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WO2010028313A2 - Analyse de phosphoprotéine de carcinomes pour l'évaluation de la sensibilité aux médicaments - Google Patents

Analyse de phosphoprotéine de carcinomes pour l'évaluation de la sensibilité aux médicaments Download PDF

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Publication number
WO2010028313A2
WO2010028313A2 PCT/US2009/056132 US2009056132W WO2010028313A2 WO 2010028313 A2 WO2010028313 A2 WO 2010028313A2 US 2009056132 W US2009056132 W US 2009056132W WO 2010028313 A2 WO2010028313 A2 WO 2010028313A2
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carcinoma
egfr
amino acid
acid residues
pathway
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PCT/US2009/056132
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WO2010028313A3 (fr
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Virginia A. Espina
Emanuel F Petricoin, Iii.
Lance A. Liotta
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George Mason Intellectual Properties, Inc.
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Publication of WO2010028313A2 publication Critical patent/WO2010028313A2/fr
Publication of WO2010028313A3 publication Critical patent/WO2010028313A3/fr

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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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • 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 invention relates generally to the treatment of cancer. More particularly, it relates to the identification of carcinomas that are likely to respond to an inhibitor of the Epidermal Growth Factor (EGF) family of receptors (the "ERBB family”), which contains at least four members, ERB1-ERB4, and their pathways (the "ERBB family pathway”) in the carcinoma cells and to the treatment of patients having such carcinomas with such inhibitors.
  • EGF Epidermal Growth Factor
  • EGFR Epidermal growth factor receptor
  • ERBB ERBB family of receptor tyrosine kinases that regulates cellular growth, survival, and proliferation.
  • EGFR has been extensively characterized regarding its kinase activity (9), amino acid sequence (10), receptor abundance (11), autophosphorylation properties (12), substrates (13-15), and mutation sites (16).
  • EGFR Over-expression of EGFR in various malignancies (17), together with the identification of specific EGFR mutations that enhance therapeutic response to small molecule inhibitors, notably in lung adenocarcinoma patients, along with the observation that patients without detectable EGFR kinase domain mutations respond to tyrosine kinase inhibitor therapy, qualifies EGFR as a promising molecular endpoint for individualized therapy (18-21).
  • Mutations in the ERBB family of protein receptors are associated with a significant proportion of carcinomas of all types. Mutations in the receptor are thought to be one way in which the ERBB family pathway becomes hyperactive and drives the growth or metastasis of the cancer cells.
  • Figure 1 shows unsupervised hierarchical clustering of LCM-procured non-small cell lung carcinoma cells analyzed by reverse phase protein microarray.
  • Each row represents a patient sample listed by known EGFR tyrosine kinase domain mutation status.
  • Each column represents a cell signaling protein endpoint.
  • the data cluster into two major clusters: Six of eight EGFR mutant samples in one cluster, and 17 wild type samples in the second cluster.
  • Figure 2 shows supervised hierarchical clustering of analyte subset determined by Wilcoxon Rank Sum analysis.
  • Each row (horizontal axis) represents a patient sample listed by known EGFR mutation status
  • each column (vertical axis) represents a protein endpoint found to be significantly different (p ⁇ 0.04, Table 1) between the wild type EGFR and mutated groups.
  • Figure 3 shows discrimination of wild type or mutated EGFR from microdissected NSCLC samples. Human non-small cell lung cancer samples of known EGFR mutation status were microdissected and key EGFR tyrosine phosphorylation residues were quantitated by reverse phase protein microarray.
  • Example ratios that were highly significantly different were Y1045:Y1068 (left panel), and Y1045: the sum of Y1068 and Yl 148 (right panel).
  • This invention relates to: 1) a method of determining if a carcinoma in a mammal is likely to respond to an ERBB family pathway inhibitor; 2) a method of treating the mammal with an ERBB family pathway inhibitor; 3) an assay for identifying such inhibitors; and 4) a kit for use in the assay.
  • the invention is not limited to these aspects; other aspects are disclosed and claimed herein.
  • the invention provides a method of determining if a carcinoma in a mammal is likely to respond to an ERBB family pathway inhibitor.
  • ERBB family pathway inhibitor means an exogenous agent (including a chemical substance, compound, molecule, or ion, such as an antibody, a drug, or a nucleic acid) which suppresses the signaling activity of the ERBB family receptors.
  • respond means clinical stabilization or inhibition of growth of the carcinoma.
  • the method comprises the steps of: a) analyzing a sample of the carcinoma to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway, and b) comparing the ratio of step (a) to a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma as the carcinoma in the mammal, wherein the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway, wherein the carcinoma is likely to respond to the inhibitor, if the ratio of step (a) is within the reference ratio range.
  • Samples are obtained by standard techniques, usually by biopsy.
  • the mammal may be any mammal, including but not limited to, humans and other primates, pets, such as dogs and cats, farm animals, and laboratory animals, such monkeys, rats, mice, rabbits, and guinea pigs.
