WO2012052757A1 - Procédé de sélection de patients selon leur phénotype tumoral - Google Patents

Procédé de sélection de patients selon leur phénotype tumoral Download PDF

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WO2012052757A1
WO2012052757A1 PCT/GB2011/052021 GB2011052021W WO2012052757A1 WO 2012052757 A1 WO2012052757 A1 WO 2012052757A1 GB 2011052021 W GB2011052021 W GB 2011052021W WO 2012052757 A1 WO2012052757 A1 WO 2012052757A1
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tumour
methyl
cancer
amino
phenotype
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PCT/GB2011/052021
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Simon Thomas Barry
Neil Robert Smith
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Astrazeneca Ab
Astrazeneca Uk Limited
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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
    • G01N33/57492—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 localized on the membrane of tumor or cancer cells
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • 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

  • tumours can generally be grouped into those that possess a tumour-vessel phenotype or a stromal-vessel phenotype; furthermore, that those with a tumour- vessel phenotype are particularly susceptible to efficacious treatment with Vascular Endothelial Growth Factors (VEGF) signalling inhibitors.
  • VEGF Vascular Endothelial Growth Factors
  • the present invention therefore opens up the possibility to stratifying cancer patients according to the cellular phenotype of the tumour they possess and identify those patients likely to respond favourably to treatment with a Vascular Endothelial Growth Factors (VEGF) signalling inhibitor.
  • VEGF Vascular Endothelial Growth Factors
  • This therefore provides opportunities, methods and tools for selecting patients for treatment with a Vascular Endothelial Growth Factors (VEGF) signalling inhibitor, based on whether the tumour to be treated possess a tumour- vessel phenotype or a stromal-vessel phenotype.
  • VEGF Vascular Endothelial Growth Factors
  • stromal fibroblast-like cells can be observed as single cells distributed through the body or mass of the tumour, as pericytes supporting vessels within the tumour and as fibrotic or desmoplastic multicellular structures surrounding nests of tumour cells (Sugimoto et al, Cancer Biol Ther, 5(12): 1640-1646, 2006). This class of stromal fibroblast-like cells are extremely adaptive and show heterogeneous phenotypes.
  • tumours can constitute significant proportions of the tumour, in particular certain human tumours such as pancreatic, breast, colorectal, and prostate (for review see Nyberg et al, Front Biosci 13:6537, 2008; and Pietras et al, Plos Med 5:el9, 10.1371/journal.pmed.0050019, 2008. Emerging data also suggest that the presence of stromal fibroblast-like cells in the tumour can be prognostic, or identify high risk populations of patients. In colorectal cancer (CRC), the presence of high levels of alpha SMA positive cells in the tumour indicate a poor outcome and high likelihood of recurrance (Tsujino et al., CCR, 13:2082, 2007).
  • CRC colorectal cancer
  • stromal infiltrate and stroma gene expression predicts clinical outcome in breast cancer (Howell et al., Breast Cancer Research 1 l(Suppl 3):S16, 2009; Van den Eynden et al., Histopathology 51 :440-451, 2007; Finak et a/., 14:518, 2008).
  • co-implantation of stromal fibroblast-like cells or recruitment to tumours results in an increase in tumour growth (E.g.
  • VEGF signalling inhibitors VEGFR- 2 and VEGFR-3 are localised to, and up-regulated on, tumour vasculature (Smith et al, Clinical Cancer Research 16: 3548, 2010). Therefore the primary clinical mechanism of action of this class of agents is through the targeting of tumour vasculature.
  • the inventors used a CD31-aSMA immuno fluorescent assay to assess both the gross morphology of the tumour relative to stroma and the distribution of the vasculature between these two compartments in in vivo and primary human tumours.
  • tumour-vessel vessels within tumour
  • stromal-vessel vessels within stroma
  • tumour-vessel vessels within tumour
  • tumour types responding to VEGFi renal cell cancer (80%), glioblastoma (96%) and hepatocellular carcinoma (93%)]
  • renal cell cancer 80%
  • glioblastoma 96%)
  • hepatocellular carcinoma 93%)
  • the latter phenotype was rare in xenografts (5%) but common in tumour types insensitive to VEGFi as a single agent
  • the inventors also demonstrated in vivo that the two phenotypes elicited different tumour responses to the VEGFR-2 inhibitor DC-101 and showed that a sub-population of CRC tumours, which exhibit the tumour- vessel phenotype, are more likely to predict patients that respond to bevacizumab.
  • tumour type e.g. renal or colorectal tumours
  • individual tumours within a tumour type individual colorectal tumours
  • the present invention provides methods of screening and preselecting patients for anti-VEGF therapy, based on the tumour phenotype.
  • a method for stratifying cancer patients comprising determining the cellular phenotype of the tumour and stratifying the patients into at least two groups according to the presence of stroma and/or location of tumour vessels within the tumour or stromal compartments of a tumour.
  • patient tumours that can be stratified in this manner are: renal cell cancer, glioblastoma, ovarian cancer, liver cancer, thyroid cancer, head and neck cancer, colorectal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer and skin cancer.
