated in the production of dihydrotestosterone (DHT) is up-regulated in poor responders and that immuno histochemistry expression of HSD17B10 on biopsy before treatment is correlated to response to chemotherapy [Salas et al, 2009].
  • DHT dihydrotestosterone
  • integrins are structurally similar heterodimers composed of a- and ⁇ -subunit transmembrane glycoproteins that are non-covalently linked. Integrins play pivotal roles in diverse cellular processes, such as migration, proliferation and attachement, angiogenesis and survival.
  • ⁇ 5 has been shown to be involved in the resistance of ovarian cancer to cisplatin [Maubant et al, 2002] and the ⁇ 3 integrin to be involved in the resistance to taxol of human breast cancer MCF7 cell line [Menendez et al, 2005].
  • the inventors have also recently demonstrated that the ⁇ 3 and ⁇ 5 integrin pathway was also largely involved in the control of hypoxia, which plays a crucial role in tumour aggressiveness and resistance to chemotherapy and radiotherapy. Indeed, they have shown that hypoxia activates RhoB and that ⁇ 3 and ⁇ 5 integrins control RhoB activation under hypoxic conditions through FAK, then controlling HIF- ⁇ expression via ⁇ 8 ⁇ -3 ⁇ [Skuli et al, 2009].
  • the invention relates to an ex vivo method for predicting the responsiveness of a patient affected with an osteosarcoma to chemotherapy, comprising the step of determining the level expression of at least one marker selected in the group consisting of ⁇ 5 integrin, FAK or ⁇ 8 ⁇ -3 ⁇ in a tumor sample from said patient.
  • the invention relate to an antagonist of ⁇ 5 integrin, FAK or ⁇ 8 ⁇ -3 ⁇ or an inhibitor of the expression of ⁇ 5 integrin, FAK or ⁇ 8 ⁇ -3 ⁇ for use in a method for enhancing clinical efficiency of chemotherapy.
  • ⁇ 5 integrin has its general meaning in the art and refers to a member of the integrin's family which are receptors that mediate attachment between a cell and the tissues surrounding it, which may be other cells or the extracellular matrix (ECM). They also play a role in cell signaling and thereby regulate cellular shape, motility, and the cell cycle.
  • FAK Fluorescence Activated kinase
  • GSK-3P or "Glycogen synthase kinase 3 beta” has its general meaning in the art and denotes a proline-directed serine-threonine kinase that was initially identified as a phosphorylating and inactivating glycogen synthase.
  • GSK3B is involved in energy metabolism, neuronal cell development, and body pattern formation.
  • the term "patient”, is intended for a human affected or likely to be affected with a tumor.
  • the invention relates to an ex vivo method for predicting the responsiveness of a patient affected with an osteosarcoma to chemotherapy, comprising the step of determining the level expression of at least one marker selected in the group consisting of ⁇ 5 integrin, FAK or GSK-3P in a tumor sample from said patient.
  • the level expression of two markers selected in the group of ⁇ 5 integrin and FAK, or ⁇ 5 integrin and GSK-3P or FAK and GSK-3P is determining.
  • the level expression of the three markers, ⁇ 5 integrin, FAK and GSK-3P is determining.
  • the potential correlation between the level expression of the three markers, ⁇ 5 integrin, FAK and GSK-3P is determining.
  • the level expression of the markers according to the invention is compared with reference level wherein a difference between said levels is indicative of the responsiveness to chemotherapy of said patient.
  • the reference level may be obtained from responder and non-responder group of patients.
  • the chemotherapy is a neoadjuvant chemotherapy.
  • the neoadjuvant chemotherapy is an association of drugs derived from SFOP OS94 regimen see for example Le Deley MC et al, 2007.
  • the tumor sample obtained from the patient is a resected ostesarcoma or a biopsy from osteosarcoma.
  • the level expression of markers may be measured for example by RT-PCR or immunohistochemistry performed on said sample.
  • the determination of the level expression of the markers ⁇ 5 integrin, FAK and GSK-3P in said sample may be performed by determining the gene expression level of the markers ⁇ 5 integrin, FAK and GSK-3p.
  • the determination comprises contacting the sample with selective reagents such as probes, primers or ligands, and thereby detecting the presence, or measuring the amount, of polypeptides or nucleic acids of interest originally in the sample.
  • Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column...
  • the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array.
  • the substrate may be a solid or semi-solid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like.
  • the substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc.
  • the contacting may be made under any condition suitable for a detectable complex, such as a nucleic acid hybrid or an antibody-antigen complex, to be formed between the reagent and the nucleic acids or polypeptides of the sample.
  • the expression level may be determined by determining the quantity of mRNA. Such method may be suitable to determine the expression level of ⁇ 5 integrin, FAK or GSK-3P gene in the sample.
  • the nucleic acid contained in the samples is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions.
  • the extracted mRNA may be then detected by hybridization (e. g., Northern blot analysis).
  • the extracted mRNA may be subjected to coupled reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that enable amplification of a region in the ⁇ 5 integrin, FAK or GSK-3P gene.
  • RT-PCR polymerase chain reaction
  • RT-PCR Preferably quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous. Extracted mRNA may be reverse-transcribed and amplified, after which amplified sequences may be detected by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.
  • LCR ligase chain reaction
  • TMA transcription- mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the method of the invention comprise the steps of providing total RNAs obtained from the sample of the patient, and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi-quantitative RT-PCR.