  • the carcinoma may be any carcinoma (any malignant neoplasm that arises from an epithelial cell). These include, but are not limited to, glioblastomas, a melanomas, or carcinomas of the lung, breast, ovary, stomach, pancreas, bladder, head, neck, colon, rectum, or kidney.
  • the carcinomas are those with a high frequency of ERBB abnormalities, such as lung, breast, melanoma, glioblastoma, bladder, head and neck.
  • the carcinoma is a lung carcinoma, such as small cell lung carcinoma or non-small cell lung carcinoma (NSCLS).
  • the carcinoma is a breast carcinoma.
  • the carcinoma sample may be analyzed by various techniques known to those skilled in the art.
  • One such technique comprises a) lysing cancerous epithelial cells isolated from the sample; and b) analyzing the lysate to determine the ratio of the relative amounts of phosphorylation of the amino acid residues.
  • the cells may be isolated by microdissection.
  • Various techniques known to those skilled in the art may be used.
  • LCM laser capture microdissection
  • the lysate is analyzed by various assay techniques in order to measure the amount of phosphorylation of the particular amino acid residues.
  • the techniques include immunoassays, enzyme-linked immunosorbent assay (ELISA), colorimetric assays, assays based on fluorescent readouts, histochemical assays, mass spectrometry, and Western blot.
  • Other techniques include mixing the lysate with beads that bind to the proteins or to nanoparticles that trap the proteins and then detecting the proteins with antibodies.
  • Luminex spectral addressable beads/cell sorter system (Austin, TX), Invitrogen's Dyna Beads (Carlsbad, CA), Ceres Nanosciences' Nanotraps (Manassas, VA), and Ambion's MagnaBeads with MALDI-TOF (Austin, TX).
  • the lysate is analyzed by reverse phase protein microarray analysis.
  • a protein microarray is an assay format that utilizes a substrate for simultaneously testing multiple samples as well as for testing multiple target proteins in the same assay.
  • the microarray format is not limited to particular embodiments but can comprise any arrangement and substrate that serves to provide a plurality of individual samples for testing.
  • the microarray comprises a flat substrate with rows and columns of individual spots, each spot comprising a sample, while in other embodiments, the microarray comprises a flat substrate with a plurality of depressions, for example, a 96-well plate, in which each depression contains one sample.
  • microarray substrates examples include nitrocellulose, derivatized glass slides, and 3- dimensional substrates, such as hydrogels.
  • nitrocellulose-coated glass slides include FAST slides (Grace BioLabs, Bend, OR or GE Healthcare), which have protein binding capacities of 75-150 ug/cm2 in a volume of 0.3-2.0 nl/spot. Nitrocellulose-coated glass slides are particularly useful, as a variety of detection methods can be used with this substrate, including chromogenic, fluorometric, and luminescent detection methods.
  • the number of samples that can be deposited onto a microarray substrate can vary.
  • the size of the substrate can often determine how many samples are located on the substrate.
  • the protein microarray comprises around 100 spots; in other embodiments, the protein microarray may comprise around 1,000 spots or around 10,000 spots. In yet other embodiments, the microarray comprises from about 1 to about 10,000 spots, about 50 to about 10,000 spots, or about 500 to about 10,000 spots. In some embodiments, the microarray comprises less than about 100,000 spots.
  • the sample volume which is deposited on each spot and used to form each spot on the microarray can also vary.
  • the volume can depend on diameter of the pin (contact printing), the inherent qualities of the pin hydrophobicity, and the method of supplying the sample.
  • the amount of sample deposited/printed can range from less than about 1 pico liter to about 100 nano liters.
  • Samples are placed or loaded onto the substrate using any one of a number of mechanisms known in the art (see Schena, "Microarray biochip technology” Eaton Pub., Natick MA, 2000, incorporated herein by reference in its entirety).
  • the samples are printed onto the microarray using a printer.
  • the printing technique can be contact or non-contact printing, and can be automated.
  • Protein microarray formats can fall into two major classes, the Forward Phase Array (FPA) and the Reverse Phase Array (RPMA), depending on whether the analyte is capture from solution phase or bound to solid substrate.
  • Forward Phase Arrays immobilize a bait molecule, such as an antibody designed to capture a specific analyte within a mixture of test sample proteins.
  • FPAs the capture molecule specific for the analyte is immobilized on a substrate. The capture molecule is then exposed to the sample, binding the analyte in the sample and immobilizing the analyte onto the substrate. The bound analyte can then be detected using a detectable label.
  • the label can bind to the analyte directly, or can be attached to a secondary "sandwich" antibody that is specific for the analyte.
  • the capture molecule can be any molecule that has specificity for an analyte and includes, but is not limited to, peptides, proteins, antibodies or fragments thereof, oligomers, DNA, RNA, and PNA. In some embodiments, the capture molecule is an antibody or fragment thereof specific for the analyte.
