WO2009098656A2 - Méthode de prédiction ou de diagnostic d'évolution de tumeurs intracrâniennes chez un sujet - Google Patents

Méthode de prédiction ou de diagnostic d'évolution de tumeurs intracrâniennes chez un sujet Download PDF

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WO2009098656A2
WO2009098656A2 PCT/IB2009/050479 IB2009050479W WO2009098656A2 WO 2009098656 A2 WO2009098656 A2 WO 2009098656A2 IB 2009050479 W IB2009050479 W IB 2009050479W WO 2009098656 A2 WO2009098656 A2 WO 2009098656A2
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gdf
subject
glioblastoma
patients
csf
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PCT/IB2009/050479
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WO2009098656A3 (fr
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Isabelle Desbaillets Hakimi
Monika Hegi
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Universite De Lausanne
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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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • 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
    • G01N2333/495—Transforming growth factor [TGF]

Definitions

  • the present invention relates to a method for diagnosing or predicting outcome of intracranial tumors in the cerebro-spinal fluid of a subject.
  • the present invention further relates to compositions and methods for treatment or prevention of intracranial tumors in a subject and to a kit useful for predicting same.
  • Glioblastoma is the most aggressive and most common type of primary brain tumors. Despite surgery, and combined chemo -radiotherapy patients ultimately recur with a median survival of 15 months. Thus, new avenues have to be taken to improve treatment strategies for this devastating disease.
  • GDF- 15 also known as Macrophage-inhibitory cytokine- 1 (MIC-I), PLAB, prostate-derived factor, PTGF- ⁇ , PTGFB, PDF, NRG-I, and NAG-I, is a divergent member of the transforming growth factor ⁇ superfamily .
  • the placenta is the only tissue that expresses large amounts of GDF- 15 under normal physiologic conditions.
  • epithelial cells including the neuroepithelium express low levels of GDF- 15 mRNA.
  • GDF- 15 protein is synthesized as a 308-amino acid propeptide which, when secreted, binds to local extracellular matrix and subsequently becomes cleaved by a furin-like protease.
  • the mature peptide which is secreted by an alternate pathway, is a 112-amino acid protein which diffuses rapidly into the circulation.
  • GDF- 15 has been shown by several groups to induce apoptosis and local GDF- 15 expression in the stroma of the malignant prostate gland, and has been linked to improved outcome.
  • GDF- 15 Human growth differentiation factor- 15 expression in cerebro-spinal fluid (CSF) of glioblastoma (GBM) patients has been evaluated and it unexpectedly results that elevated levels of GDF- 15 in the CSF of glioblastoma patients is associated with worse outcome
  • GDF- 15 in the CSF is a prognostic factor in patients with glioblastoma.
  • the present invention provides a method of diagnosis or prognosis of intracranial tumors in a subject, the method comprising detecting an elevated amount of GDF- 15 in the CSF from said subject.
  • the presence of elevated concentrations of GDF-15 in the CSF at the time of operation is associated with decreased overall survival.
  • the cut-off was set at the limit of detection of 156 pg/ml.
  • GDF-15 immunohistochemistry on glioblastoma tissue.
  • GDF-15 exhibits immunostaining on a subtype of infiltrative macrophages in human glioblastoma (A, B). Positively stained macrophages (A) here located in the perinecrotic region, close to pseudopalisades, both hallmarks of glioblastoma. Tumor cells appear negative.
  • Colon carcinoma (C) was used as positive control.
  • GDF-15 Gene expression levels of GDF-15. Expression of the GDF-15 transcript determined by qRT- PCR is relatively low in glioblastoma tissues as compared to prostate (A). Freshly isolated monocytes, and undifferentiated monocytoid cells (U937) exert low expression of GDF-15 which markedly increases upon differentiation using 65 ng/niL PMA for 48 hours. GDF-15 expression was normalized with RPOLII expression.
  • a cell includes a plurality of cells, including mixtures thereof.
  • a protein includes a plurality of proteins.
  • peptide As used herein, the terms “peptide”, “protein”, “polypeptide”, “polypeptidic” and “peptidic” are used interchangeably to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. Glioblastoma are notorious for resistance to therapy which has been attributed to DNA repair proficiency, a multitude of deregulated molecular pathways, and more recently to the particular biological behavior of tumor stem- like cells.