  • Total R can be easily extracted from the sample.
  • the sample may be treated prior to its use, e.g. in order to render nucleic acids available. Techniques of cell or protein lysis, concentration or dilution of nucleic acids, are known by the skilled person.
  • the expression level may be determined by DNA microarray analysis.
  • DNA microarray or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
  • Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs.
  • a sample from a test subject optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling.
  • Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200- 210).
  • the determination of the level expression of the markers ⁇ 5 integrin, FAK and GSK-3P in said sample may be performed by determining the level of ⁇ 5 integrin, FAK and GSK-3P proteins by immunohistochemistry.
  • Such methods comprise contacting a sample with a binding partner capable of selectively interacting with ⁇ 5 integrin, FAK and GSK-3P present in the sample.
  • the binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • immunohistochemistry a staining method based on immuno enzymatic reactions using monoclonal or polyclonal antibodies to detect cells or specific proteins such as tissue antigens.
  • immunohistochemistry protocols involve at least some of the following steps:
  • antigen retrieval eg., by pressure cooking, protease treatment, microwaving, heating in appropriate buffers, etc.
  • an amplification step may be included
  • a detection reagent e.g. chromagen, fluorescently tagged molecule or any molecule having an appropriate dynamic range to achieve the level of or sensitivity required for the assay
  • immuno enzymatic staining methods are known in the art for detecting a protein of interest. For example, immuno enzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens such as DAB, AEC, or Fast Red; or fluorescent labels such as FITC, Cy3, Cy5, Cy7, Alexafluors, etc.
  • Counterstains may include H&E, DAPI, Hoechst, so long as such stains are compatable with other detection reagents and the visualization strategy used.
  • amplification reagents may be used to intensify staining signal.
  • tyramide reagents may be used.
  • the staining methods of the present invention may be accomplished using any suitable method or system as would be apparent to one of skill in the art, including automated, semi-automated or manual systems.
  • the method of the invention may comprise a further step consisting of comparing level of ⁇ 5 integrin, FAK and GSK-3P with a reference level.
  • the "reference level” may be the level of the markers determined in a healthy patient, i.e. a patient who does not suffer from any ostesarcoma.
  • the reference level may also be a patient suffering from ostesarcoma.
  • said reference level is a healthy patient.
  • the method may further comprise a step consisting of determining the amount of cells that express ⁇ 5 integrin, FAK or GSK-3P (" ⁇ 5 integrin+ cells", “FAK+ cells,” or "GSK-3P+ cells”).
  • a percentage of ⁇ 5 integrin+ cells, FAK+ cells, or GSK- 3 ⁇ + cells of at least 20%, preferably at least 21%, more preferably at least 22%, even more preferably at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, preferably at least 28%, more preferably at least 29%, even more preferably 30% and more preferably 50% is indicative of a poor responder.
  • patients with low percentage of ⁇ 5 integrin+ cells, FAK+ cells, or GSK-3 ⁇ + is indicative of a good response of a patient affected with an osteosarcoma to chemotherapy.
  • the step of determining the amount of ⁇ 5 integrin+ cells, FAK+ cells, or GSK-3 ⁇ + cells may be combined with a step of determining the staining intensity of the ⁇ 5 integrin+ cells, FAK+ cells, or GSK-3 ⁇ + cells. Because the inventors showed a protein profile of prediction of sensitivity to chemotherapy, the present invention also relates to kits for performing the method according to the invention.
  • the present invention also relates to kits for performing the method according to the invention comprising means for determining the level of beta5 integrin, FAK and GSK-3 beta expression in tumor sample.
  • kits of the invention may comprise 3 antibodies directed against beta5 integrin, FAK and GSK-3 beta and another molecule coupled with a signalling system which binds to said antibodies.
  • kits comprise containers with the solutions ready for use. Any other forms are encompassed by the present invention and the man skilled in the art can routinely adapt the form to the use in immunohistochemistry.
  • a second object of the invention relates to a compound which is an antagonist of ⁇ 5 integrin, FAK or GSK-3 ⁇ or an inhibitor of the expression of ⁇ 5 integrin, FAK or GSK-3 ⁇ for use in a method for enhancing clinical efficiency of chemotherapy.
  • said compound according to the invention is a ⁇ 5 integrin
  • the compound according to the invention may bind to ⁇ 5 integrin, FAK or GSK-3 ⁇ and block their physiological effects.
  • a survival as MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • said ⁇ 5 integrin, FAK or GSK-3P antagonist may be a low molecular weight antagonist, e. g. a small organic molecule (natural or not).
  • small organic molecule refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.
  • Antagonists of ⁇ 5 integrin are well known in the state of the art (see for example Desgrosellier JS, et al, 2010).
  • the antagonist of ⁇ 5 integrin according to the invention is SCH221153 (see for example Kumar CC et al, 2001).
  • the antagonist of ⁇ 5 integrin according to the invention is SB-267268 (see for example Wilkinson-Berka J et al, 2006).
  • the antagonist of ⁇ 5 integrin according to the invention is the
  • Antagonists of FAK are well known in the state of the art (see for example Schultze A. et al. 2010, Expert Opin. Investig. Drugs).