  • RPMAs Reverse Phase Arrays immobilize the test sample analytes on a solid substrate.
  • the sample is placed directly on the substrate, allowing analyte in the sample to bind directly to the substrate.
  • a detection molecule specific for the analyte is then exposed to the substrate, allowing an analyte-detection molecule complex to form.
  • the detection molecule can comprise a detectable label to indicate the presence of the analyte.
  • a secondary molecule specific for the detection molecule and comprising a detectable label can be provided, allowing for an analyte-detection molecule-labeled secondary molecule complex to form.
  • RPMAs are highly sensitive and do not require a large amount of sample.
  • the high sensitivity exhibited by RPMAs is due in part to the detection molecule, which can be conjugated to a detectable label, and is also due in part to the fact that the signal from the label can be amplified independently from the immobilized analyte.
  • RPMAs can use tryamide amplification which generates high number of florescent signal on each spot, or florescent signals that are near-IR wavelength, which is outside the emission spectra for nitrocellulose.
  • Amplification chemistries that are available take advantage of methods developed for highly sensitive commercial clinical immunoassays (see, for example, King et al., J. Pathol. 183: 237-241 (1997)).
  • RPMAs can also exhibit excellent "within run” and "between run” analytical precision. RPMAs do not require direct labeling of the sample analyte and do not utilize a two-site antibody sandwich. Therefore, there is no experimental variability introduced due to labeling yield, efficiency, or epitope masking.
  • the reference ratio range is determined by techniques known to those skilled in the art.
  • samples of carcinoma cells are obtained from a collection or group of patients, each of whom has the same type of carcinoma.
  • Each sample is analyzed to determine if the ERBB family pathway in the cells is activated and if ERBB proteins in the cells have one or more mutations that affect phosphorylation of one or more amino acid residues in the proteins.
  • the carcinoma cells are analyzed to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway.
  • the relative amounts of phosphorylation can be determined by the reverse phase protein microarray techniques disclosed herein.
  • the previous steps are repeated a sufficient number of times with a sufficient number of samples from different patients to create a range of reference ratios for the amino acid residues, wherein the range is at least 80% specific for detecting carcinoma cells having one or more mutations in one or more ERBB family pathway proteins and/or having an activated ERBB family pathway.
  • the term "specific" means providing a true positive, not a false positive.
  • the method of determining the reference ratio range is repeated for different amino acid residues and for different types of carcinoma, such as NSCLC and breast carcinoma. As mentioned herein, the phosphorylation of one or more different amino acid residues is measured.
  • the relative amounts of phosphorylation of two different amino acid residues i.e., a pair, is determined, which provides a ratio.
  • a ratio In one aspect of the invention, only one pair of residues is used. However, the determination of relative phosphorylation need not be limited to one pair of amino acid residues on any of the proteins within the ERBB family pathway. Additional pairs may be measured to provide additional ratios. Also, a ratio can be determined by adding the amounts of phosphorylation of two different amino acid residues and comparing the sum to the amount of phosphorylation of a third residue.
  • the method is directed to determining if the carcinoma will respond to an EGFR pathway inhibitor.
  • EGFR family pathway inhibitor means an exogenous agent (including a chemical substance, compound, molecule, or ion, such as an antibody, a drug, or a nucleic acid) which suppresses the signaling activity of the EGFR pathway driven by one or more of the ERBB family receptors.
  • the method comprises the steps of: a) analyzing a sample of the carcinoma to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in EGFR, or one amino acid residue in EGFR and another amino acid residue in different a different protein in the EGFR pathway, such as Her2, and b) comparing the ratio of step (a) to a range of reference ratios for the same amino acid residues in carcinoma cells from a reference population of patients with the same type of carcinoma, wherein the range of reference ratios is at least 80% specific for detecting carcinoma cells having a mutation in EGFR and/or having an activated EGFR pathway. If the ratio of step (a) is within the range of reference ratios, the carcinoma is likely to respond to the inhibitor.
  • amino acid residues are measured, to provide only one ratio.
  • the amino acid residues may be any sites on an ERBB family protein that can be phosphorylated. These include, but are not limited, to Y 1248 in Her2 and S 1026, S 1070, S1071, Sl 190, S695, S991, T678, T693, Y1016, Y1045, Y1068, Y1092, Y1110, Yl 172, Yl 192, Yl 197, Y869, and Y998 in EGFR.
  • the sites evaluated are the following sites in EGFR: S1026, S1070, S1071, Sl 190, S695, S991, T678, T693, Y1016, Y1045, Y1068, Y1092, Y1110, Yl 172, Yl 192, Yl 197, Y869, and Y998.
  • the amino acid residues can be selected from the following: Y845, Y1045, Y1068, Yl 148, Yl 173, and L858R, and especially the following pairs: Y1045 and Y1068; Y1045 and Yl 148; Y1045 and Y845; Y1045 and Yl 173; and Yl 045 and L858R.