  • the present invention is useful to guide a rational choice of agents, targets, trial design, and appropriate patient selection, incorporating biomarkers defining mechanisms of response and resistance.
  • One object of the invention is to provide a method for diagnosing or predicting outcome of intracranial tumors in the cerebro-spinal fluid of a subject comprising:
  • these sequences contain at least 7, most preferably 25, more preferably 40, even more preferably 50 and still even more preferably 88 contiguous amino acids in length in common with sequence of the peptide of the invention.
  • These fragments can be prepared by a variety of methods and techniques known in the art such as for example chemical synthesis.
  • the intracranial tumor is a glioblastoma or brain metastasis and most preferably the intracranial tumor is a glioblastoma.
  • the measuring of the expression levels of GDF- 15 is preferably obtained through Western blot, immunoprecipitation, ELISA, Radio Immuno Assay, proteomics methods comprising but not limited to Mass Spectroscopy, or any other quantitative method detecting GDF- 15 that is known by the skilled in the art.
  • the method according to the invention allows evaluating the medical prognosis of said subject based on the comparison of step (b), and/or adapting the treatment of said subject.
  • adaptive the treatment generally refers to the choice of a treatment among different options, based on the specificities of the disease, concomitant pathologies or patient conditions, or the switch from one treatment to another in the course of the therapy because of the non-response, progression or resistance of the disease to the initial treatment, with the intent to offer to the patients the beast treatment for his diseases under the given circumstances.
  • the biological sample used in the method of the invention, is a bio-fluid comprising cerebrospinal fluid, blood, urine, or biopsy of brain tumor.
  • the biological sample is cerebrospinal fluid sample.
  • the subject is a mammal and preferably a human.
  • the terms "subject” or “patient” are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human.
  • the subject is a subject in need of treatment.
  • the subject can be a normal subject.
  • the present invention also relates to a method for treatment of intracranial tumors in a subject.
  • the present invention encompasses the use of modulators of expression of GDF- 15 or a biologically active fragment thereof in the preparation of a medicament for the treatment or prevention of intracranial tumors in a subject.
  • GDF- 15 may be like other members of the TGF-beta superfamily, acting as a tumor suppressor in the early stages, but acting pro-tumorigenic at the later stages of tumor progression.
  • the expression of GDF- 15 can be increased by treatment with drugs and chemicals documented to prevent tumor formation and development.
  • GDF 15 is induced by multiple types of cellular stress, such as anoxia, DNA damaging agents, and NSAIDs, independent of p53 or HIF-I alpha.
  • Applicants did not observe increased GDF 15 CSF concentrations in glioblastoma patients receiving alkylating agent chemotherapy compared to patients with measurements at the onset of surgery (chemo-na ⁇ ve).
  • NF-kappa-B nuclear factor of kappaB pathway.
  • GDF 15 may promote or suppress tumor growth in different situations, this also occurs in the brain and elevated CSF GDF 15 concentration is a marker of macrophage activation assisting tumor progression.
  • Prevention means that the administration of the modulator(s) as described results in a reduction in the likelihood that a subject at high risk for intracranial tumor, relapse and/or metastatic progression after targeted anti-tumor therapy, radiotherapy, chemotherapy, or combination thereof is applied.
  • this phrase means that the administration of the modulator(s) results in the reduction of the likelihood or probability that a subject at risk will indeed develop intracranial tumors like for example glioblastoma.
  • Biologically active means affecting any physical or biochemical properties of a living organism or biological process.
  • Bioly Active Substance refers to any molecule or mixture or complex of molecules that exerts a biological effect in vitro and/or in vivo, including pharmaceuticals, drugs, proteins, peptides, polypeptides, hormones, vitamins, steroids, polyanions, nucleosides, nucleotides, nucleic acids (e.g. DNA or RNA), nucleotides, polynucleotides, etc.
  • said modulators of expression of GDF- 15 or biologically active fragment thereof are preferably inhibitors or competitors, which preferably comprise an antibody, or an immunologically active fragment thereof, that binds to a GDF- 15 protein or biologically active fragment thereof.
  • the present invention also relates to the use of modulators of the biological activity of GDF- 15 or a biologically active fragment thereof in the preparation of a medicament for treatment or prevention of intracranial tumors in a subject.