  • the antagonist of FAK according to the invention is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • TAE226 see for example Jyotsnabaran H et al., 2007
  • 1,2,4,5-Benzenetetraamine tetrahydro chloride see for example Golubovskaya V et al, 2008.
  • Antagonists of ⁇ 8 ⁇ -3 ⁇ are well known in the state of the art (see for example Kypta R, 2005, Expert Opin. Ther. Patents or Dorronsoro I et al, 2002, Expert Opin. Ther. Patents).
  • the antagonist of ⁇ 8 ⁇ -3 ⁇ according to the invention is a thiadiazolidinone (see for example Zou Y et al., 2008).
  • the antagonist of GSK-3P according to the invention is pyrimidine and pyridine derivatives or substitutes (see for example patent applications US2009074886, WO0220495 or WO 2010012398).
  • the antagonist of GSK-3P according to the invention is the
  • ⁇ 5 integrin, FAK or GSK-3P antagonist of the invention may be an anti-P5 integrin antibody or an anti-FAK antibody or an anti-GSK-3P antibody which neutralizes ⁇ 5 integrin, FAK or GSK-3P or an anti-P5 integrin fragment thereof or an anti- FAK fragment or anti- GSK-3P fragment which neutralizes ⁇ 5 integrin, FAK or GSK-3p.
  • Antibodies directed against ⁇ 5 integrin, FAK or GSK-3P can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against ⁇ 5 integrin, FAK or GSK-3P can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al, 1983); and the EBV-hybridoma technique (Cole et al. 1985).
  • techniques described for the production of single chain antibodies can be adapted to produce anti-P5 integrin, anti-FAK or anti- GSK-3P single chain antibodies.
  • ⁇ 5 integrin, FAK or GSK-3P antagonists useful in practicing the present invention also include antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • F(ab')2 fragments which can be generated by pepsin digestion of an intact antibody molecule
  • Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to ⁇ 5 integrin, FAK or GSK- 3 ⁇ .
  • Humanized anti-P5 integrin, anti-FAK or anti-GSK-3P antibodies and antibody fragments therefrom can also be prepared according to known techniques.
  • “Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non- human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • neutralizing antibodies of ⁇ 5 integrin, FAK or GSK-3P are selected.
  • ⁇ 5 integrin, FAK or GSK-3P antagonists may be selected from aptamers.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996).
  • the compound according to the invention is an inhibitor of the ⁇ 5 integrin, FAK or GSK-3P gene expression.
  • Small inhibitory RNAs can also function as inhibitors of ⁇ 5 integrin, FAK or GSK-3P gene expression for use in the present invention.
  • ⁇ 5 integrin, FAK or GSK-3P gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that ⁇ 5 integrin, FAK or GSK-3P gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • Methods for selecting an appropriate dsRNA or dsRNA- encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T.
  • Ribozymes can also function as inhibitors of ⁇ 5 integrin, FAK or GSK-3P gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of ⁇ 5 integrin, FAK or GSK-3P mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • FAK or GSK-3 P gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis.
  • anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule.
  • DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable R A polymerase promoters such as the T7 or SP6 polymerase promoters.
  • R A polymerase promoters such as the T7 or SP6 polymerase promoters.
  • Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides siR As and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siR A or ribozyme nucleic acid to the cells and preferably cells expressing ⁇ 5 integrin, FAK or GSK-3p.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • adeno-viruses and adeno-associated viruses are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno- associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and micro encap sulation.
  • the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes
  • a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable.
  • the promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
  • Another object of the invention relates to a method for treating ostesarcoma comprising administering to a subject in need thereof a therapeutically effective amount of compound which is an antagonist of ⁇ 5 integrin, FAK or GSK-3P or an inhibitor of the expression of ⁇ 5 integrin, FAK or GSK-3P as described above.
  • the invention relates to a method for treating osteosarcoma comprising administering to a subject in need thereof a therapeutically effective amount of a ⁇ 5 integrin, FAK or GSK-3P antagonist as above described.
  • Compounds of the invention may be administered in the form of a pharmaceutical composition, as defined below.
  • a “therapeutically effective amount” is meant a sufficient amount of compound to treat and/or to prevent ostesarcoma disorder.
  • the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment and/or during phase 1 clinical try.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • Compounds according to the invention may be used for the preparation of a pharmaceutical composition for the treatment of ostesarcoma.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective dose of an antagonist of ⁇ 5 integrin, FAK or GSK-3P or an inhibitor of the ⁇ 5 integrin, FAK or GSK-3P expression, preferably a ⁇ 5 integrin, FAK or GSK-3P antagonist, according to the invention.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions for example, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • compositions of the present invention may comprise a further therapeutic active agent.
  • the present invention also relates to a kit comprising an antagonist or an inhibitor according to the invention and a further therapeutic active agent.
  • said therapeutic active agent is an anticancer agent.
  • said anticancer agents include but are not limited to fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbazine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel
  • additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photo sensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, anthracyclines, MDR inhibitors and Ca2+ ATPase inhibitors.
  • Additional anticancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
  • Additional anticancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.
  • the further therapeutic active agent can be an antiemetic agent.
  • Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron.
  • the further therapeutic active agent can be an hematopoietic colony stimulating factor.
  • Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.
  • the other therapeutic active agent can be an opioid or non-opioid analgesic agent.
  • opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene.
  • Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Illustration of immunohistochemical staining for several biological markers in controls and osteosarcomas (OS). Sections stained were photographed at x 400 magnification (B, C, D, E, F, H and J) or agnification ( A, G and I).
  • A. Colon lining cells serves as a positive control for integrin ⁇ 5 staining.
  • G. Lung adenocarcinoma serves as a positive control for RhoB staining.
  • Beta Catenin (IRS) P 0. 6297 54.8%
  • ILK (IRS) P 0 .2879
  • Betal (IRS) P 0 .9450
  • Beta3 (IRS) P 0 .9176
  • Beta5 (IRS) P 0 .1047
  • RhoB (IRS) P 0 .8494
  • TMAs tissue microarrays
  • pathology center IFR30, U563, CHU Purpan, Toulouse
  • H&E hematoxylin and eosin
  • a tissue microarray instrument (Beecher Instruments, Sun Prairie, WI, USA) was used to create holes in the different paraffin blocks and, for each tumour, three tissue cores of 1-2 mm diameter, representative of the tumour samples, were taken from the primary paraffin block and placed in a new paraffin block.
  • Serial sections of 4 ⁇ thickness were cut from the TMA blocksby using a conventional microtome. One section was stained immediately with H&E, whereas the remaining sections were stored at room temperature before immuno staining. All samples were used in compliance with the French bioethics laws regarding patient information and consent.
  • TMA sections containing samples of the 41 bone tumours were deparaffinised and rehydrated. Heat antigen retrieval was used with citrate buffer (PH 6) for ⁇ 3 integrin, Ang-2 and Ezrin. For the others antibody, sections were incubated with EDTA buffer (ImM, PH 8 or PH 9) for 40 min at 98°C to renature the antigen. Then sections were incubated for 5 min in 3% hydrogen peroxide to quench endogenous peroxidase activity.
  • citrate buffer PH 6
  • EDTA buffer ImM, PH 8 or PH 9
  • Positive controls were chosen according to the studied protein: megakaryocyte for ⁇ 3 integrin staining, basal cell of epiderm for ⁇ integrin staining, colonic crypts for ⁇ 5 integrin staining, stomach parietal cells for Ezrin staining, bone metastasis of adenocarcinoma (lung or breast) for FAK, MMP-9 RhoB, GSK-3P staining, desmoid tumor for ⁇ -catenin staining and vascular endothelium for Ang-2 and ILK staining.
  • Sensitivity which represents its ability to detect the diseased population
  • specificity which represents its ability to detect the responder population, for individual and combined marker performance
  • This index is defined as the sum of sensitivity and specificity minus one.
  • Over-expression was defined when IRS value was higher than the optimal cut-off.
  • MFS metastasis- free survival
  • OS death from any cause for overall survival
  • Table 1 lists the demographic, clinical and clinical outcomes of the 41 patients included in this study.
  • the 41 osteosarcomas were conventional high grade osteosarcoma according to the WHO classification of 2002. Among them, 8 were osteosblastic osteosarcomas, 13 were chondroblastic osteosarcomas, 12 were fibroblastic osteosarcoma and 4 could not be assigned to a specific subgroup due to unusual morphological features (such as predominant giant cells, predominant storiform pattern). Thirty-seven patients were treated by neo-adjuvant chemotherapy before surgery.
  • GSK-3P At the optimal cut-off, estimated to be 1, GSK-3P, achieved a sensitivity of 33.3%, specificity of 100.0%) and diagnostic accuracy of 65.71%). Using this cut-off, GSK-3P expression was associated to a pejorative response to treatment.
  • Rho pathways induces radio sensitization and oxygenation in human glioblastoma xenografts. Oncogene 22:8861-9, 2003.
  • Damiano JS Integrins as novel drug targets for overcoming innate drug resistance. Curr Cancer Drug Targets 2:37-43, 2002 Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer. 2010 Jan;10(l):9-22.
  • Remmele W Stegner HE: [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue].
  • IRS immunoreactive score
  • ER-ICA immunohistochemical estrogen receptor detection
  • Amittha Wickrema Growth control of multiple myeloma cells through inhibition of glycogen synthase kinase-3. Leukemia & Lymphoma, October 2008; 49(10): 1945-1953.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Hospice & Palliative Care (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Urology & Nephrology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé ex vivo permettant de prédire la réponse à une chimiothérapie d'un patient atteint d'un ostéosarcome. Ce procédé comprend l'étape consistant à déterminer le niveau d'expression d'au moins un marqueur, sélectionné dans le groupe constitué par l'intégrine β5, la FAK et la GSK-3β, dans un échantillon de tumeur provenant dudit patient. De plus, l'invention concerne un antagoniste de l'intégrine β5, de la FAK ou de la GSK-3β, ou un inhibiteur de l'expression de l'intégrine β5, de la FAK ou de la GSK-3β, utilisé dans un procédé visant à renforcer l'efficacité clinique d'une chimiothérapie.
PCT/EP2012/062147 2011-06-24 2012-06-22 Procédé pour prédire la réponse à une chimiothérapie d'un patient atteint d'un ostéosarcome Ceased WO2012175711A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP11305809 2011-06-24
EP11305809.3 2011-06-24