  • the pair is Y 1045 and Y 1068.
  • the ratio is calculated by comparing to the relative phosphorylation of one residue to the sum of the relative phosphorylations of two other residues.
  • the following residues are useful: Y1045:(Y1068+Yl 148), L858R:(Y1148+Y1068), and Her2 Y1248:(Y1148+Y1068). These ratio combinations were selected based on statistical significance within the reference population.
  • the individual epitopes were chosen because of their functional role in the coupling of the receptor to downstream signaling pathways or their influence on the kinase activity of the receptor.
  • ERBB family pathway inhibitor is an exogenous agent (including a chemical substance, compound, molecule, or ion, such as an antibody, a drug, or a nucleic acid) that suppresses the signaling activity of the ERBB family receptors. This includes small molecules that inhibit phosphorylation of one or more amino acid residues in a protein in the ERBB family pathway, large molecules, such as antibodies that bind to a receptor in the ERBB family pathway, and nucleic acid-based inhibitors.
  • Small molecule inhibitors include kinase inhibitors, especially tyrosine kinase inhibitors.
  • kinase inhibitors especially tyrosine kinase inhibitors.
  • examples include Erlotinib (Tarceva®), Gefitinib (Iressa®), Lapatinib (Tykerb®), Vandetanib (ZactimaTM), Cl- 1033 (Pfizer), EKB-569 (Wyeth), GW2016 (GlaxoSmithKline), GW572016 (Glaxo SmithKline), and PHI166 (Novartis).
  • the inhibitor is Erlotinib (Tarceva®).
  • Antibodies include polyclonal and monoclonal antibodies.
  • Monoclonal antibodies include Cetuximab (Erbitux®),
  • the monoclonal antibody is Trastuzumab (Herceptin®).
  • Nucleic acid-based inhibitors include antisense RNA, ribozymes, and interfering RNA (RNAi) molecules, including small interfering RNA (siRNA) molecules. These inhibitors block or degrade mRNA that encodes a protein in the ERBB family pathway or a microRNA (miRNA) molecule that controls expression of one or more of these proteins.
  • the invention provides a method of determining if a person with a non- small cell lung carcinoma will respond to a tyrosine kinase inhibitor.
  • the method comprises a first step of analyzing a sample of the carcinoma to determine the ratio of the relative amounts of phosphorylation of two or more different tyrosine residues in an EGFR protein in the sample, wherein the two tyrosine residues are selected from the group consisting of: Y845, Y1045, Y1068, Yl 148, Yl 173, and L858R, for example the pair Y1045 and Y1068.
  • Cancerous epithelial cells are isolated from a sample of the carcinoma by laser capture microdissection, and the cells are lysed by standard techniques.
  • the lysate is analyzed by placing it onto a reverse phase protein microarray, contacting it with phospho-antibodies to the two tyrosine residues, and determining the ratio of the relative amounts of phosphorylation by measuring the amount of phosphorylation of each of the two residues.
  • the ratio determined by the first step is compared to a reference ratio range for the same tyrosine residues in non-small cell lung carcinoma cells from a reference population of persons with non- small cell lung carcinoma.
  • the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in EGFR and/or having an activated EGFR pathway. If the ratio from the first step is within the reference ratio range, the carcinoma is likely to respond to the inhibitor.
  • Useful tyrosine kinase inhibitors include Erlotinib (Tarceva®), Gefitinib (Iressa®), and Lapatinib (Tykerb®).
  • the invention also includes a method of treating a mammal with a carcinoma.
  • the diagnostic method disclosed herein is used to determine if the carcinoma is likely to respond to an ERBB family pathway inhibitor. If it is likely to respond, then a pharmaceutically effective amount of an ERBB family pathway inhibitor is administered to the mammal.
  • the method comprises the step of administering a pharmaceutically effective amount of an ERBB family pathway inhibitor to the mammal if, in a sample of the mammal's carcinoma, the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway is within a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma as the carcinoma in the mammal.
  • the reference ratio range is at least 80% specific ( 80% probability of a true positive correct determination that a mutation or activated receptor is present) for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway.
  • the carcinoma may be any carcinoma, including but not limited to, glioblastomas, melanomas, or carcinomas of the lung, breast, ovary, stomach, pancreas, bladder, head, neck, colon, rectum, or kidney, or any malignant neoplasm considered for treatment with any of the aforementioned inhibitors.
  • the carcinoma is a lung carcinoma, such as small cell lung carcinoma and non-small cell lung carcinoma (NSCLS).
  • NSCS non-small cell lung carcinoma
  • the carcinoma is a breast carcinoma.
  • the mammal may be any mammal, including but not limited to, humans and other primates, dogs, cats, rats, mice, rabbits, guinea pigs, and farm animals.