  • a variant is a peptide having an amino acid sequence that differs to some extent from a native sequence peptide that is an amino acid sequence that varies from the native sequence by conservative amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles.
  • the amino acid sequence variants possess substitutions, deletions, side-chain modifications and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence.
  • Conservative amino acid substitutions are herein defined as exchanges within one of the following five groups:
  • Lys residues may be substituted by ornithine, homoarginine, nor-Lys, N-methyl-Lys, N, N-dimethyl-Lys and N, N, N- trimethyl-Lys.
  • Lys residues can also be replaced with synthetic basic amino acids including, but not limited to, N-I- (2-pyrazolinyl)-Arg, 2- (4-piperinyl)-Gly, 2- (4- piperinyl)-Ala, 2- [3- (2S) pyrrolininyl]-Gly and2- [3- (2S) pyrolininyl]-Ala.
  • Tyr residues may be substituted with 4- methoxy tyrosine (MeY), meta-Tyr,ortho-Tyr, nor-Tyr, 1251 -Tyr, mono -halo -Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, and nitro-Tyr.
  • MeY 4- methoxy tyrosine
  • meta-Tyr meta-Tyr,ortho-Tyr, nor-Tyr
  • 1251 -Tyr mono -halo -Tyr
  • di-halo-Tyr di-halo-Tyr
  • O-sulpho-Tyr O-phospho-Tyr
  • nitro-Tyr 4- methoxy tyrosine
  • Tyr residues may also be substituted with the 3-hydroxyl or 2-hydroxyl isomers (meta-Tyr or ortho-Tyr, respectively) and corresponding O-sulpho-and O-phospho derivatives. Tyr residues can also be replaced with synthetic hydroxyl containing amino acids including, but not limited to4-hydroxymethyl-Phe, 4-hydroxyphenyl- GIy, 2, 6-dimethyl-Tyr and 5-amino-Tyr.
  • Aliphatic amino acids may be substituted by synthetic derivatives bearing non-natural aliphatic branched or linear side chains CnH2n+2 where n is a number from 1 up to and including 8. Examples of suitable conservative substitutions by non-conventional amino acids are given in W002/064740.
  • Insertions encompass the addition of one or more naturally occurring or non conventional amino acid residues.
  • Deletion encompasses the deletion of one or more amino acid residues.
  • the physiological active protein of the invention may be prepared in order to include D-forms and/or "retro -inverso isomers" of the peptide.
  • retro-inverso isomers of short parts, variants or combinations of the physiological active protein of the invention are prepared.
  • Retro-inverso peptides are prepared for peptides of known sequence as described for example in SeIa and Zisman, in a review published in FASEB J. 1997 May;l l(6):449-56.
  • retro-inverso isomer an isomer of a linear peptide in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted; thus, there can be no end-group complementarity.
  • the invention also includes analogs in which one or more peptide bonds have been replaced with an alternative type of covalent bond (a "peptide mimetic") which is not susceptible to cleavage by peptidases.
  • a peptide mimetic an alternative type of covalent bond
  • proteolytic degradation of the peptides following injection into the subject is a problem
  • replacement of a particularly sensitive peptide bond with a noncleavable peptide mimetic will make the resulting peptide more stable and thus more useful as an active substance.
  • mimetics, and methods of incorporating them into peptides are well known in the art.
  • inhibitor refers to molecules that inhibit the function of the protein or polypeptide by binding thereto.
  • competitive inhibition refers to "inhibitors” or “antagonists” that directly inhibit the interaction between a protein or polypeptide (i.e. receptor) and its natural ligand resulting in disturbed biochemical or biological function of the receptor.
  • Competitive inhibition is a form of inhibition where binding of the inhibitor prevents binding of the ligand and vice versa.
  • the inhibitor binds to the same active site as the natural ligand, without undergoing a reaction. The ligand molecule cannot enter the active site while the inhibitor is there, and the inhibitor cannot enter the site when the ligand is there.
  • biological activity of a protein refers to the ability to carry out diverse cellular functions and to bind other molecules specifically and tightly.
  • the present invention also includes vaccines and vaccination methods.
  • methods of treating or preventing intracranial tumors in a subject can involve administering to the subject a vaccine composition comprising one or more antibody directed against GDF- 15 or a biologically active fragment thereof and/or combinations thereof or immunologically active fragments of such polypeptides.