  • the tumour can be stratified into two distinct phenotypes, a tumour-vessel and a stromal-vessel phenotype.
  • one of the stratified groups consists of patients whose tumours possess a tumour-vessel phenotype and in another embodiment, one of the stratified groups are patients whose tumour exhibit a stromal-vessel phenotype.
  • a method for stratifying a cancer patient comprising determining the phenotype of the patient's tumour and stratifying the patient into one of two groups according to the presence of stroma and/or location of tumour vessels within the tumour or stromal compartments of a tumour.
  • a tumour is defined as having a tumour- vessel phenotype if the predominant morphology (>50%, such as >60%, >70%, >80% or >90% of the tumour mass) is that of a sheet of malignant/cancerous cells (>2mm 2 ) with tumour vessels embedded within the malignant/cancerous regions such that the tumour vessels lie adjacent to malignant/cancerous cells.
  • a tumour is defined as having a stromal-vessel phenotype if the predominant morphology (>50%, such as >60%, >70%, >80% or >90% of the tumour mass) is small islands/nests of malignant/cancerous cells ( ⁇ 2mm 2 ) separated by stroma. Importantly, vessels are localised to the stroma surrounding the malignant/cancerous regions but are either absent or rarely embedded within the malignant/cancerous regions. Stromal and tumour- vessel phenotypes are shown and illustrated in Fig.l .
  • this allows the patient(s) to be classified or stratified into two distinct groups depending on whether their tumour exhibits a tumour- vessel phenotype or a stromal- vessel phenotype.
  • the cellular phenotype of a tumour sample can be determined using a tissue or cell staining technique on a tumour sample taken from the patient. Typically this involves treating the tumour sample with one or more staining agents capable of detecting or facilitating the visualisation of the presence of one or more of the following cells types:
  • tumour vessels primarily blood vessels comprising endothelial cells either embedded within the tumour or present in tumour associated stroma
  • tumour malignant/cancerous cells
  • stromal cells fibroblast- like cells and immune cells associated with the tumour.
  • the tumour sample may, for example, be a biopsy sample or surgical resection sample.
  • extraction of the sample from the patient is part of the method of the invention, in another embodiment the tumour sample has previously been isolated from the patient and the methods of the invention proceed from the point where the sample is made available.
  • the cellular phenotype of the tumour is determined by histological staining, immunohistochemistry or in situ hybridisation.
  • Histological (or tinctorial) staining is a well-established traditional technique whereby the tumour sample (e.g. a tissue slice mounted on a microscope slide) is contacted with one or more stains capable of binding to particular cellular components so as to facilitate discrimination of these different cellular components visually using a microscope, or using algorithms generated with imaging software such as Defmiens Image Analysis Software, Defmiens Tissue Studio, Aperio Genie Pro or Image J. Any suitable histological stain capable of highlighting or "showing up" the cellular compartments and vessels, in particular the tumour vessels, tumour cells and stromal cells can be employed in the methods of the invention.
  • stains that may be employed (the generic histological stain name is given first, common preparations are given in brackets) include: Haematoxylin (Ehrlich's, Delafield's, Mayer's, Harris's, Cole's, Carazzi's, Gill's) and Eosin (Eosin Y, Ethyl eosin, Eosin B) (H+E), Haematoxylins (Heidenhain's, L hinder, Verhoeff s, PTAH, Molybdenum), Trichrome stains for connective tissue (Van Gieson, Masson, MSB), elastic tissue fibre stains (Verhoeff s, Orcein, Weigert's resorcin-fuchsin and aldehyde fuchsin method), reticular fiber stains (Gordon and Sweets' and Gomori's) and immunofiurescence counterstains, DAPI, TOR3, Hoe
  • the stains listed above can generally be classified as: Haematoxylin, Eosin, Trichrome stain for connective tissue, elastic tissue fibre stain, reticular fibre stain and immunofluorescence counterstain.
  • the cellular phenotype of the tumour can also be determined using
  • immunohistochemistry using one or more antibodies capable of binding to a marker protein present in, on or associated with a vessel, tumour cells or stromal cells.
  • IHC is a widely used technique for identifying cellular or tissue constituents (marker proteins/antigens) by means of antigen/antibody interactions, the site of antibody being identified either by direct labelling of the antibody or by use of a secondary labelling method.
  • Label refers to a composition capable of producing a detectable signal indicative of the presence of the target moiety e.g. marker protein or mRNA) in an assay sample.
  • Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • IHC Two general methods of IHC are available; direct and indirect assays.
  • binding of antibody to the target antigen is determined directly.
  • This direct assay uses a labelled reagent, such as a fluorescent tag or an enzyme-labelled primary antibody, which can be visualized without further antibody interaction.
  • a labelled secondary antibody binds to the primary antibody.
  • a chromagenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.
  • the primary and/or secondary antibody used for immunohistochemistry will typically be labelled with a detectable moiety.
  • Numerous labels are available which can be generally grouped into the following categories:
  • Radioisotopes such as S , C , I , H , and I .
  • the antibody can be labelled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, New York, Pubs. (1991) for example and radioactivity can be measured using scintillation counting.