  • the principal use of the invention is expected to be to select therapeutic regimens for humans with carcinomas and then treat them with the selected regimen.
  • the invention can be used to treat pets and farm animals, and it can be used on laboratory animals to test inhibitors and to find and develop new ones.
  • the inhibitors used for treatment include small molecules that inhibit phosphorylation of one or more amino acid residues in a protein in the ERBB family pathway, antibodies that bind to a receptor in the ERBB family pathway, and interfering RNA (RNAi) molecules, including small interfering RNA (siRNA) molecules that block or degrade mRNA that encodes a protein in the ERBB family pathway.
  • RNAi interfering RNA
  • siRNA small interfering RNA
  • Examples of phosphorylation inhibitors include Erlotinib (Tarceva®), Gefitinib (Iressa®), Lapatinib (Tykerb®), Vandetanib (ZactimaTM), Cl- 1033 (Pfizer), EKB-569 (Wyeth), GW2016 (GlaxoSmithKline), GW572016 (Glaxo SmithKline), and PHI166 (Novartis).
  • Examples of monoclonal antibody inhibitors include Cetuximab (Erbitux®), (Panitumumab (Vectibix®), Trastuzumab (Herceptin®), EMD72000 (Merck), and MDX447 (Medarex/Merck). More than one of these compounds can be administered at the same time or at different times.
  • inhibitors included within the scope of the claimed invention are not limited to the specific chemical compounds and drug products disclosed herein. Any compound, molecule, or other agent that inhibits the ERBB family pathway is included.
  • the invention provides a method for treating a person with a non-small cell lung carcinoma.
  • the method comprises the step of administering a pharmaceutically effective amount of an EGFR tyrosine kinase inhibitor to the person if, in a sample of the person's carcinoma, the ratio of the relative amounts of phosphorylation of any one or more pairs of two different tyrosine residues in the EGRF protein is within a reference ratio range for the same pairs of amino acid residues in carcinoma cells from a reference population of patients with non-small cell lung carcinoma.
  • the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in EGFR and/or having an activated EGFR pathway.
  • the tyrosine residues are selected from the group consisting of: Y845, Y1045, Y1068, Yl 148, Yl 173, and L858R.
  • the residues Y1045 and Y1068 are especially useful.
  • the tyrosine kinase inhibitor is selected from the group consisting of Erlotinib (Tarceva®), Gef ⁇ tinib (Iressa®), and Lapatinib (Tykerb®).
  • the treatment methods of the invention may further include the step of administering to the mammal a pharmaceutically effective amount of an inhibitor that inhibits phosphorylation of a protein that is downstream of EGFR in the EGFR signal transduction pathway. This is a second inhibitor and is administered in addition to the first.
  • inhibitors include tyrosine kinase inhibitors, serine kinase inhibitors, threonine kinase inhibitors, monoclonal antibodies that bind to EGF or EGFR, and nucleic acid molecules that block or degrade mRNA that encodes a protein in the ERBB family pathway or an miRNA molecule that controls expression of one or more of these proteins.
  • inhibitors that may be administered: Erlotinib (Tarceva®), Gefitinib (Iressa®), Lapatinib (Tykerb®), Vandetanib (ZactimaTM),Cl-1033 (Pfizer), EKB- 569 (Wyeth), GW2016 (Glaxo SmithKline), GW572016 (GlaxoSmithKline), and PHI166 (Novartis), Cetuximab (Erbitux®), (Panitumumab (Vectibix®), Trastuzumab (Herceptin®), EMD72000 (Merck), and MDX447 (Medarex/Merck). More than one of these downstream inhibitors can be administered at the same time.
  • the dosages, routes of administration, and frequency of administration will be known to, or readily determinable by, persons skilled in the art. For FDA approved drugs, such information will be found in the package insert. More than one inhibitor can be administered at the same time or at different times.
  • the invention includes a method to identify a novel ERBB family pathway inhibitor.
  • the method comprises the steps of: (a) contacting a chemical compound with carcinoma cells, wherein the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway in the cells is within a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma, wherein the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway ; and (b) determining if a significant number of cancerous cells in the carcinoma have been killed or have had their growth suppressed.
  • the amino acid residues are in EGFR and the reference ratio range is at least 80% specific for detecting carcinoma cells having an EGFR mutation and/or having an activated EGFR pathway.
  • the inhibitor is a tyrosine kinase inhibitor.
  • the invention includes a kit for performing the assay for identifying novel ERBB family pathway inhibitors.
  • the kit comprises the carcinoma cells described herein and a chemical compound to be evaluated.
  • the cells are ones wherein the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway in the cells is within a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma, wherein the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway.
  • the compound is contacted with the carcinoma cells to determine if a significant number of cancerous cells in the carcinoma are killed by it.
  • the diagnostic and treatment methods of the invention are especially useful in personalized medicine; i.e., the design and implementation of therapeutic regimens for an individual patient.