Publications (1)

Publication Number Publication Date
WO2012175711A1 true WO2012175711A1 (fr) 2012-12-27

Family

ID=44351603

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2012/062147 Ceased WO2012175711A1 (fr) 2011-06-24 2012-06-22 Procédé pour prédire la réponse à une chimiothérapie d'un patient atteint d'un ostéosarcome

Country Status (1)

Country Link
WO (1) WO2012175711A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2930248A1 (fr) * 2014-03-31 2015-10-14 National Cancer Center Procédé pour prédire la sensibilité d'un patient présentant un ostéosarcome à un agent anticancéreux

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
WO1999032619A1 (fr) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Inhibition genetique par de l'arn double brin
WO2001036646A1 (fr) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibition d"expression genique a l"aide d"arn bicatenaire
WO2001068836A2 (fr) 2000-03-16 2001-09-20 Genetica, Inc. Procedes et compositions d'interference d'arn
WO2002020495A2 (fr) 2000-09-06 2002-03-14 Chiron Corporation Inhibiteurs de glycogene-synthase kinase 3
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
WO2006021454A2 (fr) * 2004-08-27 2006-03-02 Novartis Ag Derives de pyrimidine
WO2008042012A1 (fr) * 2006-10-05 2008-04-10 Rhode Island Hospital Compositions et procédés pour détecter et traiter une blessure rénale et une inflammation
WO2008077138A1 (fr) 2006-12-19 2008-06-26 The Board Of Trustees Of The University Of Illinois 3-benzofuranyl-4-indolyl maléimides en tant qu'inhibiteurs de gsk3 puissants pour troubles neurodégénératifs
US20090074886A1 (en) 2003-08-13 2009-03-19 Novartis Vaccines And Diagnostics Inc. Gsk-3 inhibitors
WO2009050303A1 (fr) * 2007-10-19 2009-04-23 Pharma Mar, S.A. Marqueurs moléculaires pronostiques pour le traitement par et-743
WO2010012398A1 (fr) 2008-07-30 2010-02-04 Bayer Schering Pharma Aktiengesellschaft Pyridines substituées et leur utilisation en tant qu'inhibiteurs de la gsk3
WO2011059388A1 (fr) * 2009-11-13 2011-05-19 Astrazeneca Ab Pyrazine à substitution oxazolo[4,5-c]pyridine