  • Fluorescent labels including, but are not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin, phycocyanin, or commercially available fluorophores such as SPECTRUM ORANGE ® and SPECTRUM GREEN ® and/or derivatives of any one or more of the above.
  • the fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter.
  • the enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a colour change in a substrate, which can be measured
  • the enzyme may alter the fluorescence or
  • chemiluminescence of the substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No.
  • luciferin 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, [beta]- galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • HRPO horseradish peroxidase
  • alkaline phosphatase [beta]- galactosidase
  • glucoamylase lysozyme
  • saccharide oxidases e.g., glucose oxidase, galactose
  • enzyme-substrate combinations include, for example:
  • HRPO Horseradish peroxidase
  • OPD orthophenylene diamine
  • TMB 3,3',5,5'-tetramethyl benzidine hydrochloride
  • [beta]-D-galactosidase [beta]-D-Gal) with a chromogenic substrate (e.g. p-nitrophenyl- [beta]-D-galactosidase) or fluorogenic substrate (e.g., 4-methylumbelliferyl-[beta]-D- galactosidase).
  • a chromogenic substrate e.g. p-nitrophenyl- [beta]-D-galactosidase
  • fluorogenic substrate e.g., 4-methylumbelliferyl-[beta]-D- galactosidase
  • the marker protein is present in, on or associated with a vessel and is selected from the group consisting of: CD34, vWF, CD31, VEGFPv-2, VEGFR-3, NRP1, Tie-2, CD 105 (Endoglin), ALK1, DLL4, Ang2, podoplanin, alpha-SMA, integrin alpha5betal, PDGFR-beta, NG2 and VE-Cadherin.
  • a vessel is defined as a tube comprising endothelial cells through which blood circulates.
  • the marker protein is present in, on or associated with tumour cells and the marker protein is one or several cytokeratins.
  • Cytokeratins are the intermediate filament proteins of epithelial cells.
  • the marker protein is present in, on or associated with stromal cells and is selected from the group consisting of: alpha or gamma- SMA, paladin 4Ig, podoplanin, endosialin (TEM1/CD248), stromelysin, cadherin 2, cadherin 11, integrin alphal 1, FAP, FSP, PDGFR-beta, CD68, CDl lb, neutrophil elastase, CD3, CD4, CD8, FOXP3, CD56, CD57, P4H, vimentin, desmin, collagen, tenascin, fibronectin, and laminin, MMP-3 and MMP-9.
  • the marker protein(s) can be detected using techniques well known in the art, e.g. using monoclonal or polyclonal antibodies that detect these target proteins.
  • the inventors have used CD31 and a-SMA antibodies to visualize vessels and fibroblast-like stromal cells, respectively in order to determine the tumour-vessel and stromal-vessel phenotypes of tumours.
  • antibody As used herein, the terms “antibody” and “antibodies”, also known as
  • immunoglobulins refers to a polypeptide or group of polypeptides that are comprised of at least one binding domain that is formed from the folding of polypeptide chains having three- dimensional binding spaces with internal surface shapes and charge distributions
  • monoclonal antibodies including full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two different epitope binding fragments (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments that exhibit the desired biological activity (e.g.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain at least one antigen- binding site.
  • Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art.
  • a target (marker protein) polypeptide or immunogenic fragment thereof can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • Various adjuvants may be used to increase the amount of the antigen.
  • immunological response depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
  • Polyclonal antibodies can be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized.
  • Conjugates also can be made in recombinant cell culture as fusion proteins.
  • animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining an appropriate concentration of antigen or conjugate with adjuvant and injecting the solution at multiple sites.
  • the animals are boosted with 1 ⁇ 2 to 1/10 the original amount of antigen or conjugate in adjuvant by subcutaneous injection at multiple sites.
  • the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • aggregating agents such as alum are suitably used to enhance the immune response.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma (Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), recombinant, and phage display technologies, or a combination thereof.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous or isolated antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or multiple antigenic sites in the case of multi-specific engineered antibodies. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against the same determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.
  • mice or other appropriate host animals such as hamster
  • lymphocytes may be immunized in vitro.
  • lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent or fusion partner, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59- 103 (Academic Press, 1986)).
  • the selected myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the un-fused parental cells.
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumours in an animal e.g., by i.p. injection of the cells into mice.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, affinity tags, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
  • affinity chromatography e.g., using protein A or protein G-Sepharose
  • ion-exchange chromatography e.g., affinity tags, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
  • the complex may be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular detectable label, where representative detection means include, e.g., scintillation counting, autoradiography, measurement of paramagnetism, fluorescence measurement, light absorption measurement, measurement of light scattering and the like.
  • detection means include, e.g., scintillation counting, autoradiography, measurement of paramagnetism, fluorescence measurement, light absorption measurement, measurement of light scattering and the like.
  • the cellular phenotype of the tumour can be determined visually using a microscope or computer assisted image analyser.
  • Computer assisted image analysis is performed using algorithms generated with imaging software such as Definiens Image Analysis Software, Definiens Tissue Studio, Aperio Genie Pro or Image J. There are numerous computer assisted image analysers and imaging software packages available.