  • a further application of this approach involves the use of the diagnostic method to determine which of several inhibitors, which could provide a therapeutic benefit, would actually provide the greatest benefit.
  • a sample of the carcinoma is analyzed as described herein to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway. The ratio is compared to a reference ratio range for those amino acid residues as described herein.
  • the amino acid residues are in EGFR and the reference ratio range is at least 80% specific for detecting carcinoma cells having an EGFR mutation and/or having an activated EGFR pathway; the ratio of residues is selected from the group consisting of: Y1045 and Y1068; Y1045 and Yl 148; Y1045 and Y845; Y1045 and Yl 173; and Y 1045 and L858R; and the carcinoma is non-small cell lung carcinoma.
  • EGFR signaling involves five distinct steps: 1) ligand binding, 2) conformational change in the receptor, 3) homodimerization and/or heterodimerization with other ERBB family receptors, 4) autophosphorylation of tyrosine residues, and 5) transphosphorylation of downstream kinases. Somatic mutations within the kinase domain may affect each of these steps independently or result in a cascade of altered downstream signaling events dependent on the specific receptor mutation.
  • Each individual patient's carcinoma specimen contains a variable, heterogeneous proportion of stroma, lung parenchyma, bronchial epithelium, inflammatory cells, and endothelial cells. All of these non-carcinoma subpopulations may contain EGF receptors, participate in EGFR signaling, or may contribute EGFR related ligands. Furthermore, it is unknown what proportion of the carcinoma population at any point in time is undergoing active signaling for a specific pathway.
  • LCM Laser capture microdissection
  • Tissue samples were sequenced for exons 18-21 of the EGF receptor (EGFR tyrosine kinase domain) to identify known mutations.
  • EGF receptor EGFR tyrosine kinase domain
  • the frozen section slides were fixed briefly in 70% ethanol, rinsed in water, stained with Mayer's Hematoxylin (Sigma Aldrich, St. Louis, MO), developed in Scott's Tap Water (ThermoFisher) and dehydrated in an ethanol gradient (70%, 95%, 100%) with a final rinse in xylene (Sigma).
  • the sections were allowed to air dry briefly prior to laser capture microdissection.
  • Pure tumor cell populations were microdissected with a PixCell He or Veritas LCM instrument (Molecular Devices). Microdissected cells were stored at -80 0 C prior to microarray construction.
  • Reverse Phase Protein Microarray construction The microdissected cells were subjected to lysis with a 2.5% solution of 2-mercaptoethanol (Sigma, St. Louis, MO) in T- PERTM (Pierce, Rockford, IL)/2X SDS Tris-glycine 2X SDS buffer (Invitrogen). Reverse phase protein microarrays were printed in duplicate with whole cell protein lysates as described by Petricoin et al (40).
  • the lysates were printed on glass backed nitrocellulose array slides (FAST Slides Whatman, Florham Park, NJ) using a GMS 417 arrayer (Affymetrix, Santa Clara, CA) equipped with 500 ⁇ m pins or an Aushon 2470 arrayer equipped with 350 ⁇ m pins (Aushon Biosystems, Billerica, MA). Each lysate was printed in a dilution curve representing neat, 1 :2, 1 :4, 1 :8, 1 :16 and negative control dilutions.
  • the slides were stored with desiccant (Drierite, W.A. Hammond, Xenia, OH) at -20 0 C prior to immunostaining. Reverse phase protein microarray immunostaining.
  • Immunostaining was performed on an automated slide stainer per manufacturer's instructions (Autostainer CSA kit, Dako, Carpinteria, CA). Each slide was incubated with a single primary antibody at room temperature for 30 minutes. Each array was probed with a single polyclonal or monoclonal primary antibody. The negative control slide was incubated with antibody diluent. Secondary antibody was goat anti-rabbit IgG H+L (1 :5000) (Vector Labs, Burlingame, CA) or rabbit anti-mouse IgG (1 :10) (Dako).
  • Antibody validation and phosphoprotein specificity Primary antibodies were validated prior to use by immunoblotting with complex cellular lysates such as commercial cell lysates or human tissue lysates . Criteria for antibody validation were a) a single band at the correct molecular weight, or b) if two bands were present, 80% of the signal must have been at the correct molecular weight. Specificity of the phosphoprotein- specific antibodies was verified by peptide competition on an immunoblot when the corresponding peptide/antibody pair was available. Specificity criteria were a reduction in signal intensity in the presence of the corresponding peptide compared to the antibody alone. Further specificity for phosphospecific antibodies was verified by peptide/antibody reactivity on a
  • Forward primer (AGGCCTGCTGAAAATGACTG) and reverse primer (TCCTGAGCCTGTTTTGTGTCT) were used to amplify exons 1 -4 for K- RAS sequencing.