  • an immunologically active fragment is a polypeptide that is shorter in length than the full-length naturally-occurring protein yet which induces an immune response analogous to that induced by the full-length protein.
  • an immunologically active fragment should be at least 8 residues in length and capable of stimulating an immune cell, for example, a T cell or a B cell.
  • Immune cell stimulation can be measured by detecting cell proliferation, elaboration of cytokines (e.g., IL-2), or production of an antibody. See, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual, 1998, Cold Spring Harbor Laboratory Press; and Coligan, et al, Current Protocols in Immunology, 1991-2006, John Wiley & Sons.
  • the inhibitor of the biological activity of said protein is an antibody or an immunologically fragment thereof that binds to a GDF- 15 protein or biologically active fragment thereof.
  • an antibody refers to an immunoglobulin molecule having a specific structure, that interacts (i.e., binds) only with the antigen that was used for synthesizing the antibody (i.e., the gene product of an up-regulated marker) or with an antigen closely related thereto.
  • an antibody can be a fragment of an antibody or a modified antibody, so long as it binds to one or more of the proteins encoded by the marker genes.
  • the antibody fragment can be Fab, F(ab')2, Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston J. S. et al., (1988) Proc.
  • an antibody fragment can be generated by treating an antibody with an enzyme, including papain or pepsin.
  • an enzyme including papain or pepsin.
  • a gene encoding the antibody fragment can be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co M. S. et al., (1994) J. Immunol. 152:2968-76; Better M. and Horwitz A. H. (1989) Methods Enzymol. 178:476-96.; Pluckthun A. and Skerra A. (1989) Methods Enzymol. 178:497-515.; Lamoyi E. (1986) Methods Enzymol.
  • An antibody can be modified by conjugation with a variety of molecules, for example, polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the present invention provides such modified antibodies.
  • the modified antibody can be obtained by chemically modifying an antibody. Such modification methods are conventional in the field.
  • an antibody can comprise a chimeric antibody having a variable region from a nonhuman antibody and a constant region from a human antibody, or a humanized antibody, comprising a complementarity determining region (CDR) from a nonhuman antibody, a frame work region (FR) and a constant region from a human antibody.
  • CDR complementarity determining region
  • FR frame work region
  • Such antibodies can be prepared by using known technologies. Humanization can be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (see, e.g., Verhoeyen et ah, (1988) Science 239:1534-6). Accordingly, such humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non- human species.
  • Fully human antibodies comprising human variable regions in addition to human framework and constant regions can also be used.
  • Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage (e.g., Hoogenboom & Winter, (1992) J. MoI. Biol. 227:381-8).
  • human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, e.g., in U.S. Patent Nos. 6,150,584; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016.
  • Such antibodies can be prepared by using known technologies.
  • a pharmaceutically effective amount refers to a chemical material or compound which, when administered to a human or animal organism induces a detectable pharmacologic and/or physiologic effect.
  • the respective pharmaceutically effective amount can depend on the specific patient to be treated, on the disease to be treated and on the method of administration. Further, the pharmaceutically effective amount depends on the specific protein used, especially if the protein additionally contains a drug as described or not.
  • the treatment usually comprises a multiple administration of the pharmaceutical composition, usually in intervals of several hours, days or weeks.
  • the pharmaceutically effective amount of a dosage unit of the polypeptide usually is in the range of 0.001 ng to 100 mg per kg of body weight of the patient to be treated.
  • a therapeutically effective amount or dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Initial doses can also be estimated from in vivo data, e.g. animal models, using techniques that are well known in the art.
  • One ordinarily skill in the art could readily optimise administration to humans based on animal data and will, of course, depend on the subject being treated, on the subject's weight, the severity of the disorder, the manner of administration and the judgement of the prescribing physician.
  • administering refers to contact of the pharmaceutical compositions to the subject, preferably a human.
  • the pharmaceutical composition may be dissolved or dispersed in a pharmaceutically acceptable carrier well known to those skilled in the art, for parenteral administration by, e. g., intravenous, subcutaneous or intramuscular injection or by intravenous drip infusion.
  • any conventional additives may be used such as excipients, adjuvants, binders, disintegrants, dispersing agents, lubricants, diluents, absorption enhancers, buffering agents, surfactants, solubilizing agents, preservatives, emulsif ⁇ ers, isotonizers, stabilizers, solubilizers for injection, pH adjusting agents, etc.