  • ISH in situ hybridization
  • SISH silver in situ hybridization
  • CISH chromogenic in situ hybridization
  • FISH fluorescence in situ hybridization
  • ISH is distinct from IHC, in that ISH employs one or more nucleic acid probe(s) capable of binding to a transcript encoding a marker protein whereas IHC detects proteins.
  • In situ hybridization is a method of localizing and detecting specific mRNA sequences in morphologically preserved tissues sections or cell preparations by hybridizing the complementary strand of a nucleotide probe to the sequence of interest.
  • in situ hybridization The basic principles for in situ hybridization are the same as normal hybridisation wherein a labelled nucleic acid probe is allowed to bind to matching nucleic acid in a test sample, except with ISH the probe is utilized to detect specific nucleotide sequences within cells and tissues.
  • the sensitivity of the technique is such that threshold levels of detection are in the region of 10-20 copies of mR A per cell.
  • the nucleic acid probe selected is capable of hybridising to the transcript of a marker protein present in, on or associated with (i) a vessel, wherein the marker protein is selected from the group consisting of: CD34, vWF, CD31, VEGFR-2, VEGFR-3, NRP1, Tie-2, CD 105 (Endoglin), ALK1, DLL4, Ang2, podoplanin, alpha-SMA, integrin alpha5betal, PDGFR-beta, NG2 and VE Cadherin.
  • the marker protein is selected from the group consisting of: CD34, vWF, CD31, VEGFR-2, VEGFR-3, NRP1, Tie-2, CD 105 (Endoglin), ALK1, DLL4, Ang2, podoplanin, alpha-SMA, integrin alpha5betal, PDGFR-beta, NG2 and VE Cadherin.
  • tumour cells wherein the marker protein is a cytokeratin; or (iii) stromal cells, wherein the marker protein is selected from the group consisting of: alpha or gamma-SMA, paladin 4Ig, podoplanin, endosialin (TEM1/CD248), stromelysin, cadherin 2, cadherin 11, integrin alphal 1, FAP, FSP, PDGFR- beta, CD68, CDl lb, neutrophil elastase, CD3, CD4, CD8, FOXP3, CD56, CD57, P4H, vimentin, desmin, collagen, tenascin, fibronectin, and laminin, MMP-3 and MMP-9.
  • the marker protein is selected from the group consisting of: alpha or gamma-SMA, paladin 4Ig, podoplanin, endosialin (TEM1/CD248), stromelysin, cadherin 2, cadherin 11, integrin
  • nucleic acid and amino acid sequences of the markers recited above are known and available in various publicly available databases and scientific publications. It is well within the capabilities of a person skilled in the art to prepare and use one or more nucleic acid probe(s) capable of binding to a transcript encoding a marker protein.
  • the cellular phenotype of the tumour can be determined visually using a microscope or computer assisted image analyser.
  • tumours for specific tissue origin that display a particular tumour phenotype respond differentially to treatment with a pharmaceutical drug.
  • the patient stratification methods of the invention permit identification of patients suitable for treatment with a pharmaceutical drug.
  • the patient stratification permits identification of patients for treatment with a drug targeting tumour vasculature (agents that inhibit the growth of new functional tumour vessels and/or promote the regression or disruption of existing tumour vasculature).
  • a drug targeting tumour vasculature agents that inhibit the growth of new functional tumour vessels and/or promote the regression or disruption of existing tumour vasculature.
  • the drug targeting tumour vasculature is an inhibitor of VEGF signalling pathway.
  • VEGF signalling pathway There are many examples of compounds or drugs that inhibit VEGF signalling pathway.
  • Compounds that inhibit VEGF signalling pathway typically block binding of VEGF ligand to its receptor.
  • Percy et al. (Nature Reviews Clinical Oncology, 6:569-579, 2009) provides an overview of small-molecule inhibitors of VEGFR signalling.
  • Table 1 includes: AEE788 (Novartis), axitinib (Pfizer), motesanib (Amgen), cediranib (AstraZeneca), vandetanib (AstraZeneca), sorafenib (Bayer), telatinib (Bayer), BIBF 1120 (Boehringer), brivanib alaninate (Bristol Myers Squibb), dovitinib lactate (Chiron), CP-547, 632 (Pfizer), pazopanib (Glaxo), OSI 930 (OSI), vatalanib (Novartis), semaxinib (Sugen/ Pfizer), SU6668 (Sugen/Pfizer) and sunitinib (Sugen/Pfizer).
  • VEGFR tyrosine kinase inhibitors in clinical development (see e.g. Table 23.1 therein). Any of these compounds could be used in the invention disclosed herein.
  • Large molecule (e.g. monoclonal antibody) agents that inhibit VEGF signalling pathway can also be used, such as bevacizumab, ramicirumab, aflibercept and 33C3.