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
WO1999032619A1 (fr) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Inhibition genetique par de l'arn double brin
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
WO2001036646A1 (fr) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibition d"expression genique a l"aide d"arn bicatenaire
WO2001068836A2 (fr) 2000-03-16 2001-09-20 Genetica, Inc. Procedes et compositions d'interference d'arn
WO2002020495A2 (fr) 2000-09-06 2002-03-14 Chiron Corporation Inhibiteurs de glycogene-synthase kinase 3
US20090074886A1 (en) 2003-08-13 2009-03-19 Novartis Vaccines And Diagnostics Inc. Gsk-3 inhibitors
WO2006021454A2 (fr) * 2004-08-27 2006-03-02 Novartis Ag Derives de pyrimidine
WO2008042012A1 (fr) * 2006-10-05 2008-04-10 Rhode Island Hospital Compositions et procédés pour détecter et traiter une blessure rénale et une inflammation
WO2008077138A1 (fr) 2006-12-19 2008-06-26 The Board Of Trustees Of The University Of Illinois 3-benzofuranyl-4-indolyl maléimides en tant qu'inhibiteurs de gsk3 puissants pour troubles neurodégénératifs
WO2009050303A1 (fr) * 2007-10-19 2009-04-23 Pharma Mar, S.A. Marqueurs moléculaires pronostiques pour le traitement par et-743
WO2010012398A1 (fr) 2008-07-30 2010-02-04 Bayer Schering Pharma Aktiengesellschaft Pyridines substituées et leur utilisation en tant qu'inhibiteurs de la gsk3
WO2011059388A1 (fr) * 2009-11-13 2011-05-19 Astrazeneca Ab Pyrazine à substitution oxazolo[4,5-c]pyridine