  • PCR was executed with Platinum® PCR Supermix (Invitrogen) according to manufacturer's recommendations with an annealing temperature of 55°C. Amplicons were purified using QiaQuick PCR purification kit (Qiagen). Sequencing was completed by Northwoods DNA, Inc. using forward primer (CCAACCAAGCTCTCTTGAGG) to determine presence or absence of T790M and EGFR L858R mutations and reverse primer (TGACCTGCTGTGTCGAGAAT) to determine presence or absence of K-RAS mutation.
  • EGFR mutations are associated with site-specific phosphorylation of EGF receptor in NSCL lung carcinoma cells procured by LCM.
  • LCM human lung carcinoma cells procured by LCM.
  • An advantage of this study was the collection of snap frozen tumor specimens at the time of primary surgical diagnosis. Therefore, we were not evaluating the effects of treatment on the cellular signaling pathways but rather the state of the tumor at time of procurement in treatment na ⁇ ve patients. A total of 31 cases were available for this study.
  • Criteria were: a) presence of tumor cells, b) sufficient size and quantity of tissue, and c) absence of dehydration or freeze-thaw artifacts. 1 case was judged inadequate due to freezer dehydration artifacts. 4 cases consisted of connective tissue with minimal tumor cells. One case was not analyzed due to insufficient total protein on the microarray. 83% of the cases were judged to be adequate for microdissection and array analysis. 10-15 cryosections were prepared for each case, with 1-2 sections/slide. The average number of laser pulses (shots) per slide was 1149, with an average microdissection efficiency of 95%, resulting in approximately 15,000 - 30,000 cells/sample, collected from multiple cryosections.
  • the mutant EGFR as a class appeared different from the wild type with regard to elevated phosphorylation of specific EGFR sites, while the wild type as a class had elevation in Her2 Y1248, IRS-I S612 and Smad2 S465/467.
  • EGFR mutant tissue carcinoma cell populations exhibit distinct pairwise correlations compared to wild type.
  • Spearman's rho non-parametric analysis was conducted to examine the strength of the linear relationship between pairs of endpoints among all the endpoints analyzed. Spearman's rho correlation coefficient is computed on the ranks of the data using the formula for the Pearson's correlation. The Pearson product-moment correlation coefficient measures the strength of the linear relationship between two variables.
  • Table 2 displays those correlations which have a significant p value (p ⁇ 0.01) and correlation coefficient >0.80.
  • the mutant carcinomas were found to have a correlation between pairs of phosphorylation sites on EGFR and AKT (Table 2).
  • the wild type samples lacked correlations of these same two proteins and phosphorylation sites.
  • the wild type samples were scored as having strong correlations between mTOR S2481 and IRS-I S612; Cox2 and IRS-I S612; and EGFR Y992 and 14-3-3 ⁇ (zeta/gamma/eta). These correlations were not observed in the mutant.
  • mutant samples showed a significant correlation between 11 protein pairs including Src Y527 and She Y317, as well as EGFR Y 1045 and 14- 3-3 ⁇ .
  • the point mutation specific antibody, anti-EGFR L858R did not show any statistically significant differences in our study set of 8 EGFR mutated samples and 17 wild type samples, even though there was clustering of L858R mutant samples by unsupervised hierarchical clustering (Figure 1). This is most likely due to the inclusion of multiple classes of mutations in the mutated group and the specificity of the antibody for the L858R mutation.
  • the observed signaling profiles in this study set may provide clues indicating the organization of the EGFR related protein network linkages regarding differences between the wild type and mutant adenocarcinoma samples. Comparisons of specific EGFR related protein ratios. Specific EGFR related protein ratios were evaluated to distinguish NSCLC cells harboring known EGFR mutations from wild type EGFR. Monotonic variables or ratios of phosphorylated ERBB family proteins, or total EGFR, were found to be statistically different in the EGFR mutated cohort compared to the wild type EGFR group.
  • Ratios were calculated by: a) determining the mean of each group (mutant versus wild type) for specific protein endpoints (i.e. Y1045), or b) calculating a ratio of the monotonic variable to the sum of specific endpoints (i.e. Y1068+Y1148). See Table 3. Table 1. Differentially activated proteins in microdissected NSCLC samples as a comparison of EGFR tyrosine kinase domain mutation status (wild type versus mutant).
  • IRS-1 S612 Insulin ⁇ 0.001 0.170 0.546 signaling/Growth
  • APC2 0.8809 0.004 S2481 SRC SHC 0.8809 0.004 Y527 Y317 EGFR EGFR 0.8684 0.005 Y845 Y1148 14-3-3 EGFR 0.8625 0.006 ⁇ y ⁇ Y1045
  • Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Yl 148(p ⁇ 0.044) and Y1068(p ⁇ 0.026), and decreased phosphorylation of EGFR Y1045(p ⁇ 0.002), Her2 Y1248(p ⁇ 0.015), IRS-I S612(p ⁇ 0.001), and Smad S465/467(p ⁇ 0.011), across all classes of mutated EGFR patient samples compared to wild type.