  • the inhibitor of VEGF signalling pathway is a compound or drug selected from the group consisting of: AEE788 (Novartis), axitinib
  • AEE788 (6-[4-[(4- Ethylpiperazin- 1 -yl)methyl]phenyl]-N-[(li?)- 1 -phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4- amine); axitinib - (N-Methyl-2-[[3-[(E)-2-pyridin-2-ylethenyl]-lH-indazol-6- yl]sulfanyl]benzamide); motesanib - (N-(3,3-Dimethyl-2,3-dihydro-lH-indol-6-yl)-2- [(pyridin-4-ylmethyl)amino]pyridine-3-carboxamide); cediranib - (4-[(4-fluoro-2 -methyl- ⁇ H- indol-5-yl)oxy]-6-methoxy-7-[3-(
  • the methods of the invention can also be employed to select patients suitable for treatment with a drug that targets other angiogenic pathways that are important for tumour growth such as the Ang-2-Tie-2, DLL4-Notch, a5bl-FN, Alk-l-BMP pathways.
  • tumour displays or exhibits a tumour-vessel phenotype or a stromal-vessel phenotype wherein if said tumour displays or exhibits a tumour- vessel phenotype said patient is likely to respond favourably to treatment with the inhibitor of VEGF signalling pathway whereas if the tumour exhibit or display the stromal- vessel phenotype they are unlikely to respond favourably to treatment with the inhibitor of VEGF signalling pathway.
  • a method for selecting a treatment for a patient suffering from cancer comprising (a) determining whether the patient's tumour exhibits a tumour-vessel or stromal-vessel phenotype; and, (b) selecting a treatment for the patient according to the particular cellular phenotype identified in step (a)
  • a method for selecting a patient for treatment with a drug that targets tumour vasculature comprises: a) determining the whether the patient's tumour exhibits a tumour-vessel or stromal-vessel phenotype; and b) selecting said patient for treatment with a vascular targeting agent if the tumour exhibits a tumour- vessel phenotype or selecting said patient for treatment with a stromal targeting agent e.g. an agent inhibiting the following receptors PDGFR, Alk-5, FGFR kinases, the ⁇ integrin, or an antibody drug conjugate targeting tumour fibroblasts, in combination with a vascular targeting agent if the tumour exhibits the stromal phenotype.
  • a vascular targeting agent e.g. an agent inhibiting the following receptors PDGFR, Alk-5, FGFR kinases, the ⁇ integrin, or an antibody drug conjugate targeting tumour fibroblasts, in combination with a vascular targeting agent if the tumour exhibits the stromal phenotype
  • Stromal targeting agent that target tumour modified fibroblasts are exemplified by agents that inhibit the TGF- ⁇ signalling pathway such as small molecule agents like
  • LY550410 (5-bromo-thiophene-2-sulfonic acid 2,4-dichlorobenzoylamide sodium salt) and SB-505124 (2-(5-benzo[l,3]dioxol-5-yl-2-tert-butyl-3himidazol-4-yl)-6-methylpyridine hydrochloride), or large molecule agents such as the antibodies metelimumab and
  • Inhibitors of the PDGFR signalling pathway are exemplified by small molecule agents such as imatinib (4-[(4-methylpiperazin-l-yl)methyl]-N-(4-methyl-3- ⁇ [4-(pyridin-3 -yl)pyrimidin-2-yl] amino ⁇ phenyl)benzamide), crenolanib (l-(2- ⁇ 5-[(3- Methyloxetan-3-yl)methoxy]- lH-benzimidazol- 1 -yl ⁇ quinolin-8-yl)piperidin-4-amine) and TKI258 (4-amino-5-fluor-3-[5-(4-metylpiperazin-l-yl)-lH-benzimidazol-2-yl]quinolin- 2(lH)-one), or large molecule agents.
  • imatinib 4-[(4-methylpiperazin-l-yl)methyl]-N-(4-methyl-3
  • Inhibitors of CXCR4 signalling is exemplified the peptide AMD3100 (l,l'-[l,4-Phenylenebis(methylene)]bis [1,4,8,11- tetraazacyclotetradecane]).
  • Inhibitors of FGFR signalling are specifically exemplified by AZD4547 (N-[3-[2-(3,5-dimethoxyphenyl)ethyl]-lH-pyrazol-5-yl]-4-[(3S,5R)-3,5- dimethylpiperazin-l-yl]benzamide), and non-specific inhibitors such as dovitinib (TKI258; 4- amino-5-fluoro-3-[5-(4-methylpiperazin-l-yl)-lH-benzimidazol-2-yl]quinolin-2(lH)-one) that target FGFR through broad selectivity.
  • AZD4547 N-[3-[2-(3,5-dimethoxyphenyl)ethyl]-lH-pyrazol-5-yl]-4-[(3S,5R)-3,5- dimethylpiperazin-l-yl]benzamide
  • non-specific inhibitors such as dovitinib (TKI2
  • Modulators of IL-6-Jak signalling exemplified by the small molecules 5-chloro-N2-[(lS)-l-(5-fluoro-2-pyrimidinyl)ethyl]-N4-(5-methyl-lH- pyrazol-3-yl)-2,4-pyrimidinediamine) and ruxolitinib ((3i?)-3-cyclopentyl-3-[4-(7H- pyrrolo [2,3 -d]pyrimidin-4-yl)pyrazol- 1 -yljpropanenitrile) .