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
ADER I; DELMAS C; BONNET J ET AL.: "Inhibition of Rho pathways induces radio sensitization and oxygenation in human glioblastoma xenografts", ONCOGENE, vol. 22, 2003, pages 8861 - 9
BACCI G; MERCURI M; BRICCOLI A ET AL.: "Osteogenic sarcoma of the extremity with detectable lung metastases at presentation. Results of treatment of 23 patients with chemotherapy followed by simultaneous resection of primary and metastatic lesions", CANCER, vol. 79, 1997, pages 245 - 54
CANCER TREATMENT AND RESEARCH 2009 LNKD- PUBMED:20213413, vol. 152, 2009, pages 517 - 528, ISSN: 0927-3042 *
DAMIANO JS: "Integrins as novel drug targets for overcoming innate drug resistance", CURR CANCER DRUG TARGETS, vol. 2, 2002, pages 37 - 43, XP008102581
DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; 2009, KIM SU YOUNG ET AL: "Strategies to explore new approaches in the investigation and treatment of osteosarcoma.", XP009151284, Database accession no. NLM20213413 *
DESGROSELLIER JS; CHERESH DA: "Integrins in cancer: biological implications and therapeutic opportunities", NAT REV CANCER., vol. 10, no. 1, January 2010 (2010-01-01), pages 9 - 22, XP055016659, DOI: doi:10.1038/nrc2748
DORRONSORO I ET AL., EXPERT OPIN. THER. PATENTS, 2002
DORRONSORRO I.; ANNA CASTRO; ANA MARTINEZ: "Inhibitors of glycogen synthase kinase-3: future therapy for unmet medical needs", EXPERT OPIN. THER. PATENTS, 2002
FELDING-HABERMANN B; O'TOOLE TE; SMITH JW ET AL.: "Integrin activation controls metastasis in human breast cancer", PROC NATL ACAD SCI USA, vol. 98, 2001, pages 1853 - 8, XP002484896, DOI: doi:10.1073/pnas.98.4.1853
GOLUBOVSKAYA VITA M.; CARL NYBERG; MIN ZHENG; FREDERICK KWEH; ANDREW MAGIS; DAVID OSTROV; WILLIAM G: "Cance. A small molecule inhibitor 1,2,4,5-Benzenetetraamine tetrahydrochloride, targeting the Y397 site of Focal Adhesion Kinase decreases tumor growth", J MED CHEM., 2008
HOHEISEL, NATURE REVIEWS, GENETICS, vol. 7, 2006, pages 200 - 210
HOOD JD; CHERESH DA: "Role of integrins in cell invasion and migration", NAT REV CANCER, vol. 2, 2002, pages 91 - 100
HULLINGER TG; MCCAULEY LK; DEJOODE ML ET AL.: "Effect of bone proteins on human prostate cancer cell lines in vitro", PROSTATE, vol. 36, 1998, pages 14 - 22
JENNIFER L. WILKINSON-BERKA; DARIA JONES; GEORGE TAYLOR; KASSIE JAWORSKI; DARREN J. KELLY; STEVE B. LUDBROOK; ROBERT N. WILLETTE;: "SB-267268, a Nonpeptidic Antagonist of avp3 and avp5 Integrins, Reduces Angiogenesis and VEGF Expression in a Mouse Model of Retinopathy of Prematurity", INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 2006
JYOTSNABARAN HALDER; YVONNE G. LIN; 1 WILLIAM M. MERRITT; WHITNEY A. SPANNUTH; ALPA M. NICK; TOSHIYUKI HONDA; APARNA A. KAMAT; LIZ: "Therapeutic Efficacy of a Novel Focal Adhesion Kinase Inhibitor TAE226 in Ovarian Carcinoma", AMERICAN ASSOCIATION FOR CANCER RESEARCH, 2007
KIM C; SHIN E; HONG S ET AL.: "Clinical value of ezrin expression in primary osteosarcoma", CANCER RES TREAT, vol. 41, 2009, pages 138 - 44
KONONEN J; BUBENDORFL; KALLIONIEMI A ET AL.: "Tissue microarrays for high-throughput molecular profiling of tumor specimens", NAT MED, vol. 4, 1998, pages 844 - 7, XP002927997, DOI: doi:10.1038/nm0798-844
KRAMAR A; FARAGGI D; FORTUNE A ET AL.: "mROC: a computer program for combining tumour markers in predicting disease states", COMPUT METHODS PROGRAMS BIOMED, vol. 66, 2001, pages 199 - 207
KYPTA R, EXPERT OPIN. THER. PATENTS, 2005
KYPTA ROBERT M.: "GSK-3 inhibitors and their potential in the treatment of Alzheimer's disease", EXPERT OPIN. THER. PATENTS, vol. 15, no. 10, 2005, pages 1315 - 1331
LE DELEY MC; GUINEBRETIERE JM; GENTET JC; PACQUEMENT H; PICHON F; MAREC-BERARD P; ENTZ-WERLE N; SCHMITT C; BRUGIERES L; VANEL D: "SFOP OS94: a randomised trial comparing preoperative high-dose methotrexate plus doxorubicin to high-dose methotrexate plus etoposide and ifosfamide in osteosarcoma patients", EUR J CANCER. 2007 MAR, vol. 43, no. 4, 30 January 2007 (2007-01-30), pages 752 - 61, XP005897122, DOI: doi:10.1016/j.ejca.2006.10.023
MASSABEAU C; ROUQUETTE I; LAUWERS-CANCES V ET AL.: "Basic fibroblast growth factor-2/beta3 integrin expression profile: signature of local progression after chemoradiotherapy for patients with locally advanced non-small-cell lung cancer", INT J RADIAT ONCOL BIOL PHYS, vol. 75, 2009, pages 696 - 702, XP026669270, DOI: doi:10.1016/j.ijrobp.2008.11.050
MAUBANT S; CRUET-HENNEQUART S; POULAIN L ET AL.: "Altered adhesion properties and alphav integrin expression in a cisplatin-resistant human ovarian carcinoma cell line", INT J CANCER, vol. 97, 2002, pages 186 - 94
MENENDEZ JA; VELLON L; MEHMI I ET AL.: "A novel CYR61-triggered 'CYR61- alphavbeta3 integrin loop' regulates breast cancer cell survival and chemosensitivity through activation of ERKl/ERK2 MAPK signaling pathway", ONCOGENE, vol. 24, 2005, pages 761 - 79
MEYERS PA; HELLER G; HEALEY J ET AL.: "Chemotherapy for nonmetastatic osteogenic sarcoma: the Memorial Sloan-Kettering experience", J CLIN ONCOL, vol. 10, 1992, pages 5 - 15
MONFERRAN S; SKULI N; DELMAS C ET AL.: "Alphavbeta3 and alphavbeta5 integrins control glioma cell response to ionising radiation through ILK and RhoB", INT J CANCER, vol. 123, 2008, pages 357 - 64
REARDON DA; NABORS LB; STUPP R; MIKKELSEN T: "Cilengitide: an integrin-targeting arginine-glycine-aspartic acid peptide with promising activity for glioblastoma multiforme", EXPERT OPIN INVESTIG DRUGS., vol. 17, no. 8, August 2008 (2008-08-01), pages 1225 - 35, XP002591977, DOI: doi:10.1517/13543784.17.8.1225
REISER B; FARAGGI D: "Confidence intervals for the generalized ROC criterion", BIOMETRICS, vol. 53, 1997, pages 644 - 52
REMMELE W; STEGNER HE: "Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue", PATHOLOGE, vol. 8, 1987, pages 138 - 40, XP009129710
ROSEN G; CAPARROS B; HUVOS AG ET AL.: "Preoperative chemotherapy for osteogenic sarcoma: selection of postoperative adjuvant chemotherapy based on the response of the primary tumor to preoperative chemotherapy", CANCER, vol. 49, 1982, pages 1221 - 30
SALAS S; BARTOLI C; DEVILLE JL ET AL.: "Ezrin and alpha-smooth muscle actin are immunohistochemical prognostic markers in conventional osteosarcomas", VIRCHOWS ARCH, vol. 451, 2007, pages 999 - 1007, XP019563675, DOI: doi:10.1007/s00428-007-0474-8
SALAS S; JEZEQUEL P; CAMPION L ET AL.: "Molecular characterization of the response to chemotherapy in conventional osteosarcomas: predictive value ofHSD17B10 and IFITM2", INT J CANCER, vol. 125, 2009, pages 851 - 60, XP055109879, DOI: doi:10.1002/ijc.24457
SCHULTZE ALEXANDER; WALTER FIEDLER: "Therapeutic potential and limitations of new FAK inhibitors in the treatment of cancer", EXPERT OPIN. INVESTIG. DRUGS, vol. 19, no. 6, 2010, pages 777 - 788, XP008142458, DOI: doi:10.1517/13543784.2010.489548
SKULI N; MONFERRAN S; DELMAS C ET AL.: "Alphavbeta3/alphavbeta5 integrins-FAK-RhoB: a novel pathway for hypoxia regulation in glioblastoma", CANCER RES, vol. 69, 2009, pages 3308 - 16, XP055272602, DOI: doi:10.1158/0008-5472.CAN-08-2158
YOON SO; SHIN S; MERCURIO AM: "Ras stimulation of E2F activity and a consequent E2F regulation of integrin alpha6beta4 promote the invasion of breast carcinoma cells", CANCER RES, vol. 66, 2006, pages 6288 - 95
ZHOU YING; SHAHAB UDDIN; TODD ZIMMERMAN; JEONG-AH KANG; JODIE ULASZEK; AMITTHA WICKREMA: "Growth control of multiple myeloma cells through inhibition of glycogen synthase kinase-3", LEUKEMIA & LYMPHOMA, vol. 49, no. 10, October 2008 (2008-10-01), pages 1945 - 1953