  • EGF receptor mutation status known to correlate with sensitivity to EGF pathway inhibitors, may be identified via quantitative comparison of at least two phosphorylated epitopes on the EGF receptor, or comparison of phosphorylated epitopes to total EGFR
  • b) specific ratios of protein endpoints can distinguish cells with EGFR mutation from wild type
  • c) patients exhibiting greater phosphorylation of total EGFR, Y1068, Yl 173, and/or Yl 148 compared to Y1045 may benefit from tyrosine kinase inhibitor therapy regardless of EGFR mutation status.
  • An elevated level of phosphorylation of one or more of these residues in proteins of associated downstream pathways may indicate that the patient is likely to be responsive to a) EGFR therapy (e.g. to treatment with an agent that inhibits the kinase activity of EGFR), b) a kinase inhibitor directed towards an associated downstream pathway, or c) a combination of an EGFR inhibitor and an inhibitor of a member of one of the identified downstream pathways.
  • activity of the downstream proteins indicates that the entire EGF associated pathway is active and in use in the cancer cells. Since the entire pathway is active, this may indicate that the cancer cell is more likely being driven by this pathway, and thus would be effectively treated by blocking multiple nodes along this pathway.
  • the measured ratio(s) of EGFR phosphoproteins, as well as individual or pairs of phosphoprotein data may be used to design individualized therapy regimens for single or combination agents.
  • This therapy may be a) EGFR therapy (e.g. to treatment with an agent that inhibits the kinase activity of EGFR), b) a kinase inhibitor directed towards an associated downstream pathway, or c) a combination of an EGFR inhibitor and an inhibitor of a member of one of the identified downstream pathways.
  • this information may be used to predict potential response to tyrosine kinase inhibitor therapy.
  • EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA. 101, 13306-11
  • Rapamycin induces feedback activation of Akt signaling through an IGF-lR-dependent mechanism. Oncogene.

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Abstract

La présente invention a pour objet général le traitement du cancer et plus particulièrement l’identification de carcinomes qui sont susceptibles de réagir à un inhibiteur de la voie de la famille ERBB dans les cellules de carcinome et le traitement de patients ayant de tels carcinomes avec de tels inhibiteurs. L’invention comprend, mais n’est pas limitée à : 1) un procédé permettant de déterminer si un carcinome chez un mammifère est susceptible de réagir à un inhibiteur de la voie de la famille ERBB; 2) un procédé de traitement du mammifère avec un inhibiteur de la voie de la famille ERBB; 3) un procédé pour l’identification de tels inhibiteurs; et 4) un kit destiné à être utilisé dans cette analyse.
PCT/US2009/056132 2008-09-04 2009-09-04 Analyse de phosphoprotéine de carcinomes pour l'évaluation de la sensibilité aux médicaments WO2010028313A2 (fr)

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US10317407B2 (en) 2007-08-16 2019-06-11 The Royal Institution For The Advancement Of Learning/Mcgill University Tumor cell-derived microvesicles
US10352935B2 (en) 2007-08-16 2019-07-16 The Royal Institution For The Advancement Of Learning/Mcgill University Tumor cell-derived microvesicles
WO2021064610A1 (fr) * 2019-10-01 2021-04-08 Ac Immune Sa Dosages de micro-radioliaison pour criblage de ligands

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CN105158474A (zh) * 2015-09-18 2015-12-16 安徽省立医院 一种用于检测血清her2的试剂盒及用途

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VANMETER ET AL.: 'Laser capture microdissection and protein microarray analysis of human non -small cell lung cancer: differential epidermal growth factor receptor (EGPR) phosphorylation events associated with mutated EGFR compared with wild type' MOL CELL PROTEOMICS vol. 7, no. 10, October 2008, pages 1902 - 1924 *
WULFKUHLE ET AL.: 'Multiplexed cell signaling analysis of human breast cancer applications for personalized therapy' J PROTEOME RES vol. 7, no. 4, April 2008, pages 1508 - 1517 *
ZHANG ET AL: 'Silencing the epidermal growth factor receptor gene with RNAi may be developed as a potential therapy for non small cell lung cancer' GENET VACC THERAP vol. 3, 30 June 2005, *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10317407B2 (en) 2007-08-16 2019-06-11 The Royal Institution For The Advancement Of Learning/Mcgill University Tumor cell-derived microvesicles
US10352935B2 (en) 2007-08-16 2019-07-16 The Royal Institution For The Advancement Of Learning/Mcgill University Tumor cell-derived microvesicles
US9086414B2 (en) 2008-07-08 2015-07-21 George Mason Research Foundation, Inc. Phosphorylated C-ErbB2 as a superior predictive theranostic marker for the diagnosis and treatment of cancer
WO2021064610A1 (fr) * 2019-10-01 2021-04-08 Ac Immune Sa Dosages de micro-radioliaison pour criblage de ligands

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