  • the stromal targeting agent is selected from the group consisting of: LY550410 (5-bromo-thiophene-2-sulfonic acid 2,4- dichlorobenzoylamide sodium salt), SB-505124 (2-(5-benzo[l,3]dioxol-5-yl-2-tert-butyl- 3Himidazol-4-yl)-6-methylpyridine hydrochloride), metelimumab, GC-1008, imatinib (4-[(4- methylpiperazin- 1 -yl)methyl] -N-(4-methy 1-3 - ⁇ [4-(pyridin-3 -yl)pyrimidin-2- yljamino ⁇ phenyl)benzamide), Crenolanib( 1 -(2- ⁇ 5 - [(3 -Methyloxetan-3 -yl)methoxy] - ⁇ H- benzimidazol-l-yl ⁇ quino
  • the tumour phenotype is determined using a method as described herein. Such method may include extraction of the tumour sample from the patient or may be conducted on a tumour sample previously extracted from the patient.
  • Stromal agents in the context of this invention include agents that target the fibroblast population in the tumour.
  • Vascular Targeting Agents are agents that induce rapid endothelial cell death resulting in vessel capitulation, reduction in tumour perfusion and driving necrosis within the tumour mass.
  • the drug that targets tumour vasculature is a VEGF signalling pathway inhibitor, a Tie-2-Ang-2 signalling pathway inhibitor, a Alk-1 signalling pathway inhibitor, or a DLL4 signalling pathway inhibitor.
  • the drug that targets tumour vasculature is a VEGF signalling pathway inhibitor.
  • Particularly suitable drugs are selected from the group consisting of: AEE788, axitinib, motesanib, cediranib, vandetanib, sorafenib, telatinib, BIBF-1120, brivanib alaninate, dovitinib lactate, CP-547, 632, pazopanib, OSI-930, vatalanib, semaxinib, SU6668, sunitinib, CEP-7055, E7080, SU14813, Telatinib, KRN-951, ABT-869, BMS 582664, CHR265 and ZK-304709. Vandetanib and cediranib are particularly suitable compounds for use in the present invention.
  • Agents that target Ang-2 signalling include: AMG-386 and CVX-060.
  • An agent that targets Alk-1 signalling Ang-2 is the antibody PF-03446962.
  • a method for selecting a patient for treatment with a drug that targets tumour vasculature comprises: a) determining the presence of stroma and/or location of tumour vessels within the tumour or stromal compartments of a tumour in a tumour tissue sample previously taken from the patient so as to determine whether or not the tumour exhibits a tumour-vessel or stromal-vessel phenotype; and b) selecting said patient for treatment with a vascular targeting agent if the tumour exhibits a tumour- vessel phenotype or c) selecting said patient for treatment with a stromal targeting agent in combination with a vascular targeting agent if the tumour exhibits the stromal phenotype.
  • a method for selecting a patient for treatment with a VEGF signalling pathway inhibitor comprising: a) determining the presence of stroma and/or location of tumour vessels within the tumour or stromal compartments of a tumour in a tumour tissue sample from the patient so as to determine whether or not the tumour exhibits a tumour- vessel or stromal-vessel phenotype; and, b) selecting said patient for treatment with an VEGF signalling pathway inhibitor if the tumour exhibits the tumour-vessel phenotype.
  • the tumour sample may, for example, be a sectioned sample or biopsy sample. In a particular embodiment the sample has been previously taken from the patient so that the method is not specifically practised on the human body.
  • a method for selecting a patient for combination treatment with a VEGF signalling pathway inhibitor and a stromal targeting agent comprising: a) determining the presence of stroma and/or location of tumour vessels within the tumour or stromal compartments of a tumour in a tumour tissue sample from the patient so as to determine whether or not the tumour exhibits a tumour-vessel or stromal-vessel phenotype; and (b) selecting said patient for treatment with a stromal targeting agent in combination with a VEGF signalling pathway inhibitor if the tumour exhibits the stromal-vessel phenotype.
  • the sample has been previously taken from the patient so that the method is not specifically practised on the human body.
  • a method for treating a patient suffering from cancer comprising:
  • a compound or drug capable of inhibiting VEGF signalling pathway in the preparation of a medicament for treating an individual with cancer, whose cancer cells have been determined to exhibit the tumour-vessel phenotype.
  • the medicament can be a pharmaceutical
  • the inhibitor of VEGF signalling pathway for a method or use as contemplated herein is a compound or drug selected from the group consisting of: AEE788, axitinib, motesanib, cediranib, vandetanib, sorafenib, telatinib, BIBF-1120, brivanib alaninate, dovitinib lactate, CP-547, 632, pazopanib, OSI-930, vatalanib, semaxinib, SU6668, sunitinib, CEP-7055, E7080, SU14813, Telatinib, KR -951, ABT-869, BMS 582664, CHR265 and ZK-304709.
  • the compound or drug may be administered in an effective amount to a subject in need of such treatment.
  • the compounds/drugs described herein may be useful for the treatment of cancer and other proliferative disorders.