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2930248A1 (fr) * 2014-03-31 2015-10-14 National Cancer Center Procédé pour prédire la sensibilité d'un patient présentant un ostéosarcome à un agent anticancéreux

Similar Documents

Publication Publication Date Title
Lee et al. Periostin-binding DNA aptamer inhibits breast cancer growth and metastasis
JP6599334B2 (ja) 血中循環腫瘍細胞に関する方法およびアッセイ
JP6664323B2 (ja) 神経膠芽腫を治療するための診断方法及び組成物
US7368098B2 (en) Use of biomolecular targets in the treatment and visualization of tumors
Ren et al. Annexin A1 induces oxaliplatin resistance of gastric cancer through autophagy by targeting PI3K/AKT/mTOR
CN112522394B (zh) 新型外泌体释放相关靶点及其在监测和抑制肿瘤中的应用
KR101925125B1 (ko) Nckap1을 유효성분으로 포함하는 대장암 진단 또는 대장암 전이 예후 예측용 바이오마커 조성물
EP2611933A1 (fr) Procédé de pronostic de la progression du cancer
US20090215812A1 (en) Biomarkers for evaluating likelihood of tumor sensitivity to an mtor inhibitor
Liu et al. IL1R2 promotes tumor progression via JAK2/STAT3 pathway in human clear cell renal cell carcinoma
WO2009044158A2 (fr) Inhibiteurs et utilisations
WO2017064159A1 (fr) Trpv2 en tant que biomarqueur et en tant que cible thérapeutique pour un mélanome
JP2015529808A (ja) 肺癌を有する患者における化学療法に対する耐性を検出するための新規の方法
US20180105882A1 (en) New biomarker for outcome in aml
JP7167263B2 (ja) Ｐｍｖｋを有効成分として含む放射線抵抗性癌診断用または放射線治療予後予測用バイオマーカー組成物
US20220340975A1 (en) Method of treatment and pronostic of acute myeloid leukemia
WO2011107939A1 (fr) Méthodes de prédiction de l'efficacité d'un traitement par anti-vegfa pour des tumeurs solides
WO2018055023A1 (fr) Méthodes et compositions pharmaceutiques pour le traitement du cancer du poumon
WO2012175711A1 (fr) Procédé pour prédire la réponse à une chimiothérapie d'un patient atteint d'un ostéosarcome
Rönnberg Signal transduction inhibitors
US20240229154A9 (en) Method for prognosis and treating a patient suffering from cancer
CN114045336A (zh) Cga基因作为靶点在制备用于诊断和治疗耐药实体瘤药物的应用
US20210290633A1 (en) Combination for treating cancer
EP3009147A1 (fr) Procédé de traitement de glioblastome résistant
KR20230168575A (ko) 편평형 췌장암 진단을 위한 바이오마커 및 이의 용도

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12733446

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12733446

Country of ref document: EP

Kind code of ref document: A1