  • EDTA EDTA
  • sugars such as sucrose, mannitol, trehalose or sorbitol
  • salt-forming counter-ions such as sodium
  • metal complexes e.g. Zn-protein complexes
  • non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).
  • administration of the pharmaceutical composition may be systemic or topical.
  • administration of such a pharmaceutical composition may be various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, buccal routes or via an implanted device, and may also be delivered by peristaltic means.
  • composition comprising an active ingredient of the present invention may also be incorporated or impregnated into a bioabsorbable matrix, with the matrix being administered in the form of a suspension of matrix, a gel or a solid support.
  • the matrix may be comprised of a biopolymer.
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and [gamma] ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT(TM) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished for example by filtration through sterile filtration membranes.
  • a peptide of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any and the nature of the effect desired.
  • the appropriate dosage form will depend on the disease, the protein, and the mode of administration; possibilities include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, solutions, ointments and parenteral depots.
  • the present invention further provides a method of treatment of intracranial tumors and in particular glioblastoma in a subject, said method comprising administering to said subject an effective amount of an agent which enhances or increases the activity or expression of GDF- 15.
  • an agent which enhances or increases the activity or expression of GDF- 15 comprise agents intervening in p53 and hypoxia inducible factor 1 (HIF-I) independent pathways or EGR-I related pathways, inhibitors of cyclooxygenases, or biologically active fragments of GDF- 15, and/or combinations thereof.
  • HIF-I hypoxia inducible factor 1
  • the present invention further relates to a kit useful for detecting or diagnosing the intracranial tumors in a subject or predicting outcome of a subject with a glioblastoma
  • said kit comprises a set of: a) antibodies adapted to detect GDF- 15 and/or immunogenic fragments thereof, b) a control reagent and/or a detectable label, and optionally instructions to use.
  • the detection reagents can be packaged together in the form of a kit.
  • the detection reagents can be packaged in separate containers, e.g., antibody (either bound to a solid matrix or packaged separately with reagents for binding them to the matrix), a control reagent (positive and/or negative), and/or a detectable label.
  • Instructions e.g., written, tape, VCR, CD-ROM, etc.
  • the assay format of the kit can be a sandwich ELISA, both of which are known in the art. See, for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3 rd Edition, 2001, Cold Spring Harbor Laboratory Press; and Harlow and Lane, Using Antibodies, supra.
  • An advantage of the present invention is that for patients with brain tumors, identification of diagnostic and prognostic markers in easy accessible biological material, such as plasma or cerebro-spinal fluid (CSF), greatly facilitates patient management.
  • GDF-15 growth differentiation factor 15 encodes a secreted protein of the TGF-beta superfamily and emerged as a candidate marker from Applicant's gene expression studies, with increasing expression during malignant progression of glioma.
  • GDF- 15 is likely a subpopulation of tumor infiltrating macrophages immunoreactive for GDF- 15. This is in accordance with hyper-methylation of the GDF-15 promoter in glioblastoma and increased GDF-15 expression observed upon differentiation of the macrophage-like cell-line U937.
  • GDF-15 protein measured in the CSF has a diagnostic value in patients with intracranial tumors, and has surprisingly been detected as being a prognostic marker in glioblastoma patients.
  • Cerebrospinal fluid and plasma samples Applicant has assembled a collection of 94 frozen CSF samples at the Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, for the identification of prognostic factors in brain tumors, approved by the local ethics committee (protocol 39/04).
  • a second set of 30 CSF was collected postoperative during chemotherapy of glioma patients as described (Ostermann et al., 2004). Respective plasma samples were available for 66 of the brain tumor patients.
  • GDF-15 ELISA GDF-15 protein levels were measured using a sandwich ELISA with 26G6H6 antibody as previously described (Brown et al., 2002; Moore et al., 2000). ELISA plates (Maxisorb; Nunc) were coated with 26G6H6 (1 :500) in bicarbonate buffer (pH 9.4-9.8). Recombinant GDF-15 was diluted 1 :1000 with eight doubling dilutions (1000-7.8 pg/ml) and used as the standard curve for each plate in the assay. Coated plates were washed three times and then blocked by incubation with 250 ⁇ l of 1% BSA w/v at 37°C for 1 hour.