  • Administration of the compounds/drugs, in the form of a therapeutic agent may be carried out using oral, enteral, parenteral or topical administration, including, for example, intravenous, oral, transdermal or any other mode of administration.
  • subject or patient refers to an animal or mammal including, but not limited to, human, dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rabbit, rat, mouse, etc.
  • the term "therapeutic” means an agent utilized to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a patient.
  • the methods herein for use contemplate prophylactic use as well as curative use in therapy of an existing condition.
  • the terms "therapeutically effective” or “effective”, as used herein, may be used interchangeably and refer to an amount of a therapeutic composition embodiments of the present invention.
  • a therapeutically effective amount of a composition comprising anti-VEGFR therapy is a predetermined amount calculated to achieve the desired effect, i.e., to effectively inhibit VEGF receptor signalling in an individual to whom the composition is administered.
  • the medicament or pharmaceutical composition can take the form of a solution, suspension, emulsion, tablet, pill, pellet, capsule, capsule containing liquids, powder, sustained-release formulation, suppository, emulsion, aerosol, spray, suspension, or any other form suitable for use.
  • Administration may be topical, i.e., substance is applied directly where its action is desired, enteral or oral, i.e., substance is given via the digestive tract, parenteral, i.e., substance is given by other routes than the digestive tract such as by injection.
  • the active compound and optionally another therapeutic or prophylactic agent are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to human beings.
  • the active compound for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the compositions can also include a solubilizing agent.
  • Compositions for intravenous administration can optionally include a local anaesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule.
  • the active compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • compositions for oral delivery can be in the form of tablets, lozenges, cachets, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
  • Orally administered compositions can contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavouring agents such as peppermint, oil of wintergreen, or cherry; colouring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
  • a time delay material such as glycerol monostearate or glycerol stearate can also be used.
  • Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such vehicles are of pharmaceutical grade in particular embodiments.
  • compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the VEGF signalling inhibitor compound and optionally another therapeutic or prophylactic agent and their physiologically acceptable salts and solvates can be formulated into pharmaceutical compositions for administration by inhalation or insufflation (either through the mouth or the nose) or oral, parenteral or mucosol (such as buccal, vaginal, rectal, sublingual) administration.
  • parenteral or mucosol such as buccal, vaginal, rectal, sublingual
  • local or systemic parenteral administration is used.
  • compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch or sodium
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations can also contain buffer salts, flavouring, colouring and sweetening agents as appropriate.
  • compounds or salts disclosed herein or can be administered as a pharmaceutical composition in which the pharmaceutical composition comprises between 0.1-lmg, 1-10 mg, 10-50mg, 50-100mg, 100-500mg, or 500mg to 5g of said IAP antagonist compound or salt.
  • Figure 1 shows photographs of tumour samples possessing tumour-vessel phenotype and stromal-vessel phenotype as well as a computer generated diagrams illustrating the phenotypes.
  • Figure 2 shows that the two phenotypes define monotherapy or combination therapy disease types for VEGF signalling inhibitors.
  • the y axis represents % ratio of phenotype.
  • Figure 3 shows growth curves for DC101 treated Calu-6 and Calu-3 xenograft models showing that the two phenotypes determine response to VEGF inhibition in vivo.
  • Figure 4 shows a graph of the ratio of the three RECIST outcome categories for tumour- vessel and stromal-vessel phenotypes.
  • the y-axis showing the ratio of RECIST response classifications.
  • Tumour xenograft tissue was derived from experiments conducted with licences issued under the UK Animals (Scientific Procedures) Act 1986 and after local ethical review and approval. Cell lines were maintained in the recommended growth medium and implanted subcutaneously into the left flank of immuno-compromised mice, nude, scid or scid-bg. Tumours were grown to approximately 1 cm 3 volume then collected and fixed in formalin for 24 h before being embedded in paraffin. See Table 1 for details of the preparation of each model used in this analysis.
  • Calu-6 and Calu-3 xenografts established in nude and SCID female mice, respectively were each randomised across two groups, both containing 12 animals. Once tumours reached a mean volume of ⁇ 2cm 3 (Calu-6) and ⁇ 3cm 3 (Calu-3), mice were then intraperitoneally injected twice weekly with either 15mg/kg of DC 101 (Cell Essentials Inc.) or isotype control antibody for 25 (Calu-6) and 41 (Calu-3) days.
  • DC101 is a VEGF receptor-2-specific (flk-l) monoclonal neutralizing antibody. Tumours in each group were excised and split in half; one half was snap frozen in liquid nitrogen and stored at -80°C until required, the other half was fixed in formalin for 24 hours.
  • TMA tumour microarrays
  • immunohistochemical analyses was determined by pathology assessment of tissue morphology and preservation (H&E) and the general extent of antigen preservation (CD31 and p-Tyr immunostains).
  • An immunofluorescent assay was developed to detect vessels (using an antibody to vessel endothelial cell marker, CD31) and stromal fibroblasts (using an antibody to stromal fibroblast marker, alpha-smooth muscle actin (aSMA) and tumour cells (DAPI counter stain).
  • Custom rabbit polyclonal antibody raised to C-terminus peptide of mouse CD31 CHG-CD31-P1, AstraZeneca
  • mouse monoclonal antibody to a-smooth muscle actin (aSMA; 1 A4, Sigma) were used in the development of this assay.
  • Immunofluorescence was performed on dewaxed and rehydrated FFPE sections. Antigen retrieval was carried out as above in pH 9 retrieval buffer. After blocking in 20% horse serum, sections were incubated for 1 h in a combination of 1 :20 CHG-CD31-P1 and 1 : 1000 1A4 diluted in 20% horse serum. Donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (A21206, Molecular Probes) and donkey anti-mouse IgG Alexa Fluor 555 (A31570, Molecular Probes) combined 1 :800 in serum were added for 30 min.
  • Sections were counterstained with ProLong Gold anti-fade reagent with 4',6-diamidino-2-phenylindole (DAPI; P36931, Molecular Probes), fluorescent images were scanned and captured using a MIRAX scan (Carl Zeiss) .
  • DAPI ProLong Gold anti-fade reagent with 4',6-diamidino-2-phenylindole
  • MIRAX scan Carl Zeiss
  • Example 1 Classification of tumour xenografts based on distribution of tumour stroma and vasculature
  • tumour-vessel (TV) phenotype predominant (>60% of tumour mass) morphology is that of a sheet of malignant/cancerous cell (>2mm 2 ' as detected with DAPI) with vasculature (CD31 positive) embedded within tumour.
  • stromal- vessel (SV) phenotype predominant (>60% of tumour mass) morphology is that of smaller islands/nests of malignant/cancerous cells ( ⁇ 2mm 2 as detected with DAPI) separated by stroma (aSMA positive) with vessels (CD31 positive) localised to the intervening stroma but either absent or rarely embedded within tumour.
  • tumour- vessel phenotype was common to the majority of models analysed [(40/42 (95%)] whereas the stromal- vessel phenotype was rare [(2/42 (5%], Calu-3 and NCIN87).
  • Tumour- and stromal-vessel phenotypes define disease types that are either responsive or insensitive to VEGF signalling inhibitor monotherapy
  • tumour samples formatted onto arrays were analysed for CD31-aSMA and scored for either phenotype whereby a tumour sample was classified as either TV or SV based on the predominant phenotype of the two (>60% cut off).
  • tumour- vessel phenotype was associated with tumour types classified as responding to VEGF signalling inhibitors as single agent renal cell cancer [RCC, 80% (40/50)], glioblastoma [GBM, 96% (51/53)],hepatocellular carcinoma [HCC, 93% (28/30)], thyroid cancer [ThC, 97% (35/36)] and ovarian cancer [OvC, 69%) (25/36)], whereas tumour types insensitive to these agents as monotherapy, head and neck squmaous cell carcinoma [HNSCC, 86% (42/49)], colorectal [CRC, 88% (23/26)], prostate [PC, 81% (39/48)] and lung cancer [(NSCLC, 82% (50/61)] were characterised by the stromal-vessel phenotype.
  • Figure 2 shows the ratio of the two phenotypes for RCC, GBM, HCC, CRC, PC and NSCLC.
  • VEGF inhibitors are used as combination therapy, and show limited single agent activity (defined as activity in a small percentage of patients e.g. ⁇ 15%) exhibit a stromal-vessel phenotype.
  • Example 3 Concordance between the phenotype classifications of TMA cores and matched donor tumour tissue
  • a TMA tissue core represents a small area of a larger donor specimen, this combined with erroneous sampling may mean that the core does not always represent the bulk of the tumour and therefore the predominant phenotype
  • To assess the concordance between the phenotypes of TMA cores and matched donor tumour tissue we scored 20 donor tumours and their matched TMA cores (10 CRC and 10 NSCLC) for the two phenotypes. A 90%> concordance was observed between donors and TMA cores (Table 2).
  • Table 2 concordance between the phenotype classifications of TMA cores and matched donor tumour tissue.
  • TMA CRC Donor Core
  • TMA LUNG Donor Core
  • Example 4 The effect of VEGFR-2 inhibitor, DClOl on tumour growth is determined by tumour- and stromal-vessel phenotypes in vivo.
  • tumour- vessel phenotype is more likely to predict a response to VEGFR targeted monotherapy than the stromal-vessel phenotype.
  • FOLFIRI/A vastinTM (bevacizumab) in human CRC (RECIST).

Abstract

La présente invention concerne un procédé de stratification de patients cancéreux, selon le phénotype cellulaire de la tumeur, sur la base, et en particulier sur la présence de stroma et/ou l'emplacement de vaisseaux tumoraux à l'intérieur de la tumeur ou des compartiments stromaux d'une tumeur. Cette stratification des patients permet l'identification de patients dont les tumeurs sont davantage susceptibles d'être sensibles au traitement par des médicaments anticancéreux, tels que des inhibiteurs de la signalisation par les facteurs de croissance vasculaires endothéliaux (VEGF). Par conséquent, cette invention offre des opportunités de soins de santé personnalisés dans le domaine du cancer.
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