  • the detection antibody 233-BP (1 :25,000) was added to the wells and incubated for 16 hours at 4°C. Visualization was achieved with donkey, antisheep, biotinylated IgG (Jackson's Laboratories), Streptavidin-large volume kit conjugate (Genzyme), and o-phenylenediamine substrate (Sigma). The reaction was terminated with 2N H 2 SO 4 . Absorbance was measured at 490 nm. Each sample was assayed at minimum in duplicate and the coefficients of variations were always ⁇ 10%. The limit of detection was 156 pg/ml of the diluted sample. Using this technology the limit of detection was used as cut-off for diagnosis or prediction outcome in glioblastoma patients.
  • GDF-15 Immunohistochemistry GDF-15 expression was determined by immunohistochemistry on paraffin embedded glioblastoma samples using a high temperature epitope retrieval method (5 min high pressure cooker, citrate buffer pH 6.0; anti-GDF-15, R&D systems, dilution 1 :100). Isolation of human monocytes and cell culturing.
  • Human peripheral blood monocytes were obtained from whole blood of healthy volunteers by dextran-sedimentation of the leukocytic fraction according to the Nycoprep protocol (AXIS- SHIELD, Oslo, N). Purified monocytes were cultured in Macrophage Serum Free Medium (Invitrogen). One million U937 cells were seeded on a lOcm-diameter Petri dish and differentiated using 65 ng/ml Phorbol 12-myristate 13-acetate (PMA) for 48 hours.
  • PMA Phorbol 12-myristate 13-acetate
  • GDF- 15 transcript abundance was normalized to the expression level of RPOLII (RNA Polymerase II) for all samples.
  • GDF-15 qPCR from glioblastoma tissues was performed in triplicates. Three independent experiments were carried out with cultured cells.
  • GDF- 15 CSF concentrations are increased in glioblastoma patients.
  • Applicant measured GDF- 15 protein levels in the CSF and plasma of patients with intracranial tumors, including glioblastoma, astrocytoma (WHO grade II and III), meningioma (WHO grade I and II), and metastasis, and compared them to a control cohort of patients treated in the neurology department for disorders unrelated to cancer. The distribution of the measurements is depicted in Figure 1.
  • the concentration was not significantly different from the control after correction with multiple testing.
  • Plasma concentrations of GDF- 15 were generally higher than in the CSF, but did not differentiate patients with intracranial tumors from the control cohort with a median concentration of 612 pg/ml ( Figure 1, Table 1).
  • Enhanced CSF concentrations of GDF- 15 are associated with worse outcome in glioblastoma patients.
  • Applicant evaluated whether the GDF- 15 concentration measured in the CSF of glioblastoma patients at the time of surgery was associated with overall survival.
  • MIC-I a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc Natl Acad Sci U S A 94, 11514-11519.
  • the transforming growth factor-ss superfamily cytokine macrophage inhibitory cytokine- 1 is present in high concentrations in the serum of pregnant women. J Clin Endocrinol Metab 85, 4781-4788. Ostermann, S., Csajka, C, Buclin, T., Leyvraz, S., Lejeune, F., Decosterd, L.A., and Stupp, R. (2004). Plasma and cerebrospinal fluid population pharmacokinetics of temozolomide in malignant glioma patients. Clin Cancer Res 10, 3728-3736.

Abstract

Cette invention concerne une méthode de diagnostic ou de prédiction de l'évolution de tumeurs intracrâniennes dans le liquide céphalorachidien d'un sujet. L'invention concerne par ailleurs des compositions et des méthodes de traitement ou de prévention de tumeurs intracrâniennes chez un sujet et un kit utilisé pour prédire ces tumeurs.
PCT/IB2009/050479 2008-02-05 2009-02-05 Méthode de prédiction ou de diagnostic d'évolution de tumeurs intracrâniennes chez un sujet WO2009098656A2 (fr)

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Publication number Priority date Publication date Assignee Title
US9465029B2 (en) 2004-04-16 2016-10-11 Glaxo Group Limited Methods for detecting LP-PLA2 activity and inhibition of LP-PLA2 activity
CN114438201A (zh) * 2020-11-05 2022-05-06 复旦大学附属华山医院 一种肿瘤标志物组合及其应